http://2007.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=50&target=Blent&year=&month=2007.igem.org - User contributions [en]2024-03-28T15:27:01ZFrom 2007.igem.orgMediaWiki 1.16.5http://2007.igem.org/wiki/index.php/NYMU_Taipei/Part_ListNYMU Taipei/Part List2008-01-24T09:08:45Z<p>Blent: /* Part List */</p>
<hr />
<div><p><font size="4"><br />
[[NYMU_Taipei/ProjectDescription| Project Description]] /<br />
[[NYMU_Taipei/Lab_Notes| Lab Notes]] /<br />
[[NYMU_Taipei/Part_List| Part List]] /<br />
[[NYMU_Taipei/Primer_List| Primer List]]<br />
</font><br /><hr></p><br />
<br />
==Part List==<br />
{| style="color:green;background-color:#ffffcc;" cellpadding="20" cellspacing="0" border="1"<br />
!Series<br />
!alias<br />
!Location<br />
!Description<br />
!part length<br />
!vector (antibotics)<br />
!Check<br />
|-<br />
|[http://partsregistry.org/Part:BBa_R0082 BBa_R0082]<br />
|pOmpC<br />
|1<br />
|Positively regulated, OmpR-controlled promoter<br />
|108<br />
|pSB1A2<br />
|?<br />
|-<br />
|[http://partsregistry.org/Part:BBa_C0082 BBa_C0082]<br />
|tar-envZ<br />
|1<br />
|Receptor, tar-envZ<br />
|1491<br />
|pSB2K3<br />
|-<br />
|[http://partsregistry.org/Part:BBa_B0034 BBa_B0034]<br />
|RBS<br />
|1,2,3<br />
|The Elowitz RBS (Ribosome binding site) is the definition of efficiency 1.0<br />
|12<br />
|pSB1A2<br />
|?<br />
|-<br />
|AU43-C7<br />
|[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&id=13528923 INS]<br />
|<br />
|human insulin<br />
|495<br />
|C<br />
|-<br />
|<br />
|INS_A<br />
|1<br />
|alpha chain of human insulin<br />
|63<br />
|<br />
|-<br />
|<br />
|INS_B<br />
|1<br />
|beta chain of human insulin<br />
|90<br />
|<br />
|-<br />
|[http://partsregistry.org/Part:BBa_B0014 BBa_B0014]<br />
|D-term<br />
|1,2,3<br />
|Double transcription terminator<br />
|95<br />
|pSB1AK3<br />
|OK<br />
|-<br />
|[http://partsregistry.org/Part:BBa_I14032 BBa_I14032]<br />
|pLacIQ<br />
|2<br />
|Constitutive Promoter, High Transcription<br />
|37<br />
|pSB2K3<br />
|-<br />
|<br />
|OmpRBS<br />
|2<br />
|OmpR binding sites<br />
|65<br />
|<br />
|?<br />
|-<br />
|AV88-C6<br />
|[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&id=27371195 IDE]<br />
|2<br />
|insulin degradation enzyme of mouse<br />
|3445<br />
|A<br />
|-<br />
|AU61-F6<br />
|[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&id=33879043 IGFBP3]<br />
|2<br />
|Insulin-like growth factor binding protein 3 of human<br />
|2488<br />
|C<br />
|-<br />
|AU50-C7<br />
|[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&id=38114635 IGFBP7]<br />
|2<br />
|Insulin-like growth factor binding protein 7 of human<br />
|1155<br />
|C<br />
|-<br />
|[http://partsregistry.org/Part:BBa_I15016 BBa_I15016]<br />
|B0032.ECFP<br />
|16D, plate 1<br />
|This part accepts POPS to produce ECFP under the medium RBS B0032<br />
|742<br />
|pSB1A2<br />
|OK<br />
|-<br />
|[http://partsregistry.org/Part:BBa_I15017 BBa_I15017]<br />
|B0032.EYFP<br />
|16F, plate 1<br />
|This part accepts POPS to generate EYFP under the medium strength RBS B0032<br />
|742<br />
|pSB1A2<br />
|OK<br />
|-<br />
|[http://partsregistry.org/Part:BBa_E0840 BBa_E0840]<br />
|B0030(RBS).E0040(GFP).B0015(D-term)<br />
|16E, plate 1<br />
|gfp tri-part; strong rbs<br />
|878<br />
|pSB1A2<br />
|OK<br />
|-<br />
|[http://partsregistry.org/Part:BBa_R0077 BBa_R0077]<br />
|pCinRHSL, RBS+<br />
|17L, Plate 1<br />
|CinR-HSL complex binds to this promoter and activates transcription. This part contains RBS.<br />
|231<br />
|pSB1A2<br />
|OK<br />
|-<br />
|-<br />
|[http://partsregistry.org/Part:BBa_R0078 BBa_R0078]<br />
|pCinRHSL<br />
|15D, Plate 1<br />
|CinR-HSL complex binds to this promoter and activates transcription. This part DOES NOT contain RBS.<br />
|225<br />
|pSB1A2<br />
|?<br />
|-<br />
|[http://partsregistry.org/Part:BBa_C0077 BBa_C0077]<br />
|CinR<br />
|7G, Plate 2 <br />
|cinR activator<br />
|762 <br />
|pSB2K3<br />
|OK<br />
|-<br />
|[http://partsregistry.org/Part:BBa_C0076 BBa_C0076]<br />
|HSL<br />
|7E, Plate 2<br />
|autoinducer<br />
|702<br />
|pSB2K3<br />
|OK<br />
|-<br />
|}<br />
<br />
==antibiotics keys==<br />
* A: [http://en.wikipedia.org/wiki/Ampicillin Ampicillin]<br />
** refernece at OpenWetWare: http://openwetware.org/wiki/Ampicillin<br />
** stock (100 mg/mL = 100 ug/uL) ampicillin<br />
** liquid culture (50 ug/mL = 250 ug/5mL)<br />
** 5mL liquid culture = 5 c.c. LB + 2.5uL stock ampicillin<br />
* K: [http://en.wikipedia.org/wiki/Kanamycin Kanamycin]<br />
* C: [http://en.wikipedia.org/wiki/Chloramphenicol Chloramphenicol]<br />
** 35 mg/mL<br />
** 20 mg/mL in Ethanol<br />
** we prepared 100mg in 5 mL and stored in -20 C</div>Blenthttp://2007.igem.org/wiki/index.php/NYMU_Taipei/Part_ListNYMU Taipei/Part List2008-01-24T09:07:53Z<p>Blent: /* Part List */</p>
<hr />
<div><p><font size="4"><br />
[[NYMU_Taipei/ProjectDescription| Project Description]] /<br />
[[NYMU_Taipei/Lab_Notes| Lab Notes]] /<br />
[[NYMU_Taipei/Part_List| Part List]] /<br />
[[NYMU_Taipei/Primer_List| Primer List]]<br />
</font><br /><hr></p><br />
<br />
==Part List==<br />
{| style="color:green;background-color:#ffffcc;" cellpadding="20" cellspacing="0" border="1"<br />
!Series<br />
!alias<br />
!Location<br />
!Description<br />
!part length<br />
!vector (antibotics)<br />
!Check<br />
|-<br />
|[http://partsregistry.org/Part:BBa_R0082 BBa_R0082]<br />
|pOmpC<br />
|1<br />
|Positively regulated, OmpR-controlled promoter<br />
|108<br />
|pSB1A2<br />
|?<br />
|-<br />
|[http://partsregistry.org/Part:BBa_C0082 BBa_C0082]<br />
|tar-envZ<br />
|1<br />
|Receptor, tar-envZ<br />
|1491<br />
|pSB2K3<br />
|-<br />
|[http://partsregistry.org/Part:BBa_B0034 BBa_B0034]<br />
|RBS<br />
|1,2,3<br />
|The Elowitz RBS (Ribosome binding site) is the definition of efficiency 1.0<br />
|12<br />
|pSB1A2<br />
|?<br />
|-<br />
|AU43-C7<br />
|[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&id=13528923 INS]<br />
|<br />
|human insulin<br />
|495<br />
|C<br />
|-<br />
|<br />
|INS_A<br />
|1<br />
|alpha chain of human insulin<br />
|63<br />
|<br />
|-<br />
|<br />
|INS_B<br />
|1<br />
|beta chain of human insulin<br />
|90<br />
|<br />
|-<br />
|[http://partsregistry.org/Part:BBa_B0014 BBa_B0014]<br />
|D-term<br />
|1,2,3<br />
|Double transcription terminator<br />
|95<br />
|pSB1AK3<br />
|OK<br />
|-<br />
|[http://partsregistry.org/Part:BBa_I14032 BBa_I14032]<br />
|pLacIQ<br />
|2<br />
|Constitutive Promoter, High Transcription<br />
|37<br />
|pSB2K3<br />
|-<br />
|<br />
|OmpRBS<br />
|2<br />
|OmpR binding sites<br />
|65<br />
|<br />
|?<br />
|-<br />
|AV88-C6<br />
|[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&id=27371195 IDE]<br />
|2<br />
|insulin degradation enzyme of mouse<br />
|3445<br />
|A<br />
|-<br />
|AU61-F6<br />
|[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&id=33879043 IGFBP3]<br />
|2<br />
|Insulin-like growth factor binding protein 3 of human<br />
|2488<br />
|C<br />
|-<br />
|AU50-C7<br />
|[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&id=38114635 IGFBP7]<br />
|2<br />
|Insulin-like growth factor binding protein 7 of human<br />
|1155<br />
|C<br />
|-<br />
|[http://partsregistry.org/Part:BBa_I15016 BBa_I15016]<br />
|B0032.ECFP<br />
|16D, plate 1<br />
|This part accepts POPS to produce ECFP under the medium RBS B0032<br />
|742<br />
|pSB1A2<br />
|OK<br />
|-<br />
|[http://partsregistry.org/Part:BBa_I15017 BBa_I15017]<br />
|B0032.EYFP<br />
|16F, plate 1<br />
|This part accepts POPS to generate EYFP under the medium strength RBS B0032<br />
|742<br />
|pSB1A2<br />
|OK<br />
|-<br />
|[http://partsregistry.org/Part:BBa_E0840 BBa_E0840]<br />
|B0030.E0040.B0015<br />
|16E, plate 1<br />
|gfp tri-part; strong rbs<br />
|878<br />
|pSB1A2<br />
|OK<br />
|-<br />
|[http://partsregistry.org/Part:BBa_R0077 BBa_R0077]<br />
|pCinRHSL, RBS+<br />
|17L, Plate 1<br />
|CinR-HSL complex binds to this promoter and activates transcription. This part contains RBS.<br />
|231<br />
|pSB1A2<br />
|OK<br />
|-<br />
|-<br />
|[http://partsregistry.org/Part:BBa_R0078 BBa_R0078]<br />
|pCinRHSL<br />
|15D, Plate 1<br />
|CinR-HSL complex binds to this promoter and activates transcription. This part DOES NOT contain RBS.<br />
|225<br />
|pSB1A2<br />
|?<br />
|-<br />
|[http://partsregistry.org/Part:BBa_C0077 BBa_C0077]<br />
|CinR<br />
|7G, Plate 2 <br />
|cinR activator<br />
|762 <br />
|pSB2K3<br />
|OK<br />
|-<br />
|[http://partsregistry.org/Part:BBa_C0076 BBa_C0076]<br />
|HSL<br />
|7E, Plate 2<br />
|autoinducer<br />
|702<br />
|pSB2K3<br />
|OK<br />
|-<br />
|}<br />
<br />
==antibiotics keys==<br />
* A: [http://en.wikipedia.org/wiki/Ampicillin Ampicillin]<br />
** refernece at OpenWetWare: http://openwetware.org/wiki/Ampicillin<br />
** stock (100 mg/mL = 100 ug/uL) ampicillin<br />
** liquid culture (50 ug/mL = 250 ug/5mL)<br />
** 5mL liquid culture = 5 c.c. LB + 2.5uL stock ampicillin<br />
* K: [http://en.wikipedia.org/wiki/Kanamycin Kanamycin]<br />
* C: [http://en.wikipedia.org/wiki/Chloramphenicol Chloramphenicol]<br />
** 35 mg/mL<br />
** 20 mg/mL in Ethanol<br />
** we prepared 100mg in 5 mL and stored in -20 C</div>Blenthttp://2007.igem.org/wiki/index.php/NYMU_Taipei/Lab_Notes/2007_12_12NYMU Taipei/Lab Notes/2007 12 122007-12-13T04:22:07Z<p>Blent: </p>
<hr />
<div>* pOmpC + EYFP culture<br />
** 5mL liquid culture = 5 c.c. LB + 5.0 uL stock ampicillin (after 20hr, OD600 is 0.004)<br />
* pLacIQ + OmpRBS + GFP<br />
** re-culture pLacIQ in liquid medium (Kan+)<br />
** re-culture GFP in liquid medium ()<br />
** re-culture EYFP in liquid medium ()</div>Blenthttp://2007.igem.org/wiki/index.php/NYMU_Taipei/ConstructNYMU Taipei/Construct2007-12-13T04:00:22Z<p>Blent: </p>
<hr />
<div><h2>Assembly Ordering and Progress</h2><br />
<h3>system 1</h3><br />
<table style="color: green; background-color: rgb(255,255,204)" border="1"><br />
<tr style="background-color: rgb(255,255,255)" align="center"><br />
<td colspan="14"><font color="#000000">System 1: glucose-sensing insulin generator (5 task left: 2 PCR and 3 biobrick standard assembly)<br /><br />
</font></td><br />
</tr><br />
<tr><br />
</tr><br />
<tr align="center"><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">pOmpC</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">RBS + TAT<br /><br />
</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">INSA</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">RBS + TAT<br /><br />
</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">INSB</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">RBS</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">CinR</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">RBS</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">HSL</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">D-term</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">duration</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">members</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">assembly</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">status</font></td><br />
</tr><br />
<tr align="center"><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td bgcolor="#ffb6c1" colspan="2"><font color="#000000">RBS + CinR</font></td><br />
<td bgcolor="#ffb6c1" colspan="2"><font color="#000000">RBS + HSL</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">8/30-8/31</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">翔</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">1st assembly</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">[https://2007.igem.org/NYMU_Taipei/Lab_Notes/2007_9_1 9/01, 07]<br /><br />
</font></td><br />
</tr><br />
<tr align="center"><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td bgcolor="#ffb6c1" colspan="4"><font color="#000000">CinR + HSL (RBS+)</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">9/1-9/4</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">宥,君,威</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">2nd assembly</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">[https://2007.igem.org/NYMU_Taipei/Lab_Notes/2007_9_2 9/02, 07]<br /><br />
</font></td><br />
</tr><br />
<tr align="center"><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td bgcolor="#ffb6c1" colspan="5"><font color="#000000">CinR+HSL(RBS+) + D-term</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">&nbsp;9/16-9/27</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">宥,君,</font><font color="#000000">翔,pett</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">3rd assembly</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">[https://2007.igem.org/NYMU_Taipei/Lab_Notes/2007_9_27#CinR.2BHSL.2BD-term_check 9/27 07]<br /><br />
</font></td><br />
</tr><br />
<tr align="center"><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td bgcolor="#ffb6c1" colspan="2"><font color="#000000">RBS + TATA+ INSA (PCR)</font></td><br />
<td bgcolor="#ffb6c1" colspan="2"><font color="#000000">RBS + TATB+ INSB (PCR)</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td style="background-color: rgb(255,255,255)">&nbsp;<font color="#000000">9/28-9/30</font></td><br />
<td style="background-color: rgb(255,255,255)"><br />
<p><font color="#000000">銘, pett<br /><br />
</font></p><br />
</td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">basic part (PCR)</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">[https://2007.igem.org/NYMU_Taipei/Lab_Notes/2007_9_30 9/30, 07]<br /><br />
</font></td><br />
</tr><br />
<tr align="center"><br />
<td bgcolor="#ffb6c1" colspan="3"><font color="#000000">pOmpC + TATA_INSA (failed)</font></td><br />
<td bgcolor="#ffb6c1" colspan="7"><font color="#000000">TATB_INSB + CinR+HSL+D-term (wrong assembly)<br /><br />
</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">-10/5</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">pett</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">4th assembly</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">[https://2007.igem.org/NYMU_Taipei/Lab_Notes/2007_10_5 10/5,7,8 07]<br /><br />
</font></td><br />
</tr><br />
<tr align="center"><br />
<td bgcolor="#ffb6c1" colspan="3"><font color="#000000">pOmpC + TATA_INSA</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">-10/11</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">pett</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">4th assembly</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000"><br /><br />
</font></td><br />
</tr><br />
<tr align="center"><br />
<td bgcolor="#ffb6c1" colspan="5"><font color="#000000">pOmpC + TATA_INS_A + TATB_INS_B<br /><br />
</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">-10/23</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">&nbsp;</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">4th assembly</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000"><br /><br />
</font></td><br />
</tr><br />
<tr align="center"><br />
<td bgcolor="#ffb6c1" colspan="10"><font color="#000000">pOmpC + TATA_INS_A + TATB_INS_B + CinR+HSL+D-term</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">&nbsp;</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">&nbsp;</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">Final<br /><br />
</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000"><br /><br />
</font></td><br />
</tr><br />
</table><br />
<br />
<h3>system 2</h3><br />
<table style="color: green; background-color: rgb(255,255,204)" border="1"><br />
<tr style="background-color: rgb(255,255,255)" align="center"><br />
<td colspan="9"><font color="#000000">System 2: insulin-sensing insulin destoryer (5 tasks left: 2 PCR and 3 biobrick standard assembly)<br /><br />
</font></td><br />
</tr><br />
<tr align="center"><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">pCinRHSL</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">OmpRBS</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">RBS + TATD<br /><br />
</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">IDE</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">D-term</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">duration</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">members</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">assembly</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">status</font></td><br />
</tr><br />
<tr align="center"><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td bgcolor="#ffb6c1"><font color="#000000">OmpRBS (PCR)</font></td><br />
<td bgcolor="#ffb6c1" colspan="2"><font color="#000000">RBS + TATD+ IDE (PCR)<br /><br />
</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">-102</font></td><br />
<td style="background-color: rgb(255,255,255)"><br />
<p><font color="#000000">銘</font>, <font color="#000000">茹, pett<br /><br />
</font></p><br />
</td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">basic part (PCR)</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">[https://2007.igem.org/NYMU_Taipei/Lab_Notes/2007_10_2 10/2 07]<br /><br />
</font></td><br />
</tr><br />
<tr align="center"><br />
<td bgcolor="#ffb6c1" colspan="2"><font color="#000000">pCinRHSL + OmpRBS</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">-10/5</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">pett</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">1st assembly</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">[https://2007.igem.org/NYMU_Taipei/Lab_Notes/2007_10_5 10/5 07]<br /><br />
</font></td><br />
</tr><br />
<tr align="center"><br />
<td bgcolor="#ffb6c1" colspan="4"><font color="#000000">pCinRHSL + OmpRBS + TATD_IDE (RBS+)</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">-10/11</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">&nbsp;pett</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">2nd assembly</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000"><br /><br />
</font></td><br />
</tr><br />
<tr align="center"><br />
<td bgcolor="#ffb6c1" colspan="5"><font color="#000000">pCinRHSL+OmpRBS+TATD_IDE + D-term</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">&nbsp;</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">&nbsp;</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">Final</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000"><br /><br />
</font></td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><br />
<hr /><br />
<br />
<h2>Promoter assay construct: pOmpC+(RBS+EYFP)</h2><br />
<table><br />
<tr><br />
<td>[[Image:POmpC-EYFP (plasmid).jpg|300px]]</td><br />
<td><br />
<ul><br />
<li>our first contruct for promoter activity assay under glucose stimulus</li><br />
<li>Insert size: 866bp (XbaI to SpeI)</li><br />
<li>Total vector size: 3,019bp</li><br />
<li>V-I: 2,153bp</li><br />
<li>May be checked with: BtgZI</li><br />
<li>8/27, 2007</li><br />
</ul><br />
</td><br />
</tr><br />
</table><br />
<hr /><br />
<h2>OmpRBS assay construct: pLacIQ + OmpRBS + GFP</h2><br />
<table><br />
<tr><br />
<td>[[Image:PLacIQ-OmpRBS-GFP (plasmid).jpg|300px]]</td><br />
<td><br />
<ul><br />
<li>our first construct for OmpRBS blocking assay under glucose stimulus</li><br />
<li>Insert size: 1004bp (XbaI to SpeI)</li><br />
<li>Total vector size: 5,425bp</li><br />
<li>V-I: 4,421bp</li><br />
<li>May be checked with: BtgZI</li><br />
<li>10/24, 2007</li><br />
</ul><br />
</td><br />
</tr><br />
</table><br />
<hr /><br />
<h2>OmpRBS assay construct: pLacIQ + OmpRBS</h2><br />
<table><br />
<tr><br />
<td>[[Image:PLacIQ-OmpRBS (plasmid).jpg|300px]]</td><br />
<td><br />
<ul><br />
<li>our first construct for OmpRBS blocking assay under glucose stimulus</li><br />
<li>Insert size: 120bp (XbaI to SpeI)</li><br />
<li>Total vector size: 4,541bp</li><br />
<li>V-I: 4,421bp</li><br />
<li>May be checked with:</li><br />
<li>12/13, 2007</li><br />
</ul><br />
</td><br />
</tr><br />
</table><br />
<hr /><br />
<br />
<h2>System 1: pOmpC + (TATA<font color="#ff0000">+</font>INSA) + (TATB<font color="#ff0000">+</font>INSB) + (RBS<font color="#ff0000">+</font>CinR) + (RBS<font color="#ff0000">+</font>HSL) + D-term</h2><br />
<h3>Phase 1 of system 1: RBS <font color="#ff0000">+</font> CinR</h3><br />
<table><br />
<tr><br />
<td>[[Image:RBS-CinR (plasmid).jpg|300px]]</td><br />
<td><br />
<ul><br />
<li>Insert size: 790bp</li><br />
<li>Total vector size: 2,943bp</li><br />
<li>V-I: 2,153bp</li><br />
<li>May be checked with: BsaBI</li><br />
</ul><br />
</td><br />
</tr><br />
</table><br />
<br />
<h3>Phase 1 of system 1: RBS <font color="#ff0000">+</font> HSL</h3><br />
<table><br />
<tr><br />
<td>[[Image:RBS-HSL (plasmid).jpg|300px]]</td><br />
<td><br />
<ul><br />
<li>Insert size: 730bp</li><br />
<li>Total vector size: 2,883bp</li><br />
<li>V-I: 2,153bp</li><br />
<li>May be checked with: AgeI</li><br />
</ul><br />
</td><br />
</tr><br />
</table><br />
<br />
<h3>Phase 1 of system 1: TATA <font color="#ff0000">+</font> INSA = TATA_INSA</h3><br />
<table><br />
<tr><br />
<td>[[Image:TATA INSA (insert).jpg|300px]]</td><br />
<td><br />
<ul><br />
<li>Insert size: 170bp</li><br />
<li>PCR product size: 209bp</li><br />
<li>May be checked with: RsaI</li><br />
<li>There is a extra SpeI site formed due to the addition of one extra stop codon <br />
</ul><br />
</td><br />
</tr><br />
</table><br />
<br />
<h3>Phase 1 of system 1: TATB + INSB = TATB_INSB</h3><br />
<table><br />
<tr><br />
<td>[[Image:TATB INSB (insert).jpg|300px]]</td><br />
<td><br />
<ul><br />
<li>Insert size: 220bp</li><br />
<li>PCR product size: 233bp</li><br />
<li>May be checked with AluI</li><br />
</ul><br />
</td><br />
</tr><br />
</table><br />
<br />
<h3>Phase 2 of system 1: pOmpC + TATA_INSA</h3><br />
<table><br />
<tr><br />
<td>[[Image:POmpC-TATA INSA (plasmid).jpg|300px]]</td><br />
<td><br />
<ul><br />
<li>Insert size: 292bp</li><br />
<li>Total vector size: 2,454bp</li><br />
<li>V-I: 2,162bp</li><br />
</ul><br />
</td><br />
</tr><br />
</table><br />
<br />
<h3>Phase 2 of system 1: (RBS+CinR) <font color="#ff0000">+</font> (RBS+HSL) = CinR+HSL</h3><br />
<table><br />
<tr><br />
<td>[[Image:CinR-HSL (plasmid).jpg|300px]]</td><br />
<td><br />
<ul><br />
<li>Insert size: 1,524bp</li><br />
<li>Total vector size: 3,677bp</li><br />
<li>V-I: 2153bp</li><br />
</ul><br />
</td><br />
</tr><br />
</table><br />
<br />
<h3>Phase 3 of system 1: pOmpC + TATA_INSA <font color="#ff0000">+</font> TATB_INSB</h3><br />
<table><br />
<tr><br />
<td>[[Image:POmpC-TATA INSA-TATB INSB (plasmid).jpg|300px]]</td><br />
<td><br />
<ul><br />
<li>Insert size: 502bp</li><br />
<li>Total vector size: 2,655bp</li><br />
<li>V-I: 2,153bp</li><br />
</ul><br />
</td><br />
</tr><br />
</table><br />
<br />
<h3>Phase 3 of system 1: CinR+HSL <font color="#ff0000">+</font> D-term = CinR+HSL+D-term</h3><br />
<table><br />
<tr><br />
<td>[[Image:CinR-HSL-D-term (plasmid).jpg|300px]]</td><br />
<td><br />
<ul><br />
<li>Insert size: 1,633bp</li><br />
<li>Total vector size: 4,818bp</li><br />
<li>V-I: 3,185bp</li><br />
</ul><br />
</td><br />
</tr><br />
</table><br />
<br />
<h3>Phase 4 of system 1: pOmpC + TATA_INSA + TATB_INSB <font color="#ff0000">+ </font>CinR+HSL+D-term</h3><br />
<table><br />
<tr><br />
<td>[[Image:System I (plasmid).jpg|300px]]</td><br />
<td><br />
<ul><br />
<li>Insert size: 2,139bp</li><br />
<li>Total vector size: 4,292bp</li><br />
<li>V-I: 2,153bp</li><br />
</ul><br />
</td><br />
</tr><br />
</table><br />
<hr /><br />
<br />
<h2>System 2: pCinRHSL+ OmpRBS + (TATD+IDE) + D-term</h2><br />
<h3>Phase 1 of system 2: TATD <font color="#ff0000">+</font> IDE = TATD_IDE</h3><br />
<table><br />
<tr><br />
<td>[[Image:TATD IDE (insert).jpg|300px]]</td><br />
<td><br />
<ul><br />
<li>Insert size: 3,190bp</li><br />
<li>PCR product size: 3,203bp</li><br />
</ul><br />
</td><br />
</tr><br />
</table><br />
<br />
<h3>Phase 1 of system 2: pCinRHSL <font color="#ff0000">+</font> OmpRBS</h3><br />
<table><br />
<tr><br />
<td>[[Image:PCinRHSL-OmpRBS (plasmid).jpg|300px]]</td><br />
<td><br />
<ul><br />
<li>Insert size: 308bp</li><br />
<li>Total vector size: 2,461bp</li><br />
<li>V-I: 2,153bp</li><br />
</ul><br />
</td><br />
</tr><br />
</table><br />
<br />
<h3>Phase 2 of system 2: pCinRHSL + OmpRBS <font color="#ff0000">+</font> TATD_IDE</h3><br />
<table><br />
<tr><br />
<td>[[Image:PCinRHSL-OmpRBS-TATD IDE (plasmid).jpg|300px]]</td><br />
<td><br />
<ul><br />
<li>Insert size: 3,488bp</li><br />
<li>Total vector size: 5,641bp</li><br />
<li>V-I: 2,153bp</li><br />
</ul><br />
</td><br />
</tr><br />
</table><br />
<br />
<h3>Phase 3 of system 2: pCinRHSL + OmpRBS +&nbsp; TATD_IDE <font color="#ff0000">+</font> D-term</h3><br />
<table><br />
<tr><br />
<td>[[Image:System 2 (plasmid).jpg|300px]]</td><br />
<td><br />
<ul><br />
<li>Insert size: 3,589bp</li><br />
<li>Total vector size: 5,742bp</li><br />
<li>V-I: 2,153bp</li><br />
</ul><br />
</td><br />
</tr><br />
</table><br />
<hr /><br />
<br />
<h2>Basic Constructs</h2><br />
<h3>pOmpC in pSB1A2</h3><br />
<p><br />
<table><br />
<tr><br />
<td>[[Image:POmpC (plasmid).jpg|300px]]</td><br />
<td><br />
<ul><br />
<li>Insert size: 118bp</li><br />
<li>Total vector size: 2,271bp</li><br />
<li>V-I: 2,153bp</li><br />
</ul><br />
</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<h3>pCinRHSL in pSB1A2</h3><br />
<p><br />
<table><br />
<tr><br />
<td>[[Image:PCinRHSL (plasmid).jpg|300px]]</td><br />
<td><br />
<ul><br />
<li>Insert size: 235bp</li><br />
<li>Total vector size: 2,388bp</li><br />
<li>V-I: 2,153bp</li><br />
</ul><br />
</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<h3>RBS in pSB1A2</h3><br />
<p><br />
<table><br />
<tr><br />
<td>[[Image:RBS (plasmid).jpg|300px]]</td><br />
<td><br />
<ul><br />
<li>BseR1 can be applied to check the insertion of RBS</li><br />
<li>Insert size: 22bp</li><br />
<li>Total vector size: 2,175bp</li><br />
<li>V-I: 2,153bp</li><br />
</ul><br />
</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<h3>D-term in pSB1AK3</h3><br />
<table><br />
<tr><br />
<td>[[Image:D-term (plasmid).jpg|300px]]</td><br />
<td><br />
<ul><br />
<li>Insert size: 105bp</li><br />
<li>Total vector size: 3,290bp</li><br />
<li>V-I: 3,185bp</li><br />
</ul><br />
</td><br />
</tr><br />
</table><br />
<br />
[https://2007.igem.org/Taipei Back]</div>Blenthttp://2007.igem.org/wiki/index.php/File:PLacIQ-OmpRBS_(plasmid).jpgFile:PLacIQ-OmpRBS (plasmid).jpg2007-12-13T03:57:46Z<p>Blent: </p>
<hr />
<div></div>Blenthttp://2007.igem.org/wiki/index.php/NYMU_Taipei/ConstructNYMU Taipei/Construct2007-12-13T03:55:24Z<p>Blent: </p>
<hr />
<div><h2>Assembly Ordering and Progress</h2><br />
<h3>system 1</h3><br />
<table style="color: green; background-color: rgb(255,255,204)" border="1"><br />
<tr style="background-color: rgb(255,255,255)" align="center"><br />
<td colspan="14"><font color="#000000">System 1: glucose-sensing insulin generator (5 task left: 2 PCR and 3 biobrick standard assembly)<br /><br />
</font></td><br />
</tr><br />
<tr><br />
</tr><br />
<tr align="center"><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">pOmpC</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">RBS + TAT<br /><br />
</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">INSA</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">RBS + TAT<br /><br />
</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">INSB</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">RBS</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">CinR</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">RBS</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">HSL</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">D-term</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">duration</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">members</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">assembly</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">status</font></td><br />
</tr><br />
<tr align="center"><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td bgcolor="#ffb6c1" colspan="2"><font color="#000000">RBS + CinR</font></td><br />
<td bgcolor="#ffb6c1" colspan="2"><font color="#000000">RBS + HSL</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">8/30-8/31</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">翔</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">1st assembly</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">[https://2007.igem.org/NYMU_Taipei/Lab_Notes/2007_9_1 9/01, 07]<br /><br />
</font></td><br />
</tr><br />
<tr align="center"><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td bgcolor="#ffb6c1" colspan="4"><font color="#000000">CinR + HSL (RBS+)</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">9/1-9/4</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">宥,君,威</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">2nd assembly</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">[https://2007.igem.org/NYMU_Taipei/Lab_Notes/2007_9_2 9/02, 07]<br /><br />
</font></td><br />
</tr><br />
<tr align="center"><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td bgcolor="#ffb6c1" colspan="5"><font color="#000000">CinR+HSL(RBS+) + D-term</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">&nbsp;9/16-9/27</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">宥,君,</font><font color="#000000">翔,pett</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">3rd assembly</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">[https://2007.igem.org/NYMU_Taipei/Lab_Notes/2007_9_27#CinR.2BHSL.2BD-term_check 9/27 07]<br /><br />
</font></td><br />
</tr><br />
<tr align="center"><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td bgcolor="#ffb6c1" colspan="2"><font color="#000000">RBS + TATA+ INSA (PCR)</font></td><br />
<td bgcolor="#ffb6c1" colspan="2"><font color="#000000">RBS + TATB+ INSB (PCR)</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td style="background-color: rgb(255,255,255)">&nbsp;<font color="#000000">9/28-9/30</font></td><br />
<td style="background-color: rgb(255,255,255)"><br />
<p><font color="#000000">銘, pett<br /><br />
</font></p><br />
</td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">basic part (PCR)</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">[https://2007.igem.org/NYMU_Taipei/Lab_Notes/2007_9_30 9/30, 07]<br /><br />
</font></td><br />
</tr><br />
<tr align="center"><br />
<td bgcolor="#ffb6c1" colspan="3"><font color="#000000">pOmpC + TATA_INSA (failed)</font></td><br />
<td bgcolor="#ffb6c1" colspan="7"><font color="#000000">TATB_INSB + CinR+HSL+D-term (wrong assembly)<br /><br />
</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">-10/5</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">pett</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">4th assembly</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">[https://2007.igem.org/NYMU_Taipei/Lab_Notes/2007_10_5 10/5,7,8 07]<br /><br />
</font></td><br />
</tr><br />
<tr align="center"><br />
<td bgcolor="#ffb6c1" colspan="3"><font color="#000000">pOmpC + TATA_INSA</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">-10/11</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">pett</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">4th assembly</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000"><br /><br />
</font></td><br />
</tr><br />
<tr align="center"><br />
<td bgcolor="#ffb6c1" colspan="5"><font color="#000000">pOmpC + TATA_INS_A + TATB_INS_B<br /><br />
</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">-10/23</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">&nbsp;</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">4th assembly</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000"><br /><br />
</font></td><br />
</tr><br />
<tr align="center"><br />
<td bgcolor="#ffb6c1" colspan="10"><font color="#000000">pOmpC + TATA_INS_A + TATB_INS_B + CinR+HSL+D-term</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">&nbsp;</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">&nbsp;</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">Final<br /><br />
</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000"><br /><br />
</font></td><br />
</tr><br />
</table><br />
<br />
<h3>system 2</h3><br />
<table style="color: green; background-color: rgb(255,255,204)" border="1"><br />
<tr style="background-color: rgb(255,255,255)" align="center"><br />
<td colspan="9"><font color="#000000">System 2: insulin-sensing insulin destoryer (5 tasks left: 2 PCR and 3 biobrick standard assembly)<br /><br />
</font></td><br />
</tr><br />
<tr align="center"><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">pCinRHSL</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">OmpRBS</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">RBS + TATD<br /><br />
</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">IDE</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">D-term</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">duration</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">members</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">assembly</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">status</font></td><br />
</tr><br />
<tr align="center"><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td bgcolor="#ffb6c1"><font color="#000000">OmpRBS (PCR)</font></td><br />
<td bgcolor="#ffb6c1" colspan="2"><font color="#000000">RBS + TATD+ IDE (PCR)<br /><br />
</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">-102</font></td><br />
<td style="background-color: rgb(255,255,255)"><br />
<p><font color="#000000">銘</font>, <font color="#000000">茹, pett<br /><br />
</font></p><br />
</td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">basic part (PCR)</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">[https://2007.igem.org/NYMU_Taipei/Lab_Notes/2007_10_2 10/2 07]<br /><br />
</font></td><br />
</tr><br />
<tr align="center"><br />
<td bgcolor="#ffb6c1" colspan="2"><font color="#000000">pCinRHSL + OmpRBS</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">-10/5</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">pett</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">1st assembly</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">[https://2007.igem.org/NYMU_Taipei/Lab_Notes/2007_10_5 10/5 07]<br /><br />
</font></td><br />
</tr><br />
<tr align="center"><br />
<td bgcolor="#ffb6c1" colspan="4"><font color="#000000">pCinRHSL + OmpRBS + TATD_IDE (RBS+)</font></td><br />
<td><font color="#000000">&nbsp;</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">-10/11</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">&nbsp;pett</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">2nd assembly</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000"><br /><br />
</font></td><br />
</tr><br />
<tr align="center"><br />
<td bgcolor="#ffb6c1" colspan="5"><font color="#000000">pCinRHSL+OmpRBS+TATD_IDE + D-term</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">&nbsp;</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">&nbsp;</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000">Final</font></td><br />
<td style="background-color: rgb(255,255,255)"><font color="#000000"><br /><br />
</font></td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><br />
<hr /><br />
<br />
<h2>Promoter assay construct: pOmpC+(RBS+EYFP)</h2><br />
<table><br />
<tr><br />
<td>[[Image:POmpC-EYFP (plasmid).jpg|300px]]</td><br />
<td><br />
<ul><br />
<li>our first contruct for promoter activity assay under glucose stimulus</li><br />
<li>Insert size: 866bp (XbaI to SpeI)</li><br />
<li>Total vector size: 3,019bp</li><br />
<li>V-I: 2,153bp</li><br />
<li>May be checked with: BtgZI</li><br />
<li>8/27, 2007</li><br />
</ul><br />
</td><br />
</tr><br />
</table><br />
<hr /><br />
<h2>OmpRBS assay construct: pLacIQ + OmpRBS + GFP</h2><br />
<table><br />
<tr><br />
<td>[[Image:PLacIQ-OmpRBS-GFP (plasmid).jpg|300px]]</td><br />
<td><br />
<ul><br />
<li>our first construct for OmpRBS blocking assay under glucose stimulus</li><br />
<li>Insert size: 1004bp (XbaI to SpeI)</li><br />
<li>Total vector size: 5,425bp</li><br />
<li>V-I: 4,421bp</li><br />
<li>May be checked with: BtgZI</li><br />
<li>10/24, 2007</li><br />
</ul><br />
</td><br />
</tr><br />
</table><br />
<hr /><br />
<h2>OmpRBS assay construct: pLacIQ + OmpRBS</h2><br />
<table><br />
<tr><br />
<td>[[Image:PLacIQ-OmpRBS (plasmid).jpg|300px]]</td><br />
<td><br />
<ul><br />
<li>our first construct for OmpRBS blocking assay under glucose stimulus</li><br />
<li>Insert size: 1004bp (XbaI to SpeI)</li><br />
<li>Total vector size: 4,541bp</li><br />
<li>V-I: 4,421bp</li><br />
<li>May be checked with:</li><br />
<li>12/13, 2007</li><br />
</ul><br />
</td><br />
</tr><br />
</table><br />
<hr /><br />
<br />
<h2>System 1: pOmpC + (TATA<font color="#ff0000">+</font>INSA) + (TATB<font color="#ff0000">+</font>INSB) + (RBS<font color="#ff0000">+</font>CinR) + (RBS<font color="#ff0000">+</font>HSL) + D-term</h2><br />
<h3>Phase 1 of system 1: RBS <font color="#ff0000">+</font> CinR</h3><br />
<table><br />
<tr><br />
<td>[[Image:RBS-CinR (plasmid).jpg|300px]]</td><br />
<td><br />
<ul><br />
<li>Insert size: 790bp</li><br />
<li>Total vector size: 2,943bp</li><br />
<li>V-I: 2,153bp</li><br />
<li>May be checked with: BsaBI</li><br />
</ul><br />
</td><br />
</tr><br />
</table><br />
<br />
<h3>Phase 1 of system 1: RBS <font color="#ff0000">+</font> HSL</h3><br />
<table><br />
<tr><br />
<td>[[Image:RBS-HSL (plasmid).jpg|300px]]</td><br />
<td><br />
<ul><br />
<li>Insert size: 730bp</li><br />
<li>Total vector size: 2,883bp</li><br />
<li>V-I: 2,153bp</li><br />
<li>May be checked with: AgeI</li><br />
</ul><br />
</td><br />
</tr><br />
</table><br />
<br />
<h3>Phase 1 of system 1: TATA <font color="#ff0000">+</font> INSA = TATA_INSA</h3><br />
<table><br />
<tr><br />
<td>[[Image:TATA INSA (insert).jpg|300px]]</td><br />
<td><br />
<ul><br />
<li>Insert size: 170bp</li><br />
<li>PCR product size: 209bp</li><br />
<li>May be checked with: RsaI</li><br />
<li>There is a extra SpeI site formed due to the addition of one extra stop codon <br />
</ul><br />
</td><br />
</tr><br />
</table><br />
<br />
<h3>Phase 1 of system 1: TATB + INSB = TATB_INSB</h3><br />
<table><br />
<tr><br />
<td>[[Image:TATB INSB (insert).jpg|300px]]</td><br />
<td><br />
<ul><br />
<li>Insert size: 220bp</li><br />
<li>PCR product size: 233bp</li><br />
<li>May be checked with AluI</li><br />
</ul><br />
</td><br />
</tr><br />
</table><br />
<br />
<h3>Phase 2 of system 1: pOmpC + TATA_INSA</h3><br />
<table><br />
<tr><br />
<td>[[Image:POmpC-TATA INSA (plasmid).jpg|300px]]</td><br />
<td><br />
<ul><br />
<li>Insert size: 292bp</li><br />
<li>Total vector size: 2,454bp</li><br />
<li>V-I: 2,162bp</li><br />
</ul><br />
</td><br />
</tr><br />
</table><br />
<br />
<h3>Phase 2 of system 1: (RBS+CinR) <font color="#ff0000">+</font> (RBS+HSL) = CinR+HSL</h3><br />
<table><br />
<tr><br />
<td>[[Image:CinR-HSL (plasmid).jpg|300px]]</td><br />
<td><br />
<ul><br />
<li>Insert size: 1,524bp</li><br />
<li>Total vector size: 3,677bp</li><br />
<li>V-I: 2153bp</li><br />
</ul><br />
</td><br />
</tr><br />
</table><br />
<br />
<h3>Phase 3 of system 1: pOmpC + TATA_INSA <font color="#ff0000">+</font> TATB_INSB</h3><br />
<table><br />
<tr><br />
<td>[[Image:POmpC-TATA INSA-TATB INSB (plasmid).jpg|300px]]</td><br />
<td><br />
<ul><br />
<li>Insert size: 502bp</li><br />
<li>Total vector size: 2,655bp</li><br />
<li>V-I: 2,153bp</li><br />
</ul><br />
</td><br />
</tr><br />
</table><br />
<br />
<h3>Phase 3 of system 1: CinR+HSL <font color="#ff0000">+</font> D-term = CinR+HSL+D-term</h3><br />
<table><br />
<tr><br />
<td>[[Image:CinR-HSL-D-term (plasmid).jpg|300px]]</td><br />
<td><br />
<ul><br />
<li>Insert size: 1,633bp</li><br />
<li>Total vector size: 4,818bp</li><br />
<li>V-I: 3,185bp</li><br />
</ul><br />
</td><br />
</tr><br />
</table><br />
<br />
<h3>Phase 4 of system 1: pOmpC + TATA_INSA + TATB_INSB <font color="#ff0000">+ </font>CinR+HSL+D-term</h3><br />
<table><br />
<tr><br />
<td>[[Image:System I (plasmid).jpg|300px]]</td><br />
<td><br />
<ul><br />
<li>Insert size: 2,139bp</li><br />
<li>Total vector size: 4,292bp</li><br />
<li>V-I: 2,153bp</li><br />
</ul><br />
</td><br />
</tr><br />
</table><br />
<hr /><br />
<br />
<h2>System 2: pCinRHSL+ OmpRBS + (TATD+IDE) + D-term</h2><br />
<h3>Phase 1 of system 2: TATD <font color="#ff0000">+</font> IDE = TATD_IDE</h3><br />
<table><br />
<tr><br />
<td>[[Image:TATD IDE (insert).jpg|300px]]</td><br />
<td><br />
<ul><br />
<li>Insert size: 3,190bp</li><br />
<li>PCR product size: 3,203bp</li><br />
</ul><br />
</td><br />
</tr><br />
</table><br />
<br />
<h3>Phase 1 of system 2: pCinRHSL <font color="#ff0000">+</font> OmpRBS</h3><br />
<table><br />
<tr><br />
<td>[[Image:PCinRHSL-OmpRBS (plasmid).jpg|300px]]</td><br />
<td><br />
<ul><br />
<li>Insert size: 308bp</li><br />
<li>Total vector size: 2,461bp</li><br />
<li>V-I: 2,153bp</li><br />
</ul><br />
</td><br />
</tr><br />
</table><br />
<br />
<h3>Phase 2 of system 2: pCinRHSL + OmpRBS <font color="#ff0000">+</font> TATD_IDE</h3><br />
<table><br />
<tr><br />
<td>[[Image:PCinRHSL-OmpRBS-TATD IDE (plasmid).jpg|300px]]</td><br />
<td><br />
<ul><br />
<li>Insert size: 3,488bp</li><br />
<li>Total vector size: 5,641bp</li><br />
<li>V-I: 2,153bp</li><br />
</ul><br />
</td><br />
</tr><br />
</table><br />
<br />
<h3>Phase 3 of system 2: pCinRHSL + OmpRBS +&nbsp; TATD_IDE <font color="#ff0000">+</font> D-term</h3><br />
<table><br />
<tr><br />
<td>[[Image:System 2 (plasmid).jpg|300px]]</td><br />
<td><br />
<ul><br />
<li>Insert size: 3,589bp</li><br />
<li>Total vector size: 5,742bp</li><br />
<li>V-I: 2,153bp</li><br />
</ul><br />
</td><br />
</tr><br />
</table><br />
<hr /><br />
<br />
<h2>Basic Constructs</h2><br />
<h3>pOmpC in pSB1A2</h3><br />
<p><br />
<table><br />
<tr><br />
<td>[[Image:POmpC (plasmid).jpg|300px]]</td><br />
<td><br />
<ul><br />
<li>Insert size: 118bp</li><br />
<li>Total vector size: 2,271bp</li><br />
<li>V-I: 2,153bp</li><br />
</ul><br />
</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<h3>pCinRHSL in pSB1A2</h3><br />
<p><br />
<table><br />
<tr><br />
<td>[[Image:PCinRHSL (plasmid).jpg|300px]]</td><br />
<td><br />
<ul><br />
<li>Insert size: 235bp</li><br />
<li>Total vector size: 2,388bp</li><br />
<li>V-I: 2,153bp</li><br />
</ul><br />
</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<h3>RBS in pSB1A2</h3><br />
<p><br />
<table><br />
<tr><br />
<td>[[Image:RBS (plasmid).jpg|300px]]</td><br />
<td><br />
<ul><br />
<li>BseR1 can be applied to check the insertion of RBS</li><br />
<li>Insert size: 22bp</li><br />
<li>Total vector size: 2,175bp</li><br />
<li>V-I: 2,153bp</li><br />
</ul><br />
</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<h3>D-term in pSB1AK3</h3><br />
<table><br />
<tr><br />
<td>[[Image:D-term (plasmid).jpg|300px]]</td><br />
<td><br />
<ul><br />
<li>Insert size: 105bp</li><br />
<li>Total vector size: 3,290bp</li><br />
<li>V-I: 3,185bp</li><br />
</ul><br />
</td><br />
</tr><br />
</table><br />
<br />
[https://2007.igem.org/Taipei Back]</div>Blenthttp://2007.igem.org/wiki/index.php/NYMU_Taipei/Lab_Notes/2007_12_12NYMU Taipei/Lab Notes/2007 12 122007-12-13T03:10:50Z<p>Blent: </p>
<hr />
<div>* pOmpC + EYFP culture<br />
** 5mL liquid culture = 5 c.c. LB + 5.0 uL stock ampicillin (after 20hr, OD600 is 0.004)</div>Blenthttp://2007.igem.org/wiki/index.php/NYMU_Taipei/Lab_Notes/2007_12_13NYMU Taipei/Lab Notes/2007 12 132007-12-13T03:10:21Z<p>Blent: </p>
<hr />
<div>* pOmpC + EYFP in M9 medium<br />
** 5mL liquid culture = 5 c.c. LB + 2.5uL stock ampicillin <br />
* construction of pLacIQ + OmpRBS + Reporter</div>Blenthttp://2007.igem.org/wiki/index.php/NYMU_Taipei/Part_ListNYMU Taipei/Part List2007-12-13T03:09:49Z<p>Blent: /* antibiotics keys */</p>
<hr />
<div><p><font size="4"><br />
[[NYMU_Taipei/ProjectDescription| Project Description]] /<br />
[[NYMU_Taipei/Lab_Notes| Lab Notes]] /<br />
[[NYMU_Taipei/Part_List| Part List]] /<br />
[[NYMU_Taipei/Primer_List| Primer List]]<br />
</font><br /><hr></p><br />
<br />
==Part List==<br />
{| style="color:green;background-color:#ffffcc;" cellpadding="20" cellspacing="0" border="1"<br />
!Series<br />
!alias<br />
!Location<br />
!Description<br />
!part length<br />
!vector (antibotics)<br />
!Check<br />
|-<br />
|[http://partsregistry.org/Part:BBa_R0082 BBa_R0082]<br />
|pOmpC<br />
|1<br />
|Positively regulated, OmpR-controlled promoter<br />
|108<br />
|pSB1A2<br />
|?<br />
|-<br />
|[http://partsregistry.org/Part:BBa_C0082 BBa_C0082]<br />
|tar-envZ<br />
|1<br />
|Receptor, tar-envZ<br />
|1491<br />
|pSB2K3<br />
|-<br />
|[http://partsregistry.org/Part:BBa_B0034 BBa_B0034]<br />
|RBS<br />
|1,2,3<br />
|The Elowitz RBS (Ribosome binding site) is the definition of efficiency 1.0<br />
|12<br />
|pSB1A2<br />
|?<br />
|-<br />
|AU43-C7<br />
|[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&id=13528923 INS]<br />
|<br />
|human insulin<br />
|495<br />
|C<br />
|-<br />
|<br />
|INS_A<br />
|1<br />
|alpha chain of human insulin<br />
|63<br />
|<br />
|-<br />
|<br />
|INS_B<br />
|1<br />
|beta chain of human insulin<br />
|90<br />
|<br />
|-<br />
|[http://partsregistry.org/Part:BBa_B0014 BBa_B0014]<br />
|D-term<br />
|1,2,3<br />
|Double transcription terminator<br />
|95<br />
|pSB1AK3<br />
|OK<br />
|-<br />
|[http://partsregistry.org/Part:BBa_I14032 BBa_I14032]<br />
|pLacIQ<br />
|2<br />
|Constitutive Promoter, High Transcription<br />
|37<br />
|pSB2K3<br />
|-<br />
|<br />
|OmpRBS<br />
|2<br />
|OmpR binding sites<br />
|65<br />
|<br />
|?<br />
|-<br />
|AV88-C6<br />
|[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&id=27371195 IDE]<br />
|2<br />
|insulin degradation enzyme of mouse<br />
|3445<br />
|A<br />
|-<br />
|AU61-F6<br />
|[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&id=33879043 IGFBP3]<br />
|2<br />
|Insulin-like growth factor binding protein 3 of human<br />
|2488<br />
|C<br />
|-<br />
|AU50-C7<br />
|[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&id=38114635 IGFBP7]<br />
|2<br />
|Insulin-like growth factor binding protein 7 of human<br />
|1155<br />
|C<br />
|-<br />
|[http://partsregistry.org/Part:BBa_I15016 BBa_I15016]<br />
|B0032.ECFP<br />
|16D, plate 1<br />
|This part accepts POPS to produce ECFP under the medium RBS B0032<br />
|742<br />
|pSB1A2<br />
|OK<br />
|-<br />
|[http://partsregistry.org/Part:BBa_I15017 BBa_I15017]<br />
|B0032.EYFP<br />
|16F, plate 1<br />
|This part accepts POPS to generate EYFP under the medium strength RBS B0032<br />
|742<br />
|pSB1A2<br />
|OK<br />
|-<br />
|[http://partsregistry.org/Part:BBa_R0077 BBa_R0077]<br />
|pCinRHSL, RBS+<br />
|17L, Plate 1<br />
|CinR-HSL complex binds to this promoter and activates transcription. This part contains RBS.<br />
|231<br />
|pSB1A2<br />
|OK<br />
|-<br />
|-<br />
|[http://partsregistry.org/Part:BBa_R0078 BBa_R0078]<br />
|pCinRHSL<br />
|15D, Plate 1<br />
|CinR-HSL complex binds to this promoter and activates transcription. This part DOES NOT contain RBS.<br />
|225<br />
|pSB1A2<br />
|?<br />
|-<br />
|[http://partsregistry.org/Part:BBa_C0077 BBa_C0077]<br />
|CinR<br />
|7G, Plate 2 <br />
|cinR activator<br />
|762 <br />
|pSB2K3<br />
|OK<br />
|-<br />
|[http://partsregistry.org/Part:BBa_C0076 BBa_C0076]<br />
|HSL<br />
|7E, Plate 2<br />
|autoinducer<br />
|702<br />
|pSB2K3<br />
|OK<br />
|-<br />
|}<br />
<br />
==antibiotics keys==<br />
* A: [http://en.wikipedia.org/wiki/Ampicillin Ampicillin]<br />
** refernece at OpenWetWare: http://openwetware.org/wiki/Ampicillin<br />
** stock (100 mg/mL = 100 ug/uL) ampicillin<br />
** liquid culture (50 ug/mL = 250 ug/5mL)<br />
** 5mL liquid culture = 5 c.c. LB + 2.5uL stock ampicillin<br />
* K: [http://en.wikipedia.org/wiki/Kanamycin Kanamycin]<br />
* C: [http://en.wikipedia.org/wiki/Chloramphenicol Chloramphenicol]<br />
** 35 mg/mL<br />
** 20 mg/mL in Ethanol<br />
** we prepared 100mg in 5 mL and stored in -20 C</div>Blenthttp://2007.igem.org/wiki/index.php/NYMU_Taipei/Lab_Notes/2007_12_12NYMU Taipei/Lab Notes/2007 12 122007-12-13T03:09:27Z<p>Blent: </p>
<hr />
<div>* pOmpC + EYFP culture<br />
** 5mL liquid culture = 5 c.c. LB + 5.0 uL stock ampicillin</div>Blenthttp://2007.igem.org/wiki/index.php/NYMU_Taipei/Lab_Notes/2007_12_12NYMU Taipei/Lab Notes/2007 12 122007-12-13T03:07:13Z<p>Blent: </p>
<hr />
<div>* pOmpC + EYFP culture</div>Blenthttp://2007.igem.org/wiki/index.php/NYMU_Taipei/Lab_Notes/2007_12_13NYMU Taipei/Lab Notes/2007 12 132007-12-13T03:05:25Z<p>Blent: </p>
<hr />
<div>* pOmpC + EYFP in M9 medium<br />
* construction of pLacIQ + OmpRBS + Reporter</div>Blenthttp://2007.igem.org/wiki/index.php/NYMU_Taipei/Team_scheduleNYMU Taipei/Team schedule2007-12-13T03:04:12Z<p>Blent: /* Dec 2007 */</p>
<hr />
<div><br /><hr><br />
<br />
==July 2007==<br />
<br />
<table style="text-align: left" width="50%" border="1"><br />
<br />
<tr><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Sun.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Mon.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Tue.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Wed.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Thr.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Fri.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Sat.</big></td><br />
</tr><br />
<tr><br />
<td align="center">1</td><br />
<td align="center">2</td><br />
<td align="center">3</td><br />
<td align="center">4</td><br />
<td align="center">5</td><br />
<td align="center">[[NYMU_Taipei/Regular_meeting/July_6| 6]]</td><br />
<td align="center">7</td><br />
</tr><br />
<tr><br />
<td align="center">8</td><br />
<td align="center">9</td><br />
<td align="center">10</td><br />
<td align="center">11</td><br />
<td align="center">12</td><br />
<td align="center">[[NYMU_Taipei/Regular_meeting/July_13| 13]]</td><br />
<td align="center">14</td><br />
</tr><br />
<tr><br />
<td align="center">15</td><br />
<td align="center">16</td><br />
<td align="center">17</td><br />
<td align="center">18</td><br />
<td align="center">19</td><br />
<td align="center">[[NYMU_Taipei/Regular_meeting/July_20| 20]]</td><br />
<td align="center">21</td><br />
</tr><br />
<tr><br />
<td align="center">22</td><br />
<td align="center">23</td><br />
<td align="center">24</td><br />
<td align="center">25</td><br />
<td align="center">26</td><br />
<td align="center">[[NYMU_Taipei/Regular_meeting/July_27| 27]]</td><br />
<td align="center">28</td><br />
</tr><br />
<tr><br />
<td align="center">29</td><br />
<td align="center">30</td><br />
<td align="center">31</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><br />
<br />
==Aug 2007==<br />
<table style="text-align: left" width="50%" border="1"><br />
<tr><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Sun.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Mon.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Tue.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Wed.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Thr.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Fri.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Sat.</big></td><br />
</tr><br />
<tr><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">1</td><br />
<td align="center">2</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_3| 3]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_4| 4]]</td><br />
</tr><br />
<tr><br />
<td align="center">5</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_6&7| 6]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_6&7| 7]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_8| 8]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_9| 9]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_10| 10]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_11| 11]]</td><br />
</tr><br />
<tr><br />
<td align="center">12</td><br />
<td align="center">13</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_14| 14]]</td><br />
<td align="center">15</td><br />
<td align="center">16</td><br />
<td align="center">[[NYMU_Taipei/Regular_meeting/August_17| 17]]</td><br />
<td align="center">18</td><br />
</tr><br />
<tr><br />
<td align="center">19</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_20| 20]]</td><br />
<td align="center">21</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_22| 22]]</td><br />
<br />
<td align="center">23</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_24| 24]]</td><br />
<td align="center">25</td><br />
</tr><br />
<tr><br />
<td align="center">26</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_27| 27]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_28| 28]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_29| 29]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_30| 30]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_31| 31]]</td><br />
<td align="center">&nbsp;</td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><br />
<br />
==Sep 2007==<br />
<table style="text-align: left" width="50%" border="1"><br />
<tr><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Sun.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Mon.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Tue.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Wed.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Thr.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Fri.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Sat.</big></td><br />
</tr><br />
<tr><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_1| 1]]</td><br />
</tr><br />
<tr><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_2| 2]]</td><br />
<td align="center">3</td><br />
<td align="center">4</td><br />
<td align="center">5</td><br />
<td align="center">6</td><br />
<td align="center">[[NYMU_Taipei/Regular_meeting/September_7| 7]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_8| 8]]</td><br />
</tr><br />
<tr><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_9| 9]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_10| 10]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_11| 11]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_12| 12]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_13| 13]]</td><br />
<td align="center">[[NYMU_Taipei/Regular_meeting/September_14| 14]]</td><br />
<td align="center">15</td><br />
</tr><br />
<tr><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_16| 16]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_17| 17]]</td><br />
<td align="center">18</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_19| 19]]</td><br />
<br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_20| 20]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_21| 21]]</td><br />
<td align="center">22</td><br />
</tr><br />
<tr><br />
<td align="center">23</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_24| 24]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_25| 25]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_26| 26]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_27| 27]]</td><br />
<td align="center">[[NYMU_Taipei/Regular_meeting/September_28| 28]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_29| 29]]</td><br />
</tr><br />
<tr><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_30| 30]]</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><br />
<br />
==Oct 2007==<br />
<table style="text-align: left" width="50%" border="1"><br />
<tr><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Sun.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Mon.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Tue.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Wed.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Thr.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Fri.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Oct.</big></td><br />
</tr><br />
<tr><br />
<td align="center">&nbsp;</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_1| 1]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_2| 2]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_3| 3]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_4| 4]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_5| 5]]</td><br />
<td align="center">6</td><br />
</tr><br />
<tr><br />
<td align="center">7</td><br />
<td align="center">8</td><br />
<td align="center">9</td><br />
<td align="center">10</td><br />
<td align="center">11</td><br />
<td align="center">[[NYMU_Taipei/Regular_meeting/October_12| 12]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_13| 13]]</td><br />
</tr><br />
<tr><br />
<td align="center">14</td><br />
<td align="center">15</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_16| 16]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_17| 17]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_18| 18]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_19| 19]]</td><br />
<td align="center">20</td><br />
</tr><br />
<tr><br />
<td align="center">21</td><br />
<td align="center">22</td><br />
<td align="center">23</td><br />
<td align="center">24</td><br />
<td align="center">25</td><br />
<td align="center">26</td><br />
<td align="center">27</td><br />
</tr><br />
<tr><br />
<td align="center">28</td><br />
<td align="center">29</td><br />
<td align="center">30</td><br />
<td align="center">31</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><br />
==Nov 2007==<br />
<table style="text-align: left" width="50%" border="1"><br />
<tr><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Sun.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Mon.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Tue.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Wed.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Thr.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Fri.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Oct.</big></td><br />
</tr><br />
<tr><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_11_1| 1]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_11_2| 2]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_11_3| 3]]</td><br />
</tr><br />
<tr><br />
<td align="center">4</td><br />
<td align="center">5</td><br />
<td align="center">6</td><br />
<td align="center">7</td><br />
<td align="center">8</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_11_9| 9]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_11_10| 10]]</td><br />
</tr><br />
<tr><br />
<td align="center">11</td><br />
<td align="center">12</td><br />
<td align="center">13</td><br />
<td align="center">14</td><br />
<td align="center">15</td><br />
<td align="center">16</td><br />
<td align="center">17</td><br />
</tr><br />
<tr><br />
<td align="center">18</td><br />
<td align="center">19</td><br />
<td align="center">20</td><br />
<td align="center">21</td><br />
<td align="center">22</td><br />
<td align="center">23</td><br />
<td align="center">24</td><br />
</tr><br />
<tr><br />
<td align="center">25</td><br />
<td align="center">26</td><br />
<td align="center">27</td><br />
<td align="center">28</td><br />
<td align="center">29</td><br />
<td align="center">30</td><br />
<td align="center">&nbsp;</td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><br />
==Dec 2007==<br />
<table style="text-align: left" width="50%" border="1"><br />
<tr><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Sun.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Mon.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Tue.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Wed.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Thr.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Fri.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Oct.</big></td><br />
</tr><br />
<tr><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">1</td><br />
</tr><br />
<tr><br />
<td align="center">2</td><br />
<td align="center">3</td><br />
<td align="center">4</td><br />
<td align="center">5</td><br />
<td align="center">6</td><br />
<td align="center">7</td><br />
<td align="center">8</td><br />
</tr><br />
<tr><br />
<td align="center">9</td><br />
<td align="center">10</td><br />
<td align="center">11</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_12_12| 12]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_12_13| 13]]</td><br />
<td align="center">14</td><br />
<td align="center">15</td><br />
</tr><br />
<tr><br />
<td align="center">16</td><br />
<td align="center">17</td><br />
<td align="center">18</td><br />
<td align="center">19</td><br />
<td align="center">20</td><br />
<td align="center">21</td><br />
<td align="center">22</td><br />
</tr><br />
<tr><br />
<td align="center">23</td><br />
<td align="center">24</td><br />
<td align="center">25</td><br />
<td align="center">26</td><br />
<td align="center">27</td><br />
<td align="center">28</td><br />
<td align="center">29</td><br />
</tr><br />
<tr><br />
<td align="center">30</td><br />
<td align="center">31</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><br />
<br />
<h3>System 1 assembly</h3><br />
<div align="center">&nbsp;</div><br />
<p><br />
<table border="1"><br />
<br />
<tr><br />
<td align="center"><strong>Run</strong></td><br />
<td align="center"><strong>Date and Time<br /><br />
</strong></td><br />
<td align="center"><strong>Objective</strong></td><br />
<td align="center"><strong>Attendance</strong></td><br />
</tr><br />
<tr><br />
<td style="background-color: rgb(255,255,255)" align="center">1</td><br />
<td style="background-color: rgb(255,255,255)">8 AM Sat, 9/1, 07</td><br />
<td style="background-color: rgb(255,255,255)"><br />
<ul><br />
<li>(RBS + cinR) + (RBS + HSL) = cinR + HSL</li><br />
<li>RBS + cinR ver. 2</li><br />
<li>plate culture for (cinR + HSL) and (RBS + CinR) ver.2</li><br />
</ul><br />
</td><br />
<td style="background-color: rgb(255,255,255)" align="center">茹, 翔</td><br />
</tr><br />
<tr><br />
<td style="background-color: rgb(255,255,255)" align="center">2</td><br />
<td style="background-color: rgb(255,255,255)">8 AM Sun, 9/2, 07</td><br />
<td style="background-color: rgb(255,255,255)"><br />
<ul><br />
<li>Liquid culture for (cinR + HSL) and (RBS + CinR) ver.2</li><br />
<li>[https://2007.igem.org/NYMU_Taipei/Team_schedule/classify_parts_in_box classify parts in box]</li><br />
</ul><br />
</td><br />
<td style="background-color: rgb(255,255,255)" align="center">雄, 威, 茹, 駿</td><br />
</tr><br />
<tr><br />
<td style="background-color: rgb(255,255,255)" align="center">3</td><br />
<td style="background-color: rgb(255,255,255)">8 AM Mon, 9/3, 07</td><br />
<td style="background-color: rgb(255,255,255)"><br />
<ul><br />
<li>plasmid extraction for (cinR + HSL) and (RBS + CinR) ver.2</li><br />
<li>pOmpC + (RBS + INS_A)</li><br />
<li>(RBS + cinR) + (RBS + HSL) + D-term</li><br />
</ul><br />
</td><br />
<td style="background-color: rgb(255,255,255)" align="center">宥, 君, 威</td><br />
</tr><br />
<tr><br />
<td style="background-color: rgb(255,255,255)" align="center">4</td><br />
<td style="background-color: rgb(255,255,255)">8 AM Tue, 9/4, 07</td><br />
<td style="background-color: rgb(255,255,255)"><br />
<ul><br />
<li>plate culture for ...</li><br />
<li>Measure the concentration of all excised parts<br />
<ul><br />
<li>(cinR + HSL)</li><br />
<li>(RBS + CinR) ver.2</li><br />
</ul><br />
</li><br />
<li>BsaBI digestion of cinR</li><br />
</ul><br />
</td><br />
<td style="background-color: rgb(255,255,255)" align="center">宥, 君, 威</td><br />
</tr><br />
<tr><br />
<td align="center">5</td><br />
<td>8 AM Wed, 9/5, 07</td><br />
<td><br />
<ul><br />
<li>(RBS + INS_A), PCR #1</li><br />
<li>(RBS + INS_B), PCR #1</li><br />
<li>pOmpC + (RBS + INS_A), not check yet</li><br />
<li>pOmpC + (RBS + INS_B), not check yet</li><br />
</ul><br />
</td><br />
<td align="center">威, 茹</td><br />
</tr><br />
<tr><br />
<td align="center">6</td><br />
<td>8 AM Thr, 9/6, 07</td><br />
<td><br />
<ul><br />
<li>N/A</li><br />
</ul><br />
</td><br />
<td align="center">威</td><br />
</tr><br />
<tr><br />
<td align="center">7</td><br />
<td>8 AM Fri, 9/7, 07</td><br />
<td><br />
<ul><br />
<li>N/A</li><br />
</ul><br />
</td><br />
<td align="center">儒, 威, 茹</td><br />
</tr><br />
<tr><br />
<td align="center">8</td><br />
<td>8 AM Sat, 9/8, 07</td><br />
<td><br />
<ul><br />
<li>(RBS+INS_A), PCR #2</li><br />
<li>(RBS+INS_B), PCR #2</li><br />
<li>GEL check (5 tubes for each gene)<br />
<ul><br />
<li>INS_A has two tubes A1 (0.08g), A3 (0.15g)</li><br />
<li>INS_B has five tubes B1 (0.12g), B2 (0.12g), B3 (0.16g), B4 (0.11g), B5 (0.17g)</li><br />
</ul><br />
</li><br />
<li>ligation for INS_A and pOmpC</li><br />
<li>plate culture for (pOmpC+INS_A and pOmpC+INS_B), transformation <font color="#ff0000">failed</font><br />
<ul><br />
<li>forget to add vector</li><br />
</ul><br />
</li><br />
</ul><br />
</td><br />
<td align="center">倫, 茹</td><br />
</tr><br />
<tr><br />
<td align="center">9</td><br />
<td>8 AM Sun, 9/9, 07</td><br />
<td><br />
<ul><br />
<li>plate culture <font color="#ff0000">failed</font> (possible due to absent of vector, checked by text paper record)</li><br />
<li>re-ligate INS_A insert into vector with pOmpC</li><br />
<li>re-check the concentration of insert INS_A by [http://rsb.info.nih.gov/ij/ ImageJ]</li><br />
<li>plate culture again for pOmpC+INS_A<br />
<ul><br />
<li>1uL insert + 1uL vector</li><br />
</ul><br />
</li><br />
</ul><br />
</td><br />
<td align="center">倫, 翔</td><br />
</tr><br />
<tr><br />
<td align="center">10</td><br />
<td>4 PM Mon, 9/10, 07</td><br />
<td><br />
<ul><br />
<li>plate culture is still <font color="#ff0000">failed</font></li><br />
<li>re-build pOmpC + INS_A in two different insert concentration<br />
<ul><br />
<li>2uL insert + 1uL vector</li><br />
<li>3uL insert + 1uL vector</li><br />
</ul><br />
</li><br />
</ul><br />
</td><br />
<td align="center">倫, 翔, 銘</td><br />
</tr><br />
<tr><br />
<td align="center">11</td><br />
<td>6 PM Thu, 9/11, 07</td><br />
<td><br />
<ul><br />
<li>re-measure the concentration of insert by biophotometer<br />
<ul><br />
<li>dsDNA use A260 OD to estimate, 1 A260 OD = 50 mg/mL = 0.05 ug/uL</li><br />
<li>OD ranged from 0.1 - 2.0 is located in linear region</li><br />
<li>OD close to zero is not reliable</li><br />
</ul><br />
</li><br />
<li>test for D7 and D8 (9/8 PCR #2) in box #1</li><br />
</ul><br />
</td><br />
<td align="center">銘</td><br />
</tr><br />
<tr><br />
<td align="center">12</td><br />
<td>6 PM Wed, 9/12, 07</td><br />
<td><br />
<ul><br />
<li>check (9/5 PCR) by GEL separation<br />
<ul><br />
<li>concentration of INS_A is still too low</li><br />
</ul><br />
</li><br />
<li>RE-examine the condition of PCR in different annealing temperature<br />
<ul><br />
<li>PCR INS_A in 49.8, 52.3, 55.5, 58.7 and 60.9 degree</li><br />
</ul><br />
</li><br />
</ul><br />
</td><br />
<td align="center">銘</td><br />
</tr><br />
<tr><br />
<td align="center">13</td><br />
<td>6 PM Thur, 9/13, 07</td><br />
<td><br />
<ul><br />
<li>check (9/12 PCR) by GEL separation<br />
<ul><br />
<li>band seems O.K.</li><br />
<li>best annealing temperature is around 55.5</li><br />
</ul><br />
</li><br />
<li>biophotometer concentration test<br />
<ul><br />
<li>9/5 and 9/12 PCR seem O.K.</li><br />
<li>9/8 PCR seem problemistic due to A260/A280 more than 10</li><br />
</ul><br />
</li><br />
</ul><br />
</td><br />
<td align="center">銘</td><br />
</tr><br />
<tr><br />
<td align="center">14</td><br />
<td>6 PM Sun, 9/16, 07</td><br />
<td><br />
<ul><br />
<li>digest CinR+HSL (ES) and D-term (EX)and GEL separation check<br />
<ul><br />
<li>tube #1 and #2 of CinR+HSL plasmid extraction is not correct after GEL check</li><br />
<li>first digestion of D-term (EcoRI) is not very sucessful<br />
<ul><br />
<li>thus pett re-digest D-term with double enzyme amount</li><br />
</ul><br />
</li><br />
</ul><br />
</li><br />
</ul><br />
</td><br />
<td align="center">宥, 君, 翔</td><br />
</tr><br />
<tr><br />
<td align="center">PCR</td><br />
<td>Thur, 9/27, 07</td><br />
<td><br />
<ul><br />
<li>PCR of TAT_INS_A, TAT_INS_B, OmpRBS, TAT_IDE</li><br />
</ul><br />
</td><br />
<td align="center">威</td><br />
</tr><br />
</table><br />
</p><br />
<p>&nbsp;</p><br />
[https://2007.igem.org/Taipei Back]</div>Blenthttp://2007.igem.org/wiki/index.php/NYMU_Taipei/Team_scheduleNYMU Taipei/Team schedule2007-12-13T03:02:14Z<p>Blent: /* Nov 2007 */</p>
<hr />
<div><br /><hr><br />
<br />
==July 2007==<br />
<br />
<table style="text-align: left" width="50%" border="1"><br />
<br />
<tr><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Sun.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Mon.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Tue.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Wed.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Thr.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Fri.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Sat.</big></td><br />
</tr><br />
<tr><br />
<td align="center">1</td><br />
<td align="center">2</td><br />
<td align="center">3</td><br />
<td align="center">4</td><br />
<td align="center">5</td><br />
<td align="center">[[NYMU_Taipei/Regular_meeting/July_6| 6]]</td><br />
<td align="center">7</td><br />
</tr><br />
<tr><br />
<td align="center">8</td><br />
<td align="center">9</td><br />
<td align="center">10</td><br />
<td align="center">11</td><br />
<td align="center">12</td><br />
<td align="center">[[NYMU_Taipei/Regular_meeting/July_13| 13]]</td><br />
<td align="center">14</td><br />
</tr><br />
<tr><br />
<td align="center">15</td><br />
<td align="center">16</td><br />
<td align="center">17</td><br />
<td align="center">18</td><br />
<td align="center">19</td><br />
<td align="center">[[NYMU_Taipei/Regular_meeting/July_20| 20]]</td><br />
<td align="center">21</td><br />
</tr><br />
<tr><br />
<td align="center">22</td><br />
<td align="center">23</td><br />
<td align="center">24</td><br />
<td align="center">25</td><br />
<td align="center">26</td><br />
<td align="center">[[NYMU_Taipei/Regular_meeting/July_27| 27]]</td><br />
<td align="center">28</td><br />
</tr><br />
<tr><br />
<td align="center">29</td><br />
<td align="center">30</td><br />
<td align="center">31</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><br />
<br />
==Aug 2007==<br />
<table style="text-align: left" width="50%" border="1"><br />
<tr><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Sun.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Mon.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Tue.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Wed.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Thr.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Fri.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Sat.</big></td><br />
</tr><br />
<tr><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">1</td><br />
<td align="center">2</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_3| 3]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_4| 4]]</td><br />
</tr><br />
<tr><br />
<td align="center">5</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_6&7| 6]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_6&7| 7]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_8| 8]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_9| 9]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_10| 10]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_11| 11]]</td><br />
</tr><br />
<tr><br />
<td align="center">12</td><br />
<td align="center">13</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_14| 14]]</td><br />
<td align="center">15</td><br />
<td align="center">16</td><br />
<td align="center">[[NYMU_Taipei/Regular_meeting/August_17| 17]]</td><br />
<td align="center">18</td><br />
</tr><br />
<tr><br />
<td align="center">19</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_20| 20]]</td><br />
<td align="center">21</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_22| 22]]</td><br />
<br />
<td align="center">23</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_24| 24]]</td><br />
<td align="center">25</td><br />
</tr><br />
<tr><br />
<td align="center">26</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_27| 27]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_28| 28]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_29| 29]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_30| 30]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_31| 31]]</td><br />
<td align="center">&nbsp;</td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><br />
<br />
==Sep 2007==<br />
<table style="text-align: left" width="50%" border="1"><br />
<tr><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Sun.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Mon.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Tue.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Wed.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Thr.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Fri.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Sat.</big></td><br />
</tr><br />
<tr><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_1| 1]]</td><br />
</tr><br />
<tr><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_2| 2]]</td><br />
<td align="center">3</td><br />
<td align="center">4</td><br />
<td align="center">5</td><br />
<td align="center">6</td><br />
<td align="center">[[NYMU_Taipei/Regular_meeting/September_7| 7]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_8| 8]]</td><br />
</tr><br />
<tr><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_9| 9]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_10| 10]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_11| 11]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_12| 12]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_13| 13]]</td><br />
<td align="center">[[NYMU_Taipei/Regular_meeting/September_14| 14]]</td><br />
<td align="center">15</td><br />
</tr><br />
<tr><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_16| 16]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_17| 17]]</td><br />
<td align="center">18</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_19| 19]]</td><br />
<br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_20| 20]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_21| 21]]</td><br />
<td align="center">22</td><br />
</tr><br />
<tr><br />
<td align="center">23</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_24| 24]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_25| 25]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_26| 26]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_27| 27]]</td><br />
<td align="center">[[NYMU_Taipei/Regular_meeting/September_28| 28]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_29| 29]]</td><br />
</tr><br />
<tr><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_30| 30]]</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><br />
<br />
==Oct 2007==<br />
<table style="text-align: left" width="50%" border="1"><br />
<tr><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Sun.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Mon.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Tue.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Wed.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Thr.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Fri.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Oct.</big></td><br />
</tr><br />
<tr><br />
<td align="center">&nbsp;</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_1| 1]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_2| 2]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_3| 3]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_4| 4]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_5| 5]]</td><br />
<td align="center">6</td><br />
</tr><br />
<tr><br />
<td align="center">7</td><br />
<td align="center">8</td><br />
<td align="center">9</td><br />
<td align="center">10</td><br />
<td align="center">11</td><br />
<td align="center">[[NYMU_Taipei/Regular_meeting/October_12| 12]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_13| 13]]</td><br />
</tr><br />
<tr><br />
<td align="center">14</td><br />
<td align="center">15</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_16| 16]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_17| 17]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_18| 18]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_19| 19]]</td><br />
<td align="center">20</td><br />
</tr><br />
<tr><br />
<td align="center">21</td><br />
<td align="center">22</td><br />
<td align="center">23</td><br />
<td align="center">24</td><br />
<td align="center">25</td><br />
<td align="center">26</td><br />
<td align="center">27</td><br />
</tr><br />
<tr><br />
<td align="center">28</td><br />
<td align="center">29</td><br />
<td align="center">30</td><br />
<td align="center">31</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><br />
==Nov 2007==<br />
<table style="text-align: left" width="50%" border="1"><br />
<tr><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Sun.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Mon.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Tue.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Wed.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Thr.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Fri.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Oct.</big></td><br />
</tr><br />
<tr><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_11_1| 1]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_11_2| 2]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_11_3| 3]]</td><br />
</tr><br />
<tr><br />
<td align="center">4</td><br />
<td align="center">5</td><br />
<td align="center">6</td><br />
<td align="center">7</td><br />
<td align="center">8</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_11_9| 9]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_11_10| 10]]</td><br />
</tr><br />
<tr><br />
<td align="center">11</td><br />
<td align="center">12</td><br />
<td align="center">13</td><br />
<td align="center">14</td><br />
<td align="center">15</td><br />
<td align="center">16</td><br />
<td align="center">17</td><br />
</tr><br />
<tr><br />
<td align="center">18</td><br />
<td align="center">19</td><br />
<td align="center">20</td><br />
<td align="center">21</td><br />
<td align="center">22</td><br />
<td align="center">23</td><br />
<td align="center">24</td><br />
</tr><br />
<tr><br />
<td align="center">25</td><br />
<td align="center">26</td><br />
<td align="center">27</td><br />
<td align="center">28</td><br />
<td align="center">29</td><br />
<td align="center">30</td><br />
<td align="center">&nbsp;</td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><br />
==Dec 2007==<br />
<table style="text-align: left" width="50%" border="1"><br />
<tr><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Sun.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Mon.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Tue.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Wed.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Thr.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Fri.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Oct.</big></td><br />
</tr><br />
<tr><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">1</td><br />
</tr><br />
<tr><br />
<td align="center">2</td><br />
<td align="center">3</td><br />
<td align="center">4</td><br />
<td align="center">5</td><br />
<td align="center">6</td><br />
<td align="center">7</td><br />
<td align="center">8</td><br />
</tr><br />
<tr><br />
<td align="center">9</td><br />
<td align="center">10</td><br />
<td align="center">11</td><br />
<td align="center">12</td><br />
<td align="center">13</td><br />
<td align="center">14</td><br />
<td align="center">15</td><br />
</tr><br />
<tr><br />
<td align="center">16</td><br />
<td align="center">17</td><br />
<td align="center">18</td><br />
<td align="center">19</td><br />
<td align="center">20</td><br />
<td align="center">21</td><br />
<td align="center">22</td><br />
</tr><br />
<tr><br />
<td align="center">23</td><br />
<td align="center">24</td><br />
<td align="center">25</td><br />
<td align="center">26</td><br />
<td align="center">27</td><br />
<td align="center">28</td><br />
<td align="center">29</td><br />
</tr><br />
<tr><br />
<td align="center">30</td><br />
<td align="center">31</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><br />
<br />
<h3>System 1 assembly</h3><br />
<div align="center">&nbsp;</div><br />
<p><br />
<table border="1"><br />
<br />
<tr><br />
<td align="center"><strong>Run</strong></td><br />
<td align="center"><strong>Date and Time<br /><br />
</strong></td><br />
<td align="center"><strong>Objective</strong></td><br />
<td align="center"><strong>Attendance</strong></td><br />
</tr><br />
<tr><br />
<td style="background-color: rgb(255,255,255)" align="center">1</td><br />
<td style="background-color: rgb(255,255,255)">8 AM Sat, 9/1, 07</td><br />
<td style="background-color: rgb(255,255,255)"><br />
<ul><br />
<li>(RBS + cinR) + (RBS + HSL) = cinR + HSL</li><br />
<li>RBS + cinR ver. 2</li><br />
<li>plate culture for (cinR + HSL) and (RBS + CinR) ver.2</li><br />
</ul><br />
</td><br />
<td style="background-color: rgb(255,255,255)" align="center">茹, 翔</td><br />
</tr><br />
<tr><br />
<td style="background-color: rgb(255,255,255)" align="center">2</td><br />
<td style="background-color: rgb(255,255,255)">8 AM Sun, 9/2, 07</td><br />
<td style="background-color: rgb(255,255,255)"><br />
<ul><br />
<li>Liquid culture for (cinR + HSL) and (RBS + CinR) ver.2</li><br />
<li>[https://2007.igem.org/NYMU_Taipei/Team_schedule/classify_parts_in_box classify parts in box]</li><br />
</ul><br />
</td><br />
<td style="background-color: rgb(255,255,255)" align="center">雄, 威, 茹, 駿</td><br />
</tr><br />
<tr><br />
<td style="background-color: rgb(255,255,255)" align="center">3</td><br />
<td style="background-color: rgb(255,255,255)">8 AM Mon, 9/3, 07</td><br />
<td style="background-color: rgb(255,255,255)"><br />
<ul><br />
<li>plasmid extraction for (cinR + HSL) and (RBS + CinR) ver.2</li><br />
<li>pOmpC + (RBS + INS_A)</li><br />
<li>(RBS + cinR) + (RBS + HSL) + D-term</li><br />
</ul><br />
</td><br />
<td style="background-color: rgb(255,255,255)" align="center">宥, 君, 威</td><br />
</tr><br />
<tr><br />
<td style="background-color: rgb(255,255,255)" align="center">4</td><br />
<td style="background-color: rgb(255,255,255)">8 AM Tue, 9/4, 07</td><br />
<td style="background-color: rgb(255,255,255)"><br />
<ul><br />
<li>plate culture for ...</li><br />
<li>Measure the concentration of all excised parts<br />
<ul><br />
<li>(cinR + HSL)</li><br />
<li>(RBS + CinR) ver.2</li><br />
</ul><br />
</li><br />
<li>BsaBI digestion of cinR</li><br />
</ul><br />
</td><br />
<td style="background-color: rgb(255,255,255)" align="center">宥, 君, 威</td><br />
</tr><br />
<tr><br />
<td align="center">5</td><br />
<td>8 AM Wed, 9/5, 07</td><br />
<td><br />
<ul><br />
<li>(RBS + INS_A), PCR #1</li><br />
<li>(RBS + INS_B), PCR #1</li><br />
<li>pOmpC + (RBS + INS_A), not check yet</li><br />
<li>pOmpC + (RBS + INS_B), not check yet</li><br />
</ul><br />
</td><br />
<td align="center">威, 茹</td><br />
</tr><br />
<tr><br />
<td align="center">6</td><br />
<td>8 AM Thr, 9/6, 07</td><br />
<td><br />
<ul><br />
<li>N/A</li><br />
</ul><br />
</td><br />
<td align="center">威</td><br />
</tr><br />
<tr><br />
<td align="center">7</td><br />
<td>8 AM Fri, 9/7, 07</td><br />
<td><br />
<ul><br />
<li>N/A</li><br />
</ul><br />
</td><br />
<td align="center">儒, 威, 茹</td><br />
</tr><br />
<tr><br />
<td align="center">8</td><br />
<td>8 AM Sat, 9/8, 07</td><br />
<td><br />
<ul><br />
<li>(RBS+INS_A), PCR #2</li><br />
<li>(RBS+INS_B), PCR #2</li><br />
<li>GEL check (5 tubes for each gene)<br />
<ul><br />
<li>INS_A has two tubes A1 (0.08g), A3 (0.15g)</li><br />
<li>INS_B has five tubes B1 (0.12g), B2 (0.12g), B3 (0.16g), B4 (0.11g), B5 (0.17g)</li><br />
</ul><br />
</li><br />
<li>ligation for INS_A and pOmpC</li><br />
<li>plate culture for (pOmpC+INS_A and pOmpC+INS_B), transformation <font color="#ff0000">failed</font><br />
<ul><br />
<li>forget to add vector</li><br />
</ul><br />
</li><br />
</ul><br />
</td><br />
<td align="center">倫, 茹</td><br />
</tr><br />
<tr><br />
<td align="center">9</td><br />
<td>8 AM Sun, 9/9, 07</td><br />
<td><br />
<ul><br />
<li>plate culture <font color="#ff0000">failed</font> (possible due to absent of vector, checked by text paper record)</li><br />
<li>re-ligate INS_A insert into vector with pOmpC</li><br />
<li>re-check the concentration of insert INS_A by [http://rsb.info.nih.gov/ij/ ImageJ]</li><br />
<li>plate culture again for pOmpC+INS_A<br />
<ul><br />
<li>1uL insert + 1uL vector</li><br />
</ul><br />
</li><br />
</ul><br />
</td><br />
<td align="center">倫, 翔</td><br />
</tr><br />
<tr><br />
<td align="center">10</td><br />
<td>4 PM Mon, 9/10, 07</td><br />
<td><br />
<ul><br />
<li>plate culture is still <font color="#ff0000">failed</font></li><br />
<li>re-build pOmpC + INS_A in two different insert concentration<br />
<ul><br />
<li>2uL insert + 1uL vector</li><br />
<li>3uL insert + 1uL vector</li><br />
</ul><br />
</li><br />
</ul><br />
</td><br />
<td align="center">倫, 翔, 銘</td><br />
</tr><br />
<tr><br />
<td align="center">11</td><br />
<td>6 PM Thu, 9/11, 07</td><br />
<td><br />
<ul><br />
<li>re-measure the concentration of insert by biophotometer<br />
<ul><br />
<li>dsDNA use A260 OD to estimate, 1 A260 OD = 50 mg/mL = 0.05 ug/uL</li><br />
<li>OD ranged from 0.1 - 2.0 is located in linear region</li><br />
<li>OD close to zero is not reliable</li><br />
</ul><br />
</li><br />
<li>test for D7 and D8 (9/8 PCR #2) in box #1</li><br />
</ul><br />
</td><br />
<td align="center">銘</td><br />
</tr><br />
<tr><br />
<td align="center">12</td><br />
<td>6 PM Wed, 9/12, 07</td><br />
<td><br />
<ul><br />
<li>check (9/5 PCR) by GEL separation<br />
<ul><br />
<li>concentration of INS_A is still too low</li><br />
</ul><br />
</li><br />
<li>RE-examine the condition of PCR in different annealing temperature<br />
<ul><br />
<li>PCR INS_A in 49.8, 52.3, 55.5, 58.7 and 60.9 degree</li><br />
</ul><br />
</li><br />
</ul><br />
</td><br />
<td align="center">銘</td><br />
</tr><br />
<tr><br />
<td align="center">13</td><br />
<td>6 PM Thur, 9/13, 07</td><br />
<td><br />
<ul><br />
<li>check (9/12 PCR) by GEL separation<br />
<ul><br />
<li>band seems O.K.</li><br />
<li>best annealing temperature is around 55.5</li><br />
</ul><br />
</li><br />
<li>biophotometer concentration test<br />
<ul><br />
<li>9/5 and 9/12 PCR seem O.K.</li><br />
<li>9/8 PCR seem problemistic due to A260/A280 more than 10</li><br />
</ul><br />
</li><br />
</ul><br />
</td><br />
<td align="center">銘</td><br />
</tr><br />
<tr><br />
<td align="center">14</td><br />
<td>6 PM Sun, 9/16, 07</td><br />
<td><br />
<ul><br />
<li>digest CinR+HSL (ES) and D-term (EX)and GEL separation check<br />
<ul><br />
<li>tube #1 and #2 of CinR+HSL plasmid extraction is not correct after GEL check</li><br />
<li>first digestion of D-term (EcoRI) is not very sucessful<br />
<ul><br />
<li>thus pett re-digest D-term with double enzyme amount</li><br />
</ul><br />
</li><br />
</ul><br />
</li><br />
</ul><br />
</td><br />
<td align="center">宥, 君, 翔</td><br />
</tr><br />
<tr><br />
<td align="center">PCR</td><br />
<td>Thur, 9/27, 07</td><br />
<td><br />
<ul><br />
<li>PCR of TAT_INS_A, TAT_INS_B, OmpRBS, TAT_IDE</li><br />
</ul><br />
</td><br />
<td align="center">威</td><br />
</tr><br />
</table><br />
</p><br />
<p>&nbsp;</p><br />
[https://2007.igem.org/Taipei Back]</div>Blenthttp://2007.igem.org/wiki/index.php/NYMU_Taipei/Team_scheduleNYMU Taipei/Team schedule2007-12-13T02:58:41Z<p>Blent: /* Nov 2007 */</p>
<hr />
<div><br /><hr><br />
<br />
==July 2007==<br />
<br />
<table style="text-align: left" width="50%" border="1"><br />
<br />
<tr><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Sun.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Mon.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Tue.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Wed.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Thr.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Fri.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Sat.</big></td><br />
</tr><br />
<tr><br />
<td align="center">1</td><br />
<td align="center">2</td><br />
<td align="center">3</td><br />
<td align="center">4</td><br />
<td align="center">5</td><br />
<td align="center">[[NYMU_Taipei/Regular_meeting/July_6| 6]]</td><br />
<td align="center">7</td><br />
</tr><br />
<tr><br />
<td align="center">8</td><br />
<td align="center">9</td><br />
<td align="center">10</td><br />
<td align="center">11</td><br />
<td align="center">12</td><br />
<td align="center">[[NYMU_Taipei/Regular_meeting/July_13| 13]]</td><br />
<td align="center">14</td><br />
</tr><br />
<tr><br />
<td align="center">15</td><br />
<td align="center">16</td><br />
<td align="center">17</td><br />
<td align="center">18</td><br />
<td align="center">19</td><br />
<td align="center">[[NYMU_Taipei/Regular_meeting/July_20| 20]]</td><br />
<td align="center">21</td><br />
</tr><br />
<tr><br />
<td align="center">22</td><br />
<td align="center">23</td><br />
<td align="center">24</td><br />
<td align="center">25</td><br />
<td align="center">26</td><br />
<td align="center">[[NYMU_Taipei/Regular_meeting/July_27| 27]]</td><br />
<td align="center">28</td><br />
</tr><br />
<tr><br />
<td align="center">29</td><br />
<td align="center">30</td><br />
<td align="center">31</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><br />
<br />
==Aug 2007==<br />
<table style="text-align: left" width="50%" border="1"><br />
<tr><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Sun.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Mon.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Tue.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Wed.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Thr.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Fri.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Sat.</big></td><br />
</tr><br />
<tr><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">1</td><br />
<td align="center">2</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_3| 3]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_4| 4]]</td><br />
</tr><br />
<tr><br />
<td align="center">5</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_6&7| 6]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_6&7| 7]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_8| 8]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_9| 9]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_10| 10]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_11| 11]]</td><br />
</tr><br />
<tr><br />
<td align="center">12</td><br />
<td align="center">13</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_14| 14]]</td><br />
<td align="center">15</td><br />
<td align="center">16</td><br />
<td align="center">[[NYMU_Taipei/Regular_meeting/August_17| 17]]</td><br />
<td align="center">18</td><br />
</tr><br />
<tr><br />
<td align="center">19</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_20| 20]]</td><br />
<td align="center">21</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_22| 22]]</td><br />
<br />
<td align="center">23</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_24| 24]]</td><br />
<td align="center">25</td><br />
</tr><br />
<tr><br />
<td align="center">26</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_27| 27]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_28| 28]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_29| 29]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_30| 30]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_31| 31]]</td><br />
<td align="center">&nbsp;</td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><br />
<br />
==Sep 2007==<br />
<table style="text-align: left" width="50%" border="1"><br />
<tr><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Sun.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Mon.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Tue.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Wed.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Thr.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Fri.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Sat.</big></td><br />
</tr><br />
<tr><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_1| 1]]</td><br />
</tr><br />
<tr><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_2| 2]]</td><br />
<td align="center">3</td><br />
<td align="center">4</td><br />
<td align="center">5</td><br />
<td align="center">6</td><br />
<td align="center">[[NYMU_Taipei/Regular_meeting/September_7| 7]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_8| 8]]</td><br />
</tr><br />
<tr><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_9| 9]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_10| 10]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_11| 11]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_12| 12]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_13| 13]]</td><br />
<td align="center">[[NYMU_Taipei/Regular_meeting/September_14| 14]]</td><br />
<td align="center">15</td><br />
</tr><br />
<tr><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_16| 16]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_17| 17]]</td><br />
<td align="center">18</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_19| 19]]</td><br />
<br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_20| 20]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_21| 21]]</td><br />
<td align="center">22</td><br />
</tr><br />
<tr><br />
<td align="center">23</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_24| 24]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_25| 25]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_26| 26]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_27| 27]]</td><br />
<td align="center">[[NYMU_Taipei/Regular_meeting/September_28| 28]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_29| 29]]</td><br />
</tr><br />
<tr><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_30| 30]]</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><br />
<br />
==Oct 2007==<br />
<table style="text-align: left" width="50%" border="1"><br />
<tr><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Sun.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Mon.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Tue.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Wed.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Thr.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Fri.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Oct.</big></td><br />
</tr><br />
<tr><br />
<td align="center">&nbsp;</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_1| 1]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_2| 2]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_3| 3]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_4| 4]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_5| 5]]</td><br />
<td align="center">6</td><br />
</tr><br />
<tr><br />
<td align="center">7</td><br />
<td align="center">8</td><br />
<td align="center">9</td><br />
<td align="center">10</td><br />
<td align="center">11</td><br />
<td align="center">[[NYMU_Taipei/Regular_meeting/October_12| 12]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_13| 13]]</td><br />
</tr><br />
<tr><br />
<td align="center">14</td><br />
<td align="center">15</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_16| 16]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_17| 17]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_18| 18]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_19| 19]]</td><br />
<td align="center">20</td><br />
</tr><br />
<tr><br />
<td align="center">21</td><br />
<td align="center">22</td><br />
<td align="center">23</td><br />
<td align="center">24</td><br />
<td align="center">25</td><br />
<td align="center">26</td><br />
<td align="center">27</td><br />
</tr><br />
<tr><br />
<td align="center">28</td><br />
<td align="center">29</td><br />
<td align="center">30</td><br />
<td align="center">31</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><br />
==Nov 2007==<br />
<table style="text-align: left" width="50%" border="1"><br />
<tr><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Sun.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Mon.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Tue.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Wed.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Thr.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Fri.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Oct.</big></td><br />
</tr><br />
<tr><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_11_1| 1]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_11_2| 2]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_11_3| 3]]</td><br />
</tr><br />
<tr><br />
<td align="center">4</td><br />
<td align="center">5</td><br />
<td align="center">6</td><br />
<td align="center">7</td><br />
<td align="center">8</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_11_9| 9]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_11_10| 10]]</td><br />
</tr><br />
<tr><br />
<td align="center">11</td><br />
<td align="center">12</td><br />
<td align="center">13</td><br />
<td align="center">14</td><br />
<td align="center">15</td><br />
<td align="center">16</td><br />
<td align="center">17</td><br />
</tr><br />
<tr><br />
<td align="center">18</td><br />
<td align="center">19</td><br />
<td align="center">20</td><br />
<td align="center">21</td><br />
<td align="center">22</td><br />
<td align="center">23</td><br />
<td align="center">24</td><br />
</tr><br />
<tr><br />
<td align="center">25</td><br />
<td align="center">26</td><br />
<td align="center">27</td><br />
<td align="center">28</td><br />
<td align="center">29</td><br />
<td align="center">30</td><br />
<td align="center">&nbsp;</td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><br />
<br />
<h3>System 1 assembly</h3><br />
<div align="center">&nbsp;</div><br />
<p><br />
<table border="1"><br />
<br />
<tr><br />
<td align="center"><strong>Run</strong></td><br />
<td align="center"><strong>Date and Time<br /><br />
</strong></td><br />
<td align="center"><strong>Objective</strong></td><br />
<td align="center"><strong>Attendance</strong></td><br />
</tr><br />
<tr><br />
<td style="background-color: rgb(255,255,255)" align="center">1</td><br />
<td style="background-color: rgb(255,255,255)">8 AM Sat, 9/1, 07</td><br />
<td style="background-color: rgb(255,255,255)"><br />
<ul><br />
<li>(RBS + cinR) + (RBS + HSL) = cinR + HSL</li><br />
<li>RBS + cinR ver. 2</li><br />
<li>plate culture for (cinR + HSL) and (RBS + CinR) ver.2</li><br />
</ul><br />
</td><br />
<td style="background-color: rgb(255,255,255)" align="center">茹, 翔</td><br />
</tr><br />
<tr><br />
<td style="background-color: rgb(255,255,255)" align="center">2</td><br />
<td style="background-color: rgb(255,255,255)">8 AM Sun, 9/2, 07</td><br />
<td style="background-color: rgb(255,255,255)"><br />
<ul><br />
<li>Liquid culture for (cinR + HSL) and (RBS + CinR) ver.2</li><br />
<li>[https://2007.igem.org/NYMU_Taipei/Team_schedule/classify_parts_in_box classify parts in box]</li><br />
</ul><br />
</td><br />
<td style="background-color: rgb(255,255,255)" align="center">雄, 威, 茹, 駿</td><br />
</tr><br />
<tr><br />
<td style="background-color: rgb(255,255,255)" align="center">3</td><br />
<td style="background-color: rgb(255,255,255)">8 AM Mon, 9/3, 07</td><br />
<td style="background-color: rgb(255,255,255)"><br />
<ul><br />
<li>plasmid extraction for (cinR + HSL) and (RBS + CinR) ver.2</li><br />
<li>pOmpC + (RBS + INS_A)</li><br />
<li>(RBS + cinR) + (RBS + HSL) + D-term</li><br />
</ul><br />
</td><br />
<td style="background-color: rgb(255,255,255)" align="center">宥, 君, 威</td><br />
</tr><br />
<tr><br />
<td style="background-color: rgb(255,255,255)" align="center">4</td><br />
<td style="background-color: rgb(255,255,255)">8 AM Tue, 9/4, 07</td><br />
<td style="background-color: rgb(255,255,255)"><br />
<ul><br />
<li>plate culture for ...</li><br />
<li>Measure the concentration of all excised parts<br />
<ul><br />
<li>(cinR + HSL)</li><br />
<li>(RBS + CinR) ver.2</li><br />
</ul><br />
</li><br />
<li>BsaBI digestion of cinR</li><br />
</ul><br />
</td><br />
<td style="background-color: rgb(255,255,255)" align="center">宥, 君, 威</td><br />
</tr><br />
<tr><br />
<td align="center">5</td><br />
<td>8 AM Wed, 9/5, 07</td><br />
<td><br />
<ul><br />
<li>(RBS + INS_A), PCR #1</li><br />
<li>(RBS + INS_B), PCR #1</li><br />
<li>pOmpC + (RBS + INS_A), not check yet</li><br />
<li>pOmpC + (RBS + INS_B), not check yet</li><br />
</ul><br />
</td><br />
<td align="center">威, 茹</td><br />
</tr><br />
<tr><br />
<td align="center">6</td><br />
<td>8 AM Thr, 9/6, 07</td><br />
<td><br />
<ul><br />
<li>N/A</li><br />
</ul><br />
</td><br />
<td align="center">威</td><br />
</tr><br />
<tr><br />
<td align="center">7</td><br />
<td>8 AM Fri, 9/7, 07</td><br />
<td><br />
<ul><br />
<li>N/A</li><br />
</ul><br />
</td><br />
<td align="center">儒, 威, 茹</td><br />
</tr><br />
<tr><br />
<td align="center">8</td><br />
<td>8 AM Sat, 9/8, 07</td><br />
<td><br />
<ul><br />
<li>(RBS+INS_A), PCR #2</li><br />
<li>(RBS+INS_B), PCR #2</li><br />
<li>GEL check (5 tubes for each gene)<br />
<ul><br />
<li>INS_A has two tubes A1 (0.08g), A3 (0.15g)</li><br />
<li>INS_B has five tubes B1 (0.12g), B2 (0.12g), B3 (0.16g), B4 (0.11g), B5 (0.17g)</li><br />
</ul><br />
</li><br />
<li>ligation for INS_A and pOmpC</li><br />
<li>plate culture for (pOmpC+INS_A and pOmpC+INS_B), transformation <font color="#ff0000">failed</font><br />
<ul><br />
<li>forget to add vector</li><br />
</ul><br />
</li><br />
</ul><br />
</td><br />
<td align="center">倫, 茹</td><br />
</tr><br />
<tr><br />
<td align="center">9</td><br />
<td>8 AM Sun, 9/9, 07</td><br />
<td><br />
<ul><br />
<li>plate culture <font color="#ff0000">failed</font> (possible due to absent of vector, checked by text paper record)</li><br />
<li>re-ligate INS_A insert into vector with pOmpC</li><br />
<li>re-check the concentration of insert INS_A by [http://rsb.info.nih.gov/ij/ ImageJ]</li><br />
<li>plate culture again for pOmpC+INS_A<br />
<ul><br />
<li>1uL insert + 1uL vector</li><br />
</ul><br />
</li><br />
</ul><br />
</td><br />
<td align="center">倫, 翔</td><br />
</tr><br />
<tr><br />
<td align="center">10</td><br />
<td>4 PM Mon, 9/10, 07</td><br />
<td><br />
<ul><br />
<li>plate culture is still <font color="#ff0000">failed</font></li><br />
<li>re-build pOmpC + INS_A in two different insert concentration<br />
<ul><br />
<li>2uL insert + 1uL vector</li><br />
<li>3uL insert + 1uL vector</li><br />
</ul><br />
</li><br />
</ul><br />
</td><br />
<td align="center">倫, 翔, 銘</td><br />
</tr><br />
<tr><br />
<td align="center">11</td><br />
<td>6 PM Thu, 9/11, 07</td><br />
<td><br />
<ul><br />
<li>re-measure the concentration of insert by biophotometer<br />
<ul><br />
<li>dsDNA use A260 OD to estimate, 1 A260 OD = 50 mg/mL = 0.05 ug/uL</li><br />
<li>OD ranged from 0.1 - 2.0 is located in linear region</li><br />
<li>OD close to zero is not reliable</li><br />
</ul><br />
</li><br />
<li>test for D7 and D8 (9/8 PCR #2) in box #1</li><br />
</ul><br />
</td><br />
<td align="center">銘</td><br />
</tr><br />
<tr><br />
<td align="center">12</td><br />
<td>6 PM Wed, 9/12, 07</td><br />
<td><br />
<ul><br />
<li>check (9/5 PCR) by GEL separation<br />
<ul><br />
<li>concentration of INS_A is still too low</li><br />
</ul><br />
</li><br />
<li>RE-examine the condition of PCR in different annealing temperature<br />
<ul><br />
<li>PCR INS_A in 49.8, 52.3, 55.5, 58.7 and 60.9 degree</li><br />
</ul><br />
</li><br />
</ul><br />
</td><br />
<td align="center">銘</td><br />
</tr><br />
<tr><br />
<td align="center">13</td><br />
<td>6 PM Thur, 9/13, 07</td><br />
<td><br />
<ul><br />
<li>check (9/12 PCR) by GEL separation<br />
<ul><br />
<li>band seems O.K.</li><br />
<li>best annealing temperature is around 55.5</li><br />
</ul><br />
</li><br />
<li>biophotometer concentration test<br />
<ul><br />
<li>9/5 and 9/12 PCR seem O.K.</li><br />
<li>9/8 PCR seem problemistic due to A260/A280 more than 10</li><br />
</ul><br />
</li><br />
</ul><br />
</td><br />
<td align="center">銘</td><br />
</tr><br />
<tr><br />
<td align="center">14</td><br />
<td>6 PM Sun, 9/16, 07</td><br />
<td><br />
<ul><br />
<li>digest CinR+HSL (ES) and D-term (EX)and GEL separation check<br />
<ul><br />
<li>tube #1 and #2 of CinR+HSL plasmid extraction is not correct after GEL check</li><br />
<li>first digestion of D-term (EcoRI) is not very sucessful<br />
<ul><br />
<li>thus pett re-digest D-term with double enzyme amount</li><br />
</ul><br />
</li><br />
</ul><br />
</li><br />
</ul><br />
</td><br />
<td align="center">宥, 君, 翔</td><br />
</tr><br />
<tr><br />
<td align="center">PCR</td><br />
<td>Thur, 9/27, 07</td><br />
<td><br />
<ul><br />
<li>PCR of TAT_INS_A, TAT_INS_B, OmpRBS, TAT_IDE</li><br />
</ul><br />
</td><br />
<td align="center">威</td><br />
</tr><br />
</table><br />
</p><br />
<p>&nbsp;</p><br />
[https://2007.igem.org/Taipei Back]</div>Blenthttp://2007.igem.org/wiki/index.php/NYMU_Taipei/Team_scheduleNYMU Taipei/Team schedule2007-12-13T02:51:00Z<p>Blent: /* Oct 2007 */</p>
<hr />
<div><br /><hr><br />
<br />
==July 2007==<br />
<br />
<table style="text-align: left" width="50%" border="1"><br />
<br />
<tr><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Sun.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Mon.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Tue.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Wed.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Thr.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Fri.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Sat.</big></td><br />
</tr><br />
<tr><br />
<td align="center">1</td><br />
<td align="center">2</td><br />
<td align="center">3</td><br />
<td align="center">4</td><br />
<td align="center">5</td><br />
<td align="center">[[NYMU_Taipei/Regular_meeting/July_6| 6]]</td><br />
<td align="center">7</td><br />
</tr><br />
<tr><br />
<td align="center">8</td><br />
<td align="center">9</td><br />
<td align="center">10</td><br />
<td align="center">11</td><br />
<td align="center">12</td><br />
<td align="center">[[NYMU_Taipei/Regular_meeting/July_13| 13]]</td><br />
<td align="center">14</td><br />
</tr><br />
<tr><br />
<td align="center">15</td><br />
<td align="center">16</td><br />
<td align="center">17</td><br />
<td align="center">18</td><br />
<td align="center">19</td><br />
<td align="center">[[NYMU_Taipei/Regular_meeting/July_20| 20]]</td><br />
<td align="center">21</td><br />
</tr><br />
<tr><br />
<td align="center">22</td><br />
<td align="center">23</td><br />
<td align="center">24</td><br />
<td align="center">25</td><br />
<td align="center">26</td><br />
<td align="center">[[NYMU_Taipei/Regular_meeting/July_27| 27]]</td><br />
<td align="center">28</td><br />
</tr><br />
<tr><br />
<td align="center">29</td><br />
<td align="center">30</td><br />
<td align="center">31</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><br />
<br />
==Aug 2007==<br />
<table style="text-align: left" width="50%" border="1"><br />
<tr><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Sun.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Mon.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Tue.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Wed.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Thr.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Fri.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Sat.</big></td><br />
</tr><br />
<tr><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">1</td><br />
<td align="center">2</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_3| 3]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_4| 4]]</td><br />
</tr><br />
<tr><br />
<td align="center">5</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_6&7| 6]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_6&7| 7]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_8| 8]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_9| 9]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_10| 10]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_11| 11]]</td><br />
</tr><br />
<tr><br />
<td align="center">12</td><br />
<td align="center">13</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_14| 14]]</td><br />
<td align="center">15</td><br />
<td align="center">16</td><br />
<td align="center">[[NYMU_Taipei/Regular_meeting/August_17| 17]]</td><br />
<td align="center">18</td><br />
</tr><br />
<tr><br />
<td align="center">19</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_20| 20]]</td><br />
<td align="center">21</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_22| 22]]</td><br />
<br />
<td align="center">23</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_24| 24]]</td><br />
<td align="center">25</td><br />
</tr><br />
<tr><br />
<td align="center">26</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_27| 27]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_28| 28]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_29| 29]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_30| 30]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_31| 31]]</td><br />
<td align="center">&nbsp;</td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><br />
<br />
==Sep 2007==<br />
<table style="text-align: left" width="50%" border="1"><br />
<tr><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Sun.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Mon.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Tue.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Wed.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Thr.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Fri.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Sat.</big></td><br />
</tr><br />
<tr><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_1| 1]]</td><br />
</tr><br />
<tr><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_2| 2]]</td><br />
<td align="center">3</td><br />
<td align="center">4</td><br />
<td align="center">5</td><br />
<td align="center">6</td><br />
<td align="center">[[NYMU_Taipei/Regular_meeting/September_7| 7]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_8| 8]]</td><br />
</tr><br />
<tr><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_9| 9]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_10| 10]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_11| 11]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_12| 12]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_13| 13]]</td><br />
<td align="center">[[NYMU_Taipei/Regular_meeting/September_14| 14]]</td><br />
<td align="center">15</td><br />
</tr><br />
<tr><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_16| 16]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_17| 17]]</td><br />
<td align="center">18</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_19| 19]]</td><br />
<br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_20| 20]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_21| 21]]</td><br />
<td align="center">22</td><br />
</tr><br />
<tr><br />
<td align="center">23</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_24| 24]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_25| 25]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_26| 26]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_27| 27]]</td><br />
<td align="center">[[NYMU_Taipei/Regular_meeting/September_28| 28]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_29| 29]]</td><br />
</tr><br />
<tr><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_30| 30]]</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><br />
<br />
==Oct 2007==<br />
<table style="text-align: left" width="50%" border="1"><br />
<tr><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Sun.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Mon.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Tue.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Wed.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Thr.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Fri.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Oct.</big></td><br />
</tr><br />
<tr><br />
<td align="center">&nbsp;</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_1| 1]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_2| 2]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_3| 3]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_4| 4]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_5| 5]]</td><br />
<td align="center">6</td><br />
</tr><br />
<tr><br />
<td align="center">7</td><br />
<td align="center">8</td><br />
<td align="center">9</td><br />
<td align="center">10</td><br />
<td align="center">11</td><br />
<td align="center">[[NYMU_Taipei/Regular_meeting/October_12| 12]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_13| 13]]</td><br />
</tr><br />
<tr><br />
<td align="center">14</td><br />
<td align="center">15</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_16| 16]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_17| 17]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_18| 18]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_19| 19]]</td><br />
<td align="center">20</td><br />
</tr><br />
<tr><br />
<td align="center">21</td><br />
<td align="center">22</td><br />
<td align="center">23</td><br />
<td align="center">24</td><br />
<td align="center">25</td><br />
<td align="center">26</td><br />
<td align="center">27</td><br />
</tr><br />
<tr><br />
<td align="center">28</td><br />
<td align="center">29</td><br />
<td align="center">30</td><br />
<td align="center">31</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><br />
==Nov 2007==<br />
<table style="text-align: left" width="50%" border="1"><br />
<tr><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Sun.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Mon.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Tue.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Wed.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Thr.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Fri.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Oct.</big></td><br />
</tr><br />
<tr><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_11_1| 1]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_11_2| 2]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_11_3| 3]]</td><br />
</tr><br />
<tr><br />
<td align="center">7</td><br />
<td align="center">8</td><br />
<td align="center">9</td><br />
<td align="center">10</td><br />
<td align="center">11</td><br />
<td align="center">[[NYMU_Taipei/Regular_meeting/October_12| 12]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_13| 13]]</td><br />
</tr><br />
<tr><br />
<td align="center">14</td><br />
<td align="center">15</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_16| 16]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_17| 17]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_18| 18]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_19| 19]]</td><br />
<td align="center">20</td><br />
</tr><br />
<tr><br />
<td align="center">21</td><br />
<td align="center">22</td><br />
<td align="center">23</td><br />
<td align="center">24</td><br />
<td align="center">25</td><br />
<td align="center">26</td><br />
<td align="center">27</td><br />
</tr><br />
<tr><br />
<td align="center">28</td><br />
<td align="center">29</td><br />
<td align="center">30</td><br />
<td align="center">31</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><br />
<br />
<h3>System 1 assembly</h3><br />
<div align="center">&nbsp;</div><br />
<p><br />
<table border="1"><br />
<br />
<tr><br />
<td align="center"><strong>Run</strong></td><br />
<td align="center"><strong>Date and Time<br /><br />
</strong></td><br />
<td align="center"><strong>Objective</strong></td><br />
<td align="center"><strong>Attendance</strong></td><br />
</tr><br />
<tr><br />
<td style="background-color: rgb(255,255,255)" align="center">1</td><br />
<td style="background-color: rgb(255,255,255)">8 AM Sat, 9/1, 07</td><br />
<td style="background-color: rgb(255,255,255)"><br />
<ul><br />
<li>(RBS + cinR) + (RBS + HSL) = cinR + HSL</li><br />
<li>RBS + cinR ver. 2</li><br />
<li>plate culture for (cinR + HSL) and (RBS + CinR) ver.2</li><br />
</ul><br />
</td><br />
<td style="background-color: rgb(255,255,255)" align="center">茹, 翔</td><br />
</tr><br />
<tr><br />
<td style="background-color: rgb(255,255,255)" align="center">2</td><br />
<td style="background-color: rgb(255,255,255)">8 AM Sun, 9/2, 07</td><br />
<td style="background-color: rgb(255,255,255)"><br />
<ul><br />
<li>Liquid culture for (cinR + HSL) and (RBS + CinR) ver.2</li><br />
<li>[https://2007.igem.org/NYMU_Taipei/Team_schedule/classify_parts_in_box classify parts in box]</li><br />
</ul><br />
</td><br />
<td style="background-color: rgb(255,255,255)" align="center">雄, 威, 茹, 駿</td><br />
</tr><br />
<tr><br />
<td style="background-color: rgb(255,255,255)" align="center">3</td><br />
<td style="background-color: rgb(255,255,255)">8 AM Mon, 9/3, 07</td><br />
<td style="background-color: rgb(255,255,255)"><br />
<ul><br />
<li>plasmid extraction for (cinR + HSL) and (RBS + CinR) ver.2</li><br />
<li>pOmpC + (RBS + INS_A)</li><br />
<li>(RBS + cinR) + (RBS + HSL) + D-term</li><br />
</ul><br />
</td><br />
<td style="background-color: rgb(255,255,255)" align="center">宥, 君, 威</td><br />
</tr><br />
<tr><br />
<td style="background-color: rgb(255,255,255)" align="center">4</td><br />
<td style="background-color: rgb(255,255,255)">8 AM Tue, 9/4, 07</td><br />
<td style="background-color: rgb(255,255,255)"><br />
<ul><br />
<li>plate culture for ...</li><br />
<li>Measure the concentration of all excised parts<br />
<ul><br />
<li>(cinR + HSL)</li><br />
<li>(RBS + CinR) ver.2</li><br />
</ul><br />
</li><br />
<li>BsaBI digestion of cinR</li><br />
</ul><br />
</td><br />
<td style="background-color: rgb(255,255,255)" align="center">宥, 君, 威</td><br />
</tr><br />
<tr><br />
<td align="center">5</td><br />
<td>8 AM Wed, 9/5, 07</td><br />
<td><br />
<ul><br />
<li>(RBS + INS_A), PCR #1</li><br />
<li>(RBS + INS_B), PCR #1</li><br />
<li>pOmpC + (RBS + INS_A), not check yet</li><br />
<li>pOmpC + (RBS + INS_B), not check yet</li><br />
</ul><br />
</td><br />
<td align="center">威, 茹</td><br />
</tr><br />
<tr><br />
<td align="center">6</td><br />
<td>8 AM Thr, 9/6, 07</td><br />
<td><br />
<ul><br />
<li>N/A</li><br />
</ul><br />
</td><br />
<td align="center">威</td><br />
</tr><br />
<tr><br />
<td align="center">7</td><br />
<td>8 AM Fri, 9/7, 07</td><br />
<td><br />
<ul><br />
<li>N/A</li><br />
</ul><br />
</td><br />
<td align="center">儒, 威, 茹</td><br />
</tr><br />
<tr><br />
<td align="center">8</td><br />
<td>8 AM Sat, 9/8, 07</td><br />
<td><br />
<ul><br />
<li>(RBS+INS_A), PCR #2</li><br />
<li>(RBS+INS_B), PCR #2</li><br />
<li>GEL check (5 tubes for each gene)<br />
<ul><br />
<li>INS_A has two tubes A1 (0.08g), A3 (0.15g)</li><br />
<li>INS_B has five tubes B1 (0.12g), B2 (0.12g), B3 (0.16g), B4 (0.11g), B5 (0.17g)</li><br />
</ul><br />
</li><br />
<li>ligation for INS_A and pOmpC</li><br />
<li>plate culture for (pOmpC+INS_A and pOmpC+INS_B), transformation <font color="#ff0000">failed</font><br />
<ul><br />
<li>forget to add vector</li><br />
</ul><br />
</li><br />
</ul><br />
</td><br />
<td align="center">倫, 茹</td><br />
</tr><br />
<tr><br />
<td align="center">9</td><br />
<td>8 AM Sun, 9/9, 07</td><br />
<td><br />
<ul><br />
<li>plate culture <font color="#ff0000">failed</font> (possible due to absent of vector, checked by text paper record)</li><br />
<li>re-ligate INS_A insert into vector with pOmpC</li><br />
<li>re-check the concentration of insert INS_A by [http://rsb.info.nih.gov/ij/ ImageJ]</li><br />
<li>plate culture again for pOmpC+INS_A<br />
<ul><br />
<li>1uL insert + 1uL vector</li><br />
</ul><br />
</li><br />
</ul><br />
</td><br />
<td align="center">倫, 翔</td><br />
</tr><br />
<tr><br />
<td align="center">10</td><br />
<td>4 PM Mon, 9/10, 07</td><br />
<td><br />
<ul><br />
<li>plate culture is still <font color="#ff0000">failed</font></li><br />
<li>re-build pOmpC + INS_A in two different insert concentration<br />
<ul><br />
<li>2uL insert + 1uL vector</li><br />
<li>3uL insert + 1uL vector</li><br />
</ul><br />
</li><br />
</ul><br />
</td><br />
<td align="center">倫, 翔, 銘</td><br />
</tr><br />
<tr><br />
<td align="center">11</td><br />
<td>6 PM Thu, 9/11, 07</td><br />
<td><br />
<ul><br />
<li>re-measure the concentration of insert by biophotometer<br />
<ul><br />
<li>dsDNA use A260 OD to estimate, 1 A260 OD = 50 mg/mL = 0.05 ug/uL</li><br />
<li>OD ranged from 0.1 - 2.0 is located in linear region</li><br />
<li>OD close to zero is not reliable</li><br />
</ul><br />
</li><br />
<li>test for D7 and D8 (9/8 PCR #2) in box #1</li><br />
</ul><br />
</td><br />
<td align="center">銘</td><br />
</tr><br />
<tr><br />
<td align="center">12</td><br />
<td>6 PM Wed, 9/12, 07</td><br />
<td><br />
<ul><br />
<li>check (9/5 PCR) by GEL separation<br />
<ul><br />
<li>concentration of INS_A is still too low</li><br />
</ul><br />
</li><br />
<li>RE-examine the condition of PCR in different annealing temperature<br />
<ul><br />
<li>PCR INS_A in 49.8, 52.3, 55.5, 58.7 and 60.9 degree</li><br />
</ul><br />
</li><br />
</ul><br />
</td><br />
<td align="center">銘</td><br />
</tr><br />
<tr><br />
<td align="center">13</td><br />
<td>6 PM Thur, 9/13, 07</td><br />
<td><br />
<ul><br />
<li>check (9/12 PCR) by GEL separation<br />
<ul><br />
<li>band seems O.K.</li><br />
<li>best annealing temperature is around 55.5</li><br />
</ul><br />
</li><br />
<li>biophotometer concentration test<br />
<ul><br />
<li>9/5 and 9/12 PCR seem O.K.</li><br />
<li>9/8 PCR seem problemistic due to A260/A280 more than 10</li><br />
</ul><br />
</li><br />
</ul><br />
</td><br />
<td align="center">銘</td><br />
</tr><br />
<tr><br />
<td align="center">14</td><br />
<td>6 PM Sun, 9/16, 07</td><br />
<td><br />
<ul><br />
<li>digest CinR+HSL (ES) and D-term (EX)and GEL separation check<br />
<ul><br />
<li>tube #1 and #2 of CinR+HSL plasmid extraction is not correct after GEL check</li><br />
<li>first digestion of D-term (EcoRI) is not very sucessful<br />
<ul><br />
<li>thus pett re-digest D-term with double enzyme amount</li><br />
</ul><br />
</li><br />
</ul><br />
</li><br />
</ul><br />
</td><br />
<td align="center">宥, 君, 翔</td><br />
</tr><br />
<tr><br />
<td align="center">PCR</td><br />
<td>Thur, 9/27, 07</td><br />
<td><br />
<ul><br />
<li>PCR of TAT_INS_A, TAT_INS_B, OmpRBS, TAT_IDE</li><br />
</ul><br />
</td><br />
<td align="center">威</td><br />
</tr><br />
</table><br />
</p><br />
<p>&nbsp;</p><br />
[https://2007.igem.org/Taipei Back]</div>Blenthttp://2007.igem.org/wiki/index.php/NYMU_Taipei/Team_scheduleNYMU Taipei/Team schedule2007-12-13T02:48:51Z<p>Blent: /* Oct 2007 */</p>
<hr />
<div><br /><hr><br />
<br />
==July 2007==<br />
<br />
<table style="text-align: left" width="50%" border="1"><br />
<br />
<tr><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Sun.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Mon.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Tue.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Wed.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Thr.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Fri.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Sat.</big></td><br />
</tr><br />
<tr><br />
<td align="center">1</td><br />
<td align="center">2</td><br />
<td align="center">3</td><br />
<td align="center">4</td><br />
<td align="center">5</td><br />
<td align="center">[[NYMU_Taipei/Regular_meeting/July_6| 6]]</td><br />
<td align="center">7</td><br />
</tr><br />
<tr><br />
<td align="center">8</td><br />
<td align="center">9</td><br />
<td align="center">10</td><br />
<td align="center">11</td><br />
<td align="center">12</td><br />
<td align="center">[[NYMU_Taipei/Regular_meeting/July_13| 13]]</td><br />
<td align="center">14</td><br />
</tr><br />
<tr><br />
<td align="center">15</td><br />
<td align="center">16</td><br />
<td align="center">17</td><br />
<td align="center">18</td><br />
<td align="center">19</td><br />
<td align="center">[[NYMU_Taipei/Regular_meeting/July_20| 20]]</td><br />
<td align="center">21</td><br />
</tr><br />
<tr><br />
<td align="center">22</td><br />
<td align="center">23</td><br />
<td align="center">24</td><br />
<td align="center">25</td><br />
<td align="center">26</td><br />
<td align="center">[[NYMU_Taipei/Regular_meeting/July_27| 27]]</td><br />
<td align="center">28</td><br />
</tr><br />
<tr><br />
<td align="center">29</td><br />
<td align="center">30</td><br />
<td align="center">31</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><br />
<br />
==Aug 2007==<br />
<table style="text-align: left" width="50%" border="1"><br />
<tr><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Sun.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Mon.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Tue.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Wed.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Thr.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Fri.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Sat.</big></td><br />
</tr><br />
<tr><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">1</td><br />
<td align="center">2</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_3| 3]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_4| 4]]</td><br />
</tr><br />
<tr><br />
<td align="center">5</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_6&7| 6]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_6&7| 7]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_8| 8]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_9| 9]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_10| 10]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_11| 11]]</td><br />
</tr><br />
<tr><br />
<td align="center">12</td><br />
<td align="center">13</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_14| 14]]</td><br />
<td align="center">15</td><br />
<td align="center">16</td><br />
<td align="center">[[NYMU_Taipei/Regular_meeting/August_17| 17]]</td><br />
<td align="center">18</td><br />
</tr><br />
<tr><br />
<td align="center">19</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_20| 20]]</td><br />
<td align="center">21</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_22| 22]]</td><br />
<br />
<td align="center">23</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_24| 24]]</td><br />
<td align="center">25</td><br />
</tr><br />
<tr><br />
<td align="center">26</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_27| 27]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_28| 28]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_29| 29]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_30| 30]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_8_31| 31]]</td><br />
<td align="center">&nbsp;</td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><br />
<br />
==Sep 2007==<br />
<table style="text-align: left" width="50%" border="1"><br />
<tr><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Sun.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Mon.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Tue.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Wed.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Thr.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Fri.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Sat.</big></td><br />
</tr><br />
<tr><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_1| 1]]</td><br />
</tr><br />
<tr><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_2| 2]]</td><br />
<td align="center">3</td><br />
<td align="center">4</td><br />
<td align="center">5</td><br />
<td align="center">6</td><br />
<td align="center">[[NYMU_Taipei/Regular_meeting/September_7| 7]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_8| 8]]</td><br />
</tr><br />
<tr><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_9| 9]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_10| 10]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_11| 11]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_12| 12]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_13| 13]]</td><br />
<td align="center">[[NYMU_Taipei/Regular_meeting/September_14| 14]]</td><br />
<td align="center">15</td><br />
</tr><br />
<tr><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_16| 16]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_17| 17]]</td><br />
<td align="center">18</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_19| 19]]</td><br />
<br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_20| 20]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_21| 21]]</td><br />
<td align="center">22</td><br />
</tr><br />
<tr><br />
<td align="center">23</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_24| 24]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_25| 25]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_26| 26]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_27| 27]]</td><br />
<td align="center">[[NYMU_Taipei/Regular_meeting/September_28| 28]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_29| 29]]</td><br />
</tr><br />
<tr><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_9_30| 30]]</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><br />
<br />
==Oct 2007==<br />
<table style="text-align: left" width="50%" border="1"><br />
<tr><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Sun.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Mon.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Tue.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Wed.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Thr.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Fri.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Oct.</big></td><br />
</tr><br />
<tr><br />
<td align="center">&nbsp;</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_1| 1]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_2| 2]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_3| 3]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_4| 4]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_5| 5]]</td><br />
<td align="center">6</td><br />
</tr><br />
<tr><br />
<td align="center">7</td><br />
<td align="center">8</td><br />
<td align="center">9</td><br />
<td align="center">10</td><br />
<td align="center">11</td><br />
<td align="center">[[NYMU_Taipei/Regular_meeting/October_12| 12]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_13| 13]]</td><br />
</tr><br />
<tr><br />
<td align="center">14</td><br />
<td align="center">15</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_16| 16]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_17| 17]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_18| 18]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_19| 19]]</td><br />
<td align="center">20</td><br />
</tr><br />
<tr><br />
<td align="center">21</td><br />
<td align="center">22</td><br />
<td align="center">23</td><br />
<td align="center">24</td><br />
<td align="center">25</td><br />
<td align="center">26</td><br />
<td align="center">27</td><br />
</tr><br />
<tr><br />
<td align="center">28</td><br />
<td align="center">29</td><br />
<td align="center">30</td><br />
<td align="center">31</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><br />
==Oct 2007==<br />
<table style="text-align: left" width="50%" border="1"><br />
<tr><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Sun.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Mon.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Tue.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Wed.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Thr.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Fri.</big></td><br />
<td style="font-weight: bold; background-color: rgb(204,255,204); text-align: center"><big>Oct.</big></td><br />
</tr><br />
<tr><br />
<td align="center">&nbsp;</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_1| 1]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_2| 2]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_3| 3]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_4| 4]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_5| 5]]</td><br />
<td align="center">6</td><br />
</tr><br />
<tr><br />
<td align="center">7</td><br />
<td align="center">8</td><br />
<td align="center">9</td><br />
<td align="center">10</td><br />
<td align="center">11</td><br />
<td align="center">[[NYMU_Taipei/Regular_meeting/October_12| 12]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_13| 13]]</td><br />
</tr><br />
<tr><br />
<td align="center">14</td><br />
<td align="center">15</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_16| 16]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_17| 17]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_18| 18]]</td><br />
<td align="center">[[NYMU_Taipei/Lab_Notes/2007_10_19| 19]]</td><br />
<td align="center">20</td><br />
</tr><br />
<tr><br />
<td align="center">21</td><br />
<td align="center">22</td><br />
<td align="center">23</td><br />
<td align="center">24</td><br />
<td align="center">25</td><br />
<td align="center">26</td><br />
<td align="center">27</td><br />
</tr><br />
<tr><br />
<td align="center">28</td><br />
<td align="center">29</td><br />
<td align="center">30</td><br />
<td align="center">31</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
<td align="center">&nbsp;</td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><br />
<br />
<h3>System 1 assembly</h3><br />
<div align="center">&nbsp;</div><br />
<p><br />
<table border="1"><br />
<br />
<tr><br />
<td align="center"><strong>Run</strong></td><br />
<td align="center"><strong>Date and Time<br /><br />
</strong></td><br />
<td align="center"><strong>Objective</strong></td><br />
<td align="center"><strong>Attendance</strong></td><br />
</tr><br />
<tr><br />
<td style="background-color: rgb(255,255,255)" align="center">1</td><br />
<td style="background-color: rgb(255,255,255)">8 AM Sat, 9/1, 07</td><br />
<td style="background-color: rgb(255,255,255)"><br />
<ul><br />
<li>(RBS + cinR) + (RBS + HSL) = cinR + HSL</li><br />
<li>RBS + cinR ver. 2</li><br />
<li>plate culture for (cinR + HSL) and (RBS + CinR) ver.2</li><br />
</ul><br />
</td><br />
<td style="background-color: rgb(255,255,255)" align="center">茹, 翔</td><br />
</tr><br />
<tr><br />
<td style="background-color: rgb(255,255,255)" align="center">2</td><br />
<td style="background-color: rgb(255,255,255)">8 AM Sun, 9/2, 07</td><br />
<td style="background-color: rgb(255,255,255)"><br />
<ul><br />
<li>Liquid culture for (cinR + HSL) and (RBS + CinR) ver.2</li><br />
<li>[https://2007.igem.org/NYMU_Taipei/Team_schedule/classify_parts_in_box classify parts in box]</li><br />
</ul><br />
</td><br />
<td style="background-color: rgb(255,255,255)" align="center">雄, 威, 茹, 駿</td><br />
</tr><br />
<tr><br />
<td style="background-color: rgb(255,255,255)" align="center">3</td><br />
<td style="background-color: rgb(255,255,255)">8 AM Mon, 9/3, 07</td><br />
<td style="background-color: rgb(255,255,255)"><br />
<ul><br />
<li>plasmid extraction for (cinR + HSL) and (RBS + CinR) ver.2</li><br />
<li>pOmpC + (RBS + INS_A)</li><br />
<li>(RBS + cinR) + (RBS + HSL) + D-term</li><br />
</ul><br />
</td><br />
<td style="background-color: rgb(255,255,255)" align="center">宥, 君, 威</td><br />
</tr><br />
<tr><br />
<td style="background-color: rgb(255,255,255)" align="center">4</td><br />
<td style="background-color: rgb(255,255,255)">8 AM Tue, 9/4, 07</td><br />
<td style="background-color: rgb(255,255,255)"><br />
<ul><br />
<li>plate culture for ...</li><br />
<li>Measure the concentration of all excised parts<br />
<ul><br />
<li>(cinR + HSL)</li><br />
<li>(RBS + CinR) ver.2</li><br />
</ul><br />
</li><br />
<li>BsaBI digestion of cinR</li><br />
</ul><br />
</td><br />
<td style="background-color: rgb(255,255,255)" align="center">宥, 君, 威</td><br />
</tr><br />
<tr><br />
<td align="center">5</td><br />
<td>8 AM Wed, 9/5, 07</td><br />
<td><br />
<ul><br />
<li>(RBS + INS_A), PCR #1</li><br />
<li>(RBS + INS_B), PCR #1</li><br />
<li>pOmpC + (RBS + INS_A), not check yet</li><br />
<li>pOmpC + (RBS + INS_B), not check yet</li><br />
</ul><br />
</td><br />
<td align="center">威, 茹</td><br />
</tr><br />
<tr><br />
<td align="center">6</td><br />
<td>8 AM Thr, 9/6, 07</td><br />
<td><br />
<ul><br />
<li>N/A</li><br />
</ul><br />
</td><br />
<td align="center">威</td><br />
</tr><br />
<tr><br />
<td align="center">7</td><br />
<td>8 AM Fri, 9/7, 07</td><br />
<td><br />
<ul><br />
<li>N/A</li><br />
</ul><br />
</td><br />
<td align="center">儒, 威, 茹</td><br />
</tr><br />
<tr><br />
<td align="center">8</td><br />
<td>8 AM Sat, 9/8, 07</td><br />
<td><br />
<ul><br />
<li>(RBS+INS_A), PCR #2</li><br />
<li>(RBS+INS_B), PCR #2</li><br />
<li>GEL check (5 tubes for each gene)<br />
<ul><br />
<li>INS_A has two tubes A1 (0.08g), A3 (0.15g)</li><br />
<li>INS_B has five tubes B1 (0.12g), B2 (0.12g), B3 (0.16g), B4 (0.11g), B5 (0.17g)</li><br />
</ul><br />
</li><br />
<li>ligation for INS_A and pOmpC</li><br />
<li>plate culture for (pOmpC+INS_A and pOmpC+INS_B), transformation <font color="#ff0000">failed</font><br />
<ul><br />
<li>forget to add vector</li><br />
</ul><br />
</li><br />
</ul><br />
</td><br />
<td align="center">倫, 茹</td><br />
</tr><br />
<tr><br />
<td align="center">9</td><br />
<td>8 AM Sun, 9/9, 07</td><br />
<td><br />
<ul><br />
<li>plate culture <font color="#ff0000">failed</font> (possible due to absent of vector, checked by text paper record)</li><br />
<li>re-ligate INS_A insert into vector with pOmpC</li><br />
<li>re-check the concentration of insert INS_A by [http://rsb.info.nih.gov/ij/ ImageJ]</li><br />
<li>plate culture again for pOmpC+INS_A<br />
<ul><br />
<li>1uL insert + 1uL vector</li><br />
</ul><br />
</li><br />
</ul><br />
</td><br />
<td align="center">倫, 翔</td><br />
</tr><br />
<tr><br />
<td align="center">10</td><br />
<td>4 PM Mon, 9/10, 07</td><br />
<td><br />
<ul><br />
<li>plate culture is still <font color="#ff0000">failed</font></li><br />
<li>re-build pOmpC + INS_A in two different insert concentration<br />
<ul><br />
<li>2uL insert + 1uL vector</li><br />
<li>3uL insert + 1uL vector</li><br />
</ul><br />
</li><br />
</ul><br />
</td><br />
<td align="center">倫, 翔, 銘</td><br />
</tr><br />
<tr><br />
<td align="center">11</td><br />
<td>6 PM Thu, 9/11, 07</td><br />
<td><br />
<ul><br />
<li>re-measure the concentration of insert by biophotometer<br />
<ul><br />
<li>dsDNA use A260 OD to estimate, 1 A260 OD = 50 mg/mL = 0.05 ug/uL</li><br />
<li>OD ranged from 0.1 - 2.0 is located in linear region</li><br />
<li>OD close to zero is not reliable</li><br />
</ul><br />
</li><br />
<li>test for D7 and D8 (9/8 PCR #2) in box #1</li><br />
</ul><br />
</td><br />
<td align="center">銘</td><br />
</tr><br />
<tr><br />
<td align="center">12</td><br />
<td>6 PM Wed, 9/12, 07</td><br />
<td><br />
<ul><br />
<li>check (9/5 PCR) by GEL separation<br />
<ul><br />
<li>concentration of INS_A is still too low</li><br />
</ul><br />
</li><br />
<li>RE-examine the condition of PCR in different annealing temperature<br />
<ul><br />
<li>PCR INS_A in 49.8, 52.3, 55.5, 58.7 and 60.9 degree</li><br />
</ul><br />
</li><br />
</ul><br />
</td><br />
<td align="center">銘</td><br />
</tr><br />
<tr><br />
<td align="center">13</td><br />
<td>6 PM Thur, 9/13, 07</td><br />
<td><br />
<ul><br />
<li>check (9/12 PCR) by GEL separation<br />
<ul><br />
<li>band seems O.K.</li><br />
<li>best annealing temperature is around 55.5</li><br />
</ul><br />
</li><br />
<li>biophotometer concentration test<br />
<ul><br />
<li>9/5 and 9/12 PCR seem O.K.</li><br />
<li>9/8 PCR seem problemistic due to A260/A280 more than 10</li><br />
</ul><br />
</li><br />
</ul><br />
</td><br />
<td align="center">銘</td><br />
</tr><br />
<tr><br />
<td align="center">14</td><br />
<td>6 PM Sun, 9/16, 07</td><br />
<td><br />
<ul><br />
<li>digest CinR+HSL (ES) and D-term (EX)and GEL separation check<br />
<ul><br />
<li>tube #1 and #2 of CinR+HSL plasmid extraction is not correct after GEL check</li><br />
<li>first digestion of D-term (EcoRI) is not very sucessful<br />
<ul><br />
<li>thus pett re-digest D-term with double enzyme amount</li><br />
</ul><br />
</li><br />
</ul><br />
</li><br />
</ul><br />
</td><br />
<td align="center">宥, 君, 翔</td><br />
</tr><br />
<tr><br />
<td align="center">PCR</td><br />
<td>Thur, 9/27, 07</td><br />
<td><br />
<ul><br />
<li>PCR of TAT_INS_A, TAT_INS_B, OmpRBS, TAT_IDE</li><br />
</ul><br />
</td><br />
<td align="center">威</td><br />
</tr><br />
</table><br />
</p><br />
<p>&nbsp;</p><br />
[https://2007.igem.org/Taipei Back]</div>Blenthttp://2007.igem.org/wiki/index.php/NYMU_Taipei/ProjectDescriptionNYMU Taipei/ProjectDescription2007-10-27T10:22:47Z<p>Blent: /* Mammalian Reporting System */</p>
<hr />
<div><p><font size="4"><br />
[[NYMU_Taipei/ProjectDescription| Project Description]] /<br />
[[NYMU_Taipei/Lab_Notes| Lab Notes]] /<br />
[[NYMU_Taipei/Part_List| Part List]] /<br />
[[NYMU_Taipei/Primer_List| Primer List]]<br />
</font><br /><hr></p><br />
<br />
==<font size=5 color=red>GlucOperon</font>==<br />
* A '''Gluc'''ose-based synthetic '''Operon''' system<br />
* An automatic system in E.coli that operates blood sugar wonderfully!<br />
* Insulin secretion system that promotes quality of life for diabetic patients<br />
----<br />
<br />
==Summary==<br />
* Diabetes mellitus is a significant problem especially in developed countries and leads to several severe long-term complications. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Controlling blood sugar in a reasonable level and avoiding severe emergency as diabetes ketoacidosis (DKA) are very important clinical issues. Thus, the NYMU_Taipei iGEM 2007 team is designing a biological system to sense environmental glucose concentration and decrease the level of glucose by releasing insulin. Besides, life-protection functions for removing toxic ketoacids produced during DKA and preventing hypoglycemia status will also be established. This system will be a convenient and safe design for those patients with diabetes mellitus, and further improve their quality of life by avoiding them from diabetes-related morbidity and mortality. <br />
----<br />
==Motivation==<br />
* Over 7 percent of adults in the United States (US) are known to have diabetes mellitus. However, because of the associated microvascular and macrovascular disease, diabetes accounts for almost 14 percent of US health care expenditures, at least one-half of which are related to complications such as myocardial infarction, stroke, end-stage renal disease, retinopathy, and foot ulcers. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus, one of the most common chronic diseases in childhood, is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Data from the Center of Disease Control (CDC) show that more than 13,000 children and adolescents (19 years of age) are diagnosed with type 1 disease each year with a prevalence of 1.7 per 1000. Control of blood sugar and prevention of severe complications, such;as diabetic ketoacidosis (DKA), are important issues nowadays. <br />
* Diabetic ketoacidosis (DKA) and hyperosmolar hyperglycemic state (HHS, also known as nonketotic hyperglycemia) are two of the most serious acute complications of diabetes. They are part of the spectrum of hyperglycemia and each represents an extreme in the spectrum. DKA is characterized by the triad of hyperglycemia, anion gap metabolic acidosis, and ketonemia. Metabolic acidosis is often the major finding. The serum glucose concentration is usually greater than 500 mg/dL (27.8 mmol/L) and less than 800 mg/dL (44.4 mmol/L). Three ketone bodies are produced in DKA: two ketoacids (beta-hydroxybutyric acid and acetoacetic acid), and one neutral ketone (acetone). The treatment of DKA and HHS is similar, including the administration of insulin and correction of the fluid and electrolyte abnormalities that may be present, including hyperglycemia and hyperosmolality, hypovolemia, metabolic acidosis (in DKA), and potassium depletion. <br />
* Existed works (see References below) are focusing on how to take advantages from properties of mammalian cells to express and secrete insulin in a trail-and-error manner. However, from the perspective of synthetic biology, it is possible to engineer a cell works as we want, no matter what type of cells we apply to. <br />
----<br />
==Goal and System Design==<br />
* For the iGEM 2007 project, our team focuses on the development of a prokaryotic system to <br />
** express insulin regulated by external glucose concentration and <br />
** clean the ketone bodies accumulated by mamalian cells due to insufficient intake of glucose <br />
** balance between glucose and insulin to stablize glucose level and prevent low blood glucose condition <br />
* system specification is shown below: <br />
[[Image:GAI-t curve.jpg|center|500px]]<br />
* System diagram (initial state)<br />
[[Image:IGEM2007 insulin circuit diagram.png|center]]<br />
* System under high glucose condition (insulin generating circuit is activated)<br />
[[Image:High_glucose.png|center]]<br />
* System under transition state (insulin increases glucose uptake; whole system shifts from insulin generation to insulin degradation)<br />
[[Image:Trans_glucose.png|center]]<br />
* System under low glucose condition (when glucose is low enough, the insulin generation turns off and insulin degradation turns on)<br />
[[Image:Low_glucose.png|center]]<br />
* System after insulin degradation (both circuits turn off and wait IDE to be naturally degraded; back to initial state)<br />
[[Image:Post_glucose.png|center]]<br />
* Circuit #1: high glucose-triggered insulin generator <br />
** The basic architecture for our insulin secretion design is shown in the figure below: <br />
** External glucose can be sensed by GSD (Glucose Sensing Domain) from gene RcsC and transmits response to ERD (Responsive Domain from EnvZ gene) <br />
** TCS phosphorylation relay activates the expression of insulin (both alpha and beta chains) <br />
** With signal peptide (SP), the assembled insulin (by disulfide bonding) can be exported outside the membrane of the prokaryotic cells <br />
[[Image:Insulin basic2.png|center|500px]] <br />
* Circuit #2: low glucose and insulin triggered insulin inhibitor/destroyer<br />
[[Image:NYMU_Taipei_insulin_inhibitor_2.png|center|500px]] <br />
[[Image:NYMU_Taipei_insulin_inhibitor.png|center|700px]] <br />
----<br />
<br />
==Implementation==<br />
* [[NYMU_Taipei/Part_List | Part List]]<br />
* [[NYMU_Taipei/Lab_Notes | Lab notes]]<br />
* [[NYMU_Taipei/Protocols| Protocols]]<br />
* [[NYMU_Taipei/Primer_List | Primer List]]<br />
----<br />
==Verification==<br />
===pOmpC characterization by testing circuit #1 pOmpC + EYFP===<br />
* first trail: glucose can induce expression pOmpC operon when glucose concentration higher than 1% after 14h culture in the LB medium<br />
[[Image:POmpC-EYFP test1.jpg|center]]<br />
<br /><br />
* second trail: 1% glucose induction will activate the insulin expression after 4hr induction<br />
[[Image:POmpC-EYFP test2.jpg|center]]<br />
<br />
===OmpRBS characterization by testing circuit #2 pLacIQ + OmpRBS + GFP===<br />
===Mammalian Reporting System===<br />
* cellular growth effect to E.coli-free medium (EFM) after 15hr or 8 hr culture (12 replicates)<br />
** samples are as follows: no EFM, only insulin, 10-50% total volume of EFM, and LPS (toxic to cell)<br />
[[Image:GWE_15h.jpg|center|500px]]<br />
<br /><br />
* insulin induces glucose uptake in L6 cell (8 replicates)<br />
** samples are as follows: induced after 10, 20, and 30 minutes<br />
[[Image:L6_INS_10-30.jpg|center|500px]]<br />
----<br />
<br />
==References==<br />
* Engineered enteroendocrine cells secrete insulin in response to glucose and reverse hyperglycemia in diabetic mice, 2007 Mol Ther. <br />
* Development of genetically engineered human intestinal cells for regulated insulin secretion using rAAV-mediated gene transfer, 2003 Biochem Biophys Res Commun <br />
* http://www.utdol.com/utd/content/topic.do <br />
* Recombinant DNA Technology in the Synthesis of Human Insulin <br />
* Glucose-Dependent Insulin Release from Genetically Engineered K Cells, 2000 science <br />
* [http://www.nature.com/gt/journal/v7/n21/abs/3301306a.html Glucose-stimulated and self-limiting insulin production by glucose 6-phosphatase promoter driven insulin expression in hepatoma cells, 2000 Gene Therapy]<br />
[https://2007.igem.org/Taipei Back]</div>Blenthttp://2007.igem.org/wiki/index.php/NYMU_Taipei/ProjectDescriptionNYMU Taipei/ProjectDescription2007-10-27T04:39:52Z<p>Blent: /* Mammalian Reporting System */</p>
<hr />
<div><p><font size="4"><br />
[[NYMU_Taipei/ProjectDescription| Project Description]] /<br />
[[NYMU_Taipei/Lab_Notes| Lab Notes]] /<br />
[[NYMU_Taipei/Part_List| Part List]] /<br />
[[NYMU_Taipei/Primer_List| Primer List]]<br />
</font><br /><hr></p><br />
<br />
==<font size=5 color=red>GlucOperon</font>==<br />
* A '''Gluc'''ose-based synthetic '''Operon''' system<br />
* An automatic system in E.coli that operates blood sugar wonderfully!<br />
* Insulin secretion system that promotes quality of life for diabetic patients<br />
----<br />
<br />
==Summary==<br />
* Diabetes mellitus is a significant problem especially in developed countries and leads to several severe long-term complications. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Controlling blood sugar in a reasonable level and avoiding severe emergency as diabetes ketoacidosis (DKA) are very important clinical issues. Thus, the NYMU_Taipei iGEM 2007 team is designing a biological system to sense environmental glucose concentration and decrease the level of glucose by releasing insulin. Besides, life-protection functions for removing toxic ketoacids produced during DKA and preventing hypoglycemia status will also be established. This system will be a convenient and safe design for those patients with diabetes mellitus, and further improve their quality of life by avoiding them from diabetes-related morbidity and mortality. <br />
----<br />
==Motivation==<br />
* Over 7 percent of adults in the United States (US) are known to have diabetes mellitus. However, because of the associated microvascular and macrovascular disease, diabetes accounts for almost 14 percent of US health care expenditures, at least one-half of which are related to complications such as myocardial infarction, stroke, end-stage renal disease, retinopathy, and foot ulcers. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus, one of the most common chronic diseases in childhood, is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Data from the Center of Disease Control (CDC) show that more than 13,000 children and adolescents (19 years of age) are diagnosed with type 1 disease each year with a prevalence of 1.7 per 1000. Control of blood sugar and prevention of severe complications, such;as diabetic ketoacidosis (DKA), are important issues nowadays. <br />
* Diabetic ketoacidosis (DKA) and hyperosmolar hyperglycemic state (HHS, also known as nonketotic hyperglycemia) are two of the most serious acute complications of diabetes. They are part of the spectrum of hyperglycemia and each represents an extreme in the spectrum. DKA is characterized by the triad of hyperglycemia, anion gap metabolic acidosis, and ketonemia. Metabolic acidosis is often the major finding. The serum glucose concentration is usually greater than 500 mg/dL (27.8 mmol/L) and less than 800 mg/dL (44.4 mmol/L). Three ketone bodies are produced in DKA: two ketoacids (beta-hydroxybutyric acid and acetoacetic acid), and one neutral ketone (acetone). The treatment of DKA and HHS is similar, including the administration of insulin and correction of the fluid and electrolyte abnormalities that may be present, including hyperglycemia and hyperosmolality, hypovolemia, metabolic acidosis (in DKA), and potassium depletion. <br />
* Existed works (see References below) are focusing on how to take advantages from properties of mammalian cells to express and secrete insulin in a trail-and-error manner. However, from the perspective of synthetic biology, it is possible to engineer a cell works as we want, no matter what type of cells we apply to. <br />
----<br />
==Goal and System Design==<br />
* For the iGEM 2007 project, our team focuses on the development of a prokaryotic system to <br />
** express insulin regulated by external glucose concentration and <br />
** clean the ketone bodies accumulated by mamalian cells due to insufficient intake of glucose <br />
** balance between glucose and insulin to stablize glucose level and prevent low blood glucose condition <br />
* system specification is shown below: <br />
[[Image:GAI-t curve.jpg|center|500px]]<br />
* System diagram (initial state)<br />
[[Image:IGEM2007 insulin circuit diagram.png|center]]<br />
* System under high glucose condition (insulin generating circuit is activated)<br />
[[Image:High_glucose.png|center]]<br />
* System under transition state (insulin increases glucose uptake; whole system shifts from insulin generation to insulin degradation)<br />
[[Image:Trans_glucose.png|center]]<br />
* System under low glucose condition (when glucose is low enough, the insulin generation turns off and insulin degradation turns on)<br />
[[Image:Low_glucose.png|center]]<br />
* System after insulin degradation (both circuits turn off and wait IDE to be naturally degraded; back to initial state)<br />
[[Image:Post_glucose.png|center]]<br />
* Circuit #1: high glucose-triggered insulin generator <br />
** The basic architecture for our insulin secretion design is shown in the figure below: <br />
** External glucose can be sensed by GSD (Glucose Sensing Domain) from gene RcsC and transmits response to ERD (Responsive Domain from EnvZ gene) <br />
** TCS phosphorylation relay activates the expression of insulin (both alpha and beta chains) <br />
** With signal peptide (SP), the assembled insulin (by disulfide bonding) can be exported outside the membrane of the prokaryotic cells <br />
[[Image:Insulin basic2.png|center|500px]] <br />
* Circuit #2: low glucose and insulin triggered insulin inhibitor/destroyer<br />
[[Image:NYMU_Taipei_insulin_inhibitor_2.png|center|500px]] <br />
[[Image:NYMU_Taipei_insulin_inhibitor.png|center|700px]] <br />
----<br />
<br />
==Implementation==<br />
* [[NYMU_Taipei/Part_List | Part List]]<br />
* [[NYMU_Taipei/Lab_Notes | Lab notes]]<br />
* [[NYMU_Taipei/Protocols| Protocols]]<br />
* [[NYMU_Taipei/Primer_List | Primer List]]<br />
----<br />
==Verification==<br />
===pOmpC characterization by testing circuit #1 pOmpC + EYFP===<br />
* first trail: glucose can induce expression pOmpC operon when glucose concentration higher than 1% after 14h culture in the LB medium<br />
[[Image:POmpC-EYFP test1.jpg|center]]<br />
<br /><br />
* second trail: 1% glucose induction will activate the insulin expression after 4hr induction<br />
[[Image:POmpC-EYFP test2.jpg|center]]<br />
<br />
===OmpRBS characterization by testing circuit #2 pLacIQ + OmpRBS + GFP===<br />
===Mammalian Reporting System===<br />
* cellular growth effect to E.coli-free medium (EFM) after 15hr culture (12 replicates)<br />
** samples are as follows: no EFM, only insulin, 10-50% total volume of EFM, and LPS (toxic to cell)<br />
[[Image:GWE_15h.jpg|center|500px]]<br />
<br /><br />
* insulin induces glucose uptake in L6 cell (8 replicates)<br />
** samples are as follows: induced after 10, 20, and 30 minutes<br />
[[Image:L6_INS_10-30.jpg|center|500px]]<br />
----<br />
<br />
==References==<br />
* Engineered enteroendocrine cells secrete insulin in response to glucose and reverse hyperglycemia in diabetic mice, 2007 Mol Ther. <br />
* Development of genetically engineered human intestinal cells for regulated insulin secretion using rAAV-mediated gene transfer, 2003 Biochem Biophys Res Commun <br />
* http://www.utdol.com/utd/content/topic.do <br />
* Recombinant DNA Technology in the Synthesis of Human Insulin <br />
* Glucose-Dependent Insulin Release from Genetically Engineered K Cells, 2000 science <br />
* [http://www.nature.com/gt/journal/v7/n21/abs/3301306a.html Glucose-stimulated and self-limiting insulin production by glucose 6-phosphatase promoter driven insulin expression in hepatoma cells, 2000 Gene Therapy]<br />
[https://2007.igem.org/Taipei Back]</div>Blenthttp://2007.igem.org/wiki/index.php/NYMU_Taipei/ProjectDescriptionNYMU Taipei/ProjectDescription2007-10-27T04:39:10Z<p>Blent: /* Mammalian Reporting System */</p>
<hr />
<div><p><font size="4"><br />
[[NYMU_Taipei/ProjectDescription| Project Description]] /<br />
[[NYMU_Taipei/Lab_Notes| Lab Notes]] /<br />
[[NYMU_Taipei/Part_List| Part List]] /<br />
[[NYMU_Taipei/Primer_List| Primer List]]<br />
</font><br /><hr></p><br />
<br />
==<font size=5 color=red>GlucOperon</font>==<br />
* A '''Gluc'''ose-based synthetic '''Operon''' system<br />
* An automatic system in E.coli that operates blood sugar wonderfully!<br />
* Insulin secretion system that promotes quality of life for diabetic patients<br />
----<br />
<br />
==Summary==<br />
* Diabetes mellitus is a significant problem especially in developed countries and leads to several severe long-term complications. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Controlling blood sugar in a reasonable level and avoiding severe emergency as diabetes ketoacidosis (DKA) are very important clinical issues. Thus, the NYMU_Taipei iGEM 2007 team is designing a biological system to sense environmental glucose concentration and decrease the level of glucose by releasing insulin. Besides, life-protection functions for removing toxic ketoacids produced during DKA and preventing hypoglycemia status will also be established. This system will be a convenient and safe design for those patients with diabetes mellitus, and further improve their quality of life by avoiding them from diabetes-related morbidity and mortality. <br />
----<br />
==Motivation==<br />
* Over 7 percent of adults in the United States (US) are known to have diabetes mellitus. However, because of the associated microvascular and macrovascular disease, diabetes accounts for almost 14 percent of US health care expenditures, at least one-half of which are related to complications such as myocardial infarction, stroke, end-stage renal disease, retinopathy, and foot ulcers. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus, one of the most common chronic diseases in childhood, is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Data from the Center of Disease Control (CDC) show that more than 13,000 children and adolescents (19 years of age) are diagnosed with type 1 disease each year with a prevalence of 1.7 per 1000. Control of blood sugar and prevention of severe complications, such;as diabetic ketoacidosis (DKA), are important issues nowadays. <br />
* Diabetic ketoacidosis (DKA) and hyperosmolar hyperglycemic state (HHS, also known as nonketotic hyperglycemia) are two of the most serious acute complications of diabetes. They are part of the spectrum of hyperglycemia and each represents an extreme in the spectrum. DKA is characterized by the triad of hyperglycemia, anion gap metabolic acidosis, and ketonemia. Metabolic acidosis is often the major finding. The serum glucose concentration is usually greater than 500 mg/dL (27.8 mmol/L) and less than 800 mg/dL (44.4 mmol/L). Three ketone bodies are produced in DKA: two ketoacids (beta-hydroxybutyric acid and acetoacetic acid), and one neutral ketone (acetone). The treatment of DKA and HHS is similar, including the administration of insulin and correction of the fluid and electrolyte abnormalities that may be present, including hyperglycemia and hyperosmolality, hypovolemia, metabolic acidosis (in DKA), and potassium depletion. <br />
* Existed works (see References below) are focusing on how to take advantages from properties of mammalian cells to express and secrete insulin in a trail-and-error manner. However, from the perspective of synthetic biology, it is possible to engineer a cell works as we want, no matter what type of cells we apply to. <br />
----<br />
==Goal and System Design==<br />
* For the iGEM 2007 project, our team focuses on the development of a prokaryotic system to <br />
** express insulin regulated by external glucose concentration and <br />
** clean the ketone bodies accumulated by mamalian cells due to insufficient intake of glucose <br />
** balance between glucose and insulin to stablize glucose level and prevent low blood glucose condition <br />
* system specification is shown below: <br />
[[Image:GAI-t curve.jpg|center|500px]]<br />
* System diagram (initial state)<br />
[[Image:IGEM2007 insulin circuit diagram.png|center]]<br />
* System under high glucose condition (insulin generating circuit is activated)<br />
[[Image:High_glucose.png|center]]<br />
* System under transition state (insulin increases glucose uptake; whole system shifts from insulin generation to insulin degradation)<br />
[[Image:Trans_glucose.png|center]]<br />
* System under low glucose condition (when glucose is low enough, the insulin generation turns off and insulin degradation turns on)<br />
[[Image:Low_glucose.png|center]]<br />
* System after insulin degradation (both circuits turn off and wait IDE to be naturally degraded; back to initial state)<br />
[[Image:Post_glucose.png|center]]<br />
* Circuit #1: high glucose-triggered insulin generator <br />
** The basic architecture for our insulin secretion design is shown in the figure below: <br />
** External glucose can be sensed by GSD (Glucose Sensing Domain) from gene RcsC and transmits response to ERD (Responsive Domain from EnvZ gene) <br />
** TCS phosphorylation relay activates the expression of insulin (both alpha and beta chains) <br />
** With signal peptide (SP), the assembled insulin (by disulfide bonding) can be exported outside the membrane of the prokaryotic cells <br />
[[Image:Insulin basic2.png|center|500px]] <br />
* Circuit #2: low glucose and insulin triggered insulin inhibitor/destroyer<br />
[[Image:NYMU_Taipei_insulin_inhibitor_2.png|center|500px]] <br />
[[Image:NYMU_Taipei_insulin_inhibitor.png|center|700px]] <br />
----<br />
<br />
==Implementation==<br />
* [[NYMU_Taipei/Part_List | Part List]]<br />
* [[NYMU_Taipei/Lab_Notes | Lab notes]]<br />
* [[NYMU_Taipei/Protocols| Protocols]]<br />
* [[NYMU_Taipei/Primer_List | Primer List]]<br />
----<br />
==Verification==<br />
===pOmpC characterization by testing circuit #1 pOmpC + EYFP===<br />
* first trail: glucose can induce expression pOmpC operon when glucose concentration higher than 1% after 14h culture in the LB medium<br />
[[Image:POmpC-EYFP test1.jpg|center]]<br />
<br /><br />
* second trail: 1% glucose induction will activate the insulin expression after 4hr induction<br />
[[Image:POmpC-EYFP test2.jpg|center]]<br />
<br />
===OmpRBS characterization by testing circuit #2 pLacIQ + OmpRBS + GFP===<br />
===Mammalian Reporting System===<br />
* cellular growth effect to E.coli-free medium (EFM) after 15hr culture (12 replicates)<br />
[[Image:GWE_15h.jpg|center|500px]]<br />
* samples are as follows: no EFM, only insulin, 10-50% total volume of EFM, and LPS (toxic to cell)<br />
<br /><br />
* insulin induces glucose uptake in L6 cell (8 replicates)<br />
[[Image:L6_INS_10-30.jpg|center|500px]]<br />
* samples are as follows: induced after 10, 20, and 30 minutes<br />
----<br />
<br />
==References==<br />
* Engineered enteroendocrine cells secrete insulin in response to glucose and reverse hyperglycemia in diabetic mice, 2007 Mol Ther. <br />
* Development of genetically engineered human intestinal cells for regulated insulin secretion using rAAV-mediated gene transfer, 2003 Biochem Biophys Res Commun <br />
* http://www.utdol.com/utd/content/topic.do <br />
* Recombinant DNA Technology in the Synthesis of Human Insulin <br />
* Glucose-Dependent Insulin Release from Genetically Engineered K Cells, 2000 science <br />
* [http://www.nature.com/gt/journal/v7/n21/abs/3301306a.html Glucose-stimulated and self-limiting insulin production by glucose 6-phosphatase promoter driven insulin expression in hepatoma cells, 2000 Gene Therapy]<br />
[https://2007.igem.org/Taipei Back]</div>Blenthttp://2007.igem.org/wiki/index.php/NYMU_Taipei/ProjectDescriptionNYMU Taipei/ProjectDescription2007-10-27T04:34:39Z<p>Blent: /* Mammalian Reporting System */</p>
<hr />
<div><p><font size="4"><br />
[[NYMU_Taipei/ProjectDescription| Project Description]] /<br />
[[NYMU_Taipei/Lab_Notes| Lab Notes]] /<br />
[[NYMU_Taipei/Part_List| Part List]] /<br />
[[NYMU_Taipei/Primer_List| Primer List]]<br />
</font><br /><hr></p><br />
<br />
==<font size=5 color=red>GlucOperon</font>==<br />
* A '''Gluc'''ose-based synthetic '''Operon''' system<br />
* An automatic system in E.coli that operates blood sugar wonderfully!<br />
* Insulin secretion system that promotes quality of life for diabetic patients<br />
----<br />
<br />
==Summary==<br />
* Diabetes mellitus is a significant problem especially in developed countries and leads to several severe long-term complications. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Controlling blood sugar in a reasonable level and avoiding severe emergency as diabetes ketoacidosis (DKA) are very important clinical issues. Thus, the NYMU_Taipei iGEM 2007 team is designing a biological system to sense environmental glucose concentration and decrease the level of glucose by releasing insulin. Besides, life-protection functions for removing toxic ketoacids produced during DKA and preventing hypoglycemia status will also be established. This system will be a convenient and safe design for those patients with diabetes mellitus, and further improve their quality of life by avoiding them from diabetes-related morbidity and mortality. <br />
----<br />
==Motivation==<br />
* Over 7 percent of adults in the United States (US) are known to have diabetes mellitus. However, because of the associated microvascular and macrovascular disease, diabetes accounts for almost 14 percent of US health care expenditures, at least one-half of which are related to complications such as myocardial infarction, stroke, end-stage renal disease, retinopathy, and foot ulcers. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus, one of the most common chronic diseases in childhood, is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Data from the Center of Disease Control (CDC) show that more than 13,000 children and adolescents (19 years of age) are diagnosed with type 1 disease each year with a prevalence of 1.7 per 1000. Control of blood sugar and prevention of severe complications, such;as diabetic ketoacidosis (DKA), are important issues nowadays. <br />
* Diabetic ketoacidosis (DKA) and hyperosmolar hyperglycemic state (HHS, also known as nonketotic hyperglycemia) are two of the most serious acute complications of diabetes. They are part of the spectrum of hyperglycemia and each represents an extreme in the spectrum. DKA is characterized by the triad of hyperglycemia, anion gap metabolic acidosis, and ketonemia. Metabolic acidosis is often the major finding. The serum glucose concentration is usually greater than 500 mg/dL (27.8 mmol/L) and less than 800 mg/dL (44.4 mmol/L). Three ketone bodies are produced in DKA: two ketoacids (beta-hydroxybutyric acid and acetoacetic acid), and one neutral ketone (acetone). The treatment of DKA and HHS is similar, including the administration of insulin and correction of the fluid and electrolyte abnormalities that may be present, including hyperglycemia and hyperosmolality, hypovolemia, metabolic acidosis (in DKA), and potassium depletion. <br />
* Existed works (see References below) are focusing on how to take advantages from properties of mammalian cells to express and secrete insulin in a trail-and-error manner. However, from the perspective of synthetic biology, it is possible to engineer a cell works as we want, no matter what type of cells we apply to. <br />
----<br />
==Goal and System Design==<br />
* For the iGEM 2007 project, our team focuses on the development of a prokaryotic system to <br />
** express insulin regulated by external glucose concentration and <br />
** clean the ketone bodies accumulated by mamalian cells due to insufficient intake of glucose <br />
** balance between glucose and insulin to stablize glucose level and prevent low blood glucose condition <br />
* system specification is shown below: <br />
[[Image:GAI-t curve.jpg|center|500px]]<br />
* System diagram (initial state)<br />
[[Image:IGEM2007 insulin circuit diagram.png|center]]<br />
* System under high glucose condition (insulin generating circuit is activated)<br />
[[Image:High_glucose.png|center]]<br />
* System under transition state (insulin increases glucose uptake; whole system shifts from insulin generation to insulin degradation)<br />
[[Image:Trans_glucose.png|center]]<br />
* System under low glucose condition (when glucose is low enough, the insulin generation turns off and insulin degradation turns on)<br />
[[Image:Low_glucose.png|center]]<br />
* System after insulin degradation (both circuits turn off and wait IDE to be naturally degraded; back to initial state)<br />
[[Image:Post_glucose.png|center]]<br />
* Circuit #1: high glucose-triggered insulin generator <br />
** The basic architecture for our insulin secretion design is shown in the figure below: <br />
** External glucose can be sensed by GSD (Glucose Sensing Domain) from gene RcsC and transmits response to ERD (Responsive Domain from EnvZ gene) <br />
** TCS phosphorylation relay activates the expression of insulin (both alpha and beta chains) <br />
** With signal peptide (SP), the assembled insulin (by disulfide bonding) can be exported outside the membrane of the prokaryotic cells <br />
[[Image:Insulin basic2.png|center|500px]] <br />
* Circuit #2: low glucose and insulin triggered insulin inhibitor/destroyer<br />
[[Image:NYMU_Taipei_insulin_inhibitor_2.png|center|500px]] <br />
[[Image:NYMU_Taipei_insulin_inhibitor.png|center|700px]] <br />
----<br />
<br />
==Implementation==<br />
* [[NYMU_Taipei/Part_List | Part List]]<br />
* [[NYMU_Taipei/Lab_Notes | Lab notes]]<br />
* [[NYMU_Taipei/Protocols| Protocols]]<br />
* [[NYMU_Taipei/Primer_List | Primer List]]<br />
----<br />
==Verification==<br />
===pOmpC characterization by testing circuit #1 pOmpC + EYFP===<br />
* first trail: glucose can induce expression pOmpC operon when glucose concentration higher than 1% after 14h culture in the LB medium<br />
[[Image:POmpC-EYFP test1.jpg|center]]<br />
<br /><br />
* second trail: 1% glucose induction will activate the insulin expression after 4hr induction<br />
[[Image:POmpC-EYFP test2.jpg|center]]<br />
<br />
===OmpRBS characterization by testing circuit #2 pLacIQ + OmpRBS + GFP===<br />
===Mammalian Reporting System===<br />
* insulin induces glucose uptake in L6 cell (8 replicates)<br />
[[Image:L6_INS_10-30.jpg|center|500px]]<br />
<br/><br />
* cellular growth effect to E.coli-free medium after 15hr culture (12 replicates)<br />
[[Image:GWE_15h.jpg|center|500px]]<br />
----<br />
<br />
==References==<br />
* Engineered enteroendocrine cells secrete insulin in response to glucose and reverse hyperglycemia in diabetic mice, 2007 Mol Ther. <br />
* Development of genetically engineered human intestinal cells for regulated insulin secretion using rAAV-mediated gene transfer, 2003 Biochem Biophys Res Commun <br />
* http://www.utdol.com/utd/content/topic.do <br />
* Recombinant DNA Technology in the Synthesis of Human Insulin <br />
* Glucose-Dependent Insulin Release from Genetically Engineered K Cells, 2000 science <br />
* [http://www.nature.com/gt/journal/v7/n21/abs/3301306a.html Glucose-stimulated and self-limiting insulin production by glucose 6-phosphatase promoter driven insulin expression in hepatoma cells, 2000 Gene Therapy]<br />
[https://2007.igem.org/Taipei Back]</div>Blenthttp://2007.igem.org/wiki/index.php/File:L6_INS_10-30.jpgFile:L6 INS 10-30.jpg2007-10-27T04:32:29Z<p>Blent: </p>
<hr />
<div></div>Blenthttp://2007.igem.org/wiki/index.php/NYMU_Taipei/ProjectDescriptionNYMU Taipei/ProjectDescription2007-10-27T04:32:08Z<p>Blent: /* Mammalian Reporting System */</p>
<hr />
<div><p><font size="4"><br />
[[NYMU_Taipei/ProjectDescription| Project Description]] /<br />
[[NYMU_Taipei/Lab_Notes| Lab Notes]] /<br />
[[NYMU_Taipei/Part_List| Part List]] /<br />
[[NYMU_Taipei/Primer_List| Primer List]]<br />
</font><br /><hr></p><br />
<br />
==<font size=5 color=red>GlucOperon</font>==<br />
* A '''Gluc'''ose-based synthetic '''Operon''' system<br />
* An automatic system in E.coli that operates blood sugar wonderfully!<br />
* Insulin secretion system that promotes quality of life for diabetic patients<br />
----<br />
<br />
==Summary==<br />
* Diabetes mellitus is a significant problem especially in developed countries and leads to several severe long-term complications. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Controlling blood sugar in a reasonable level and avoiding severe emergency as diabetes ketoacidosis (DKA) are very important clinical issues. Thus, the NYMU_Taipei iGEM 2007 team is designing a biological system to sense environmental glucose concentration and decrease the level of glucose by releasing insulin. Besides, life-protection functions for removing toxic ketoacids produced during DKA and preventing hypoglycemia status will also be established. This system will be a convenient and safe design for those patients with diabetes mellitus, and further improve their quality of life by avoiding them from diabetes-related morbidity and mortality. <br />
----<br />
==Motivation==<br />
* Over 7 percent of adults in the United States (US) are known to have diabetes mellitus. However, because of the associated microvascular and macrovascular disease, diabetes accounts for almost 14 percent of US health care expenditures, at least one-half of which are related to complications such as myocardial infarction, stroke, end-stage renal disease, retinopathy, and foot ulcers. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus, one of the most common chronic diseases in childhood, is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Data from the Center of Disease Control (CDC) show that more than 13,000 children and adolescents (19 years of age) are diagnosed with type 1 disease each year with a prevalence of 1.7 per 1000. Control of blood sugar and prevention of severe complications, such;as diabetic ketoacidosis (DKA), are important issues nowadays. <br />
* Diabetic ketoacidosis (DKA) and hyperosmolar hyperglycemic state (HHS, also known as nonketotic hyperglycemia) are two of the most serious acute complications of diabetes. They are part of the spectrum of hyperglycemia and each represents an extreme in the spectrum. DKA is characterized by the triad of hyperglycemia, anion gap metabolic acidosis, and ketonemia. Metabolic acidosis is often the major finding. The serum glucose concentration is usually greater than 500 mg/dL (27.8 mmol/L) and less than 800 mg/dL (44.4 mmol/L). Three ketone bodies are produced in DKA: two ketoacids (beta-hydroxybutyric acid and acetoacetic acid), and one neutral ketone (acetone). The treatment of DKA and HHS is similar, including the administration of insulin and correction of the fluid and electrolyte abnormalities that may be present, including hyperglycemia and hyperosmolality, hypovolemia, metabolic acidosis (in DKA), and potassium depletion. <br />
* Existed works (see References below) are focusing on how to take advantages from properties of mammalian cells to express and secrete insulin in a trail-and-error manner. However, from the perspective of synthetic biology, it is possible to engineer a cell works as we want, no matter what type of cells we apply to. <br />
----<br />
==Goal and System Design==<br />
* For the iGEM 2007 project, our team focuses on the development of a prokaryotic system to <br />
** express insulin regulated by external glucose concentration and <br />
** clean the ketone bodies accumulated by mamalian cells due to insufficient intake of glucose <br />
** balance between glucose and insulin to stablize glucose level and prevent low blood glucose condition <br />
* system specification is shown below: <br />
[[Image:GAI-t curve.jpg|center|500px]]<br />
* System diagram (initial state)<br />
[[Image:IGEM2007 insulin circuit diagram.png|center]]<br />
* System under high glucose condition (insulin generating circuit is activated)<br />
[[Image:High_glucose.png|center]]<br />
* System under transition state (insulin increases glucose uptake; whole system shifts from insulin generation to insulin degradation)<br />
[[Image:Trans_glucose.png|center]]<br />
* System under low glucose condition (when glucose is low enough, the insulin generation turns off and insulin degradation turns on)<br />
[[Image:Low_glucose.png|center]]<br />
* System after insulin degradation (both circuits turn off and wait IDE to be naturally degraded; back to initial state)<br />
[[Image:Post_glucose.png|center]]<br />
* Circuit #1: high glucose-triggered insulin generator <br />
** The basic architecture for our insulin secretion design is shown in the figure below: <br />
** External glucose can be sensed by GSD (Glucose Sensing Domain) from gene RcsC and transmits response to ERD (Responsive Domain from EnvZ gene) <br />
** TCS phosphorylation relay activates the expression of insulin (both alpha and beta chains) <br />
** With signal peptide (SP), the assembled insulin (by disulfide bonding) can be exported outside the membrane of the prokaryotic cells <br />
[[Image:Insulin basic2.png|center|500px]] <br />
* Circuit #2: low glucose and insulin triggered insulin inhibitor/destroyer<br />
[[Image:NYMU_Taipei_insulin_inhibitor_2.png|center|500px]] <br />
[[Image:NYMU_Taipei_insulin_inhibitor.png|center|700px]] <br />
----<br />
<br />
==Implementation==<br />
* [[NYMU_Taipei/Part_List | Part List]]<br />
* [[NYMU_Taipei/Lab_Notes | Lab notes]]<br />
* [[NYMU_Taipei/Protocols| Protocols]]<br />
* [[NYMU_Taipei/Primer_List | Primer List]]<br />
----<br />
==Verification==<br />
===pOmpC characterization by testing circuit #1 pOmpC + EYFP===<br />
* first trail: glucose can induce expression pOmpC operon when glucose concentration higher than 1% after 14h culture in the LB medium<br />
[[Image:POmpC-EYFP test1.jpg|center]]<br />
<br /><br />
* second trail: 1% glucose induction will activate the insulin expression after 4hr induction<br />
[[Image:POmpC-EYFP test2.jpg|center]]<br />
<br />
===OmpRBS characterization by testing circuit #2 pLacIQ + OmpRBS + GFP===<br />
===Mammalian Reporting System===<br />
* insulin induces glucose uptake<br />
[[Image:L6_INS_10-30.jpg|center|500px]]<br />
* cellular growth effect to E.coli-free medium after 15hr culture<br />
[[Image:GWE_15h.jpg|center|500px]]<br />
----<br />
<br />
==References==<br />
* Engineered enteroendocrine cells secrete insulin in response to glucose and reverse hyperglycemia in diabetic mice, 2007 Mol Ther. <br />
* Development of genetically engineered human intestinal cells for regulated insulin secretion using rAAV-mediated gene transfer, 2003 Biochem Biophys Res Commun <br />
* http://www.utdol.com/utd/content/topic.do <br />
* Recombinant DNA Technology in the Synthesis of Human Insulin <br />
* Glucose-Dependent Insulin Release from Genetically Engineered K Cells, 2000 science <br />
* [http://www.nature.com/gt/journal/v7/n21/abs/3301306a.html Glucose-stimulated and self-limiting insulin production by glucose 6-phosphatase promoter driven insulin expression in hepatoma cells, 2000 Gene Therapy]<br />
[https://2007.igem.org/Taipei Back]</div>Blenthttp://2007.igem.org/wiki/index.php/NYMU_Taipei/ProjectDescriptionNYMU Taipei/ProjectDescription2007-10-27T04:28:18Z<p>Blent: /* Mammalian Reporting System */</p>
<hr />
<div><p><font size="4"><br />
[[NYMU_Taipei/ProjectDescription| Project Description]] /<br />
[[NYMU_Taipei/Lab_Notes| Lab Notes]] /<br />
[[NYMU_Taipei/Part_List| Part List]] /<br />
[[NYMU_Taipei/Primer_List| Primer List]]<br />
</font><br /><hr></p><br />
<br />
==<font size=5 color=red>GlucOperon</font>==<br />
* A '''Gluc'''ose-based synthetic '''Operon''' system<br />
* An automatic system in E.coli that operates blood sugar wonderfully!<br />
* Insulin secretion system that promotes quality of life for diabetic patients<br />
----<br />
<br />
==Summary==<br />
* Diabetes mellitus is a significant problem especially in developed countries and leads to several severe long-term complications. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Controlling blood sugar in a reasonable level and avoiding severe emergency as diabetes ketoacidosis (DKA) are very important clinical issues. Thus, the NYMU_Taipei iGEM 2007 team is designing a biological system to sense environmental glucose concentration and decrease the level of glucose by releasing insulin. Besides, life-protection functions for removing toxic ketoacids produced during DKA and preventing hypoglycemia status will also be established. This system will be a convenient and safe design for those patients with diabetes mellitus, and further improve their quality of life by avoiding them from diabetes-related morbidity and mortality. <br />
----<br />
==Motivation==<br />
* Over 7 percent of adults in the United States (US) are known to have diabetes mellitus. However, because of the associated microvascular and macrovascular disease, diabetes accounts for almost 14 percent of US health care expenditures, at least one-half of which are related to complications such as myocardial infarction, stroke, end-stage renal disease, retinopathy, and foot ulcers. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus, one of the most common chronic diseases in childhood, is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Data from the Center of Disease Control (CDC) show that more than 13,000 children and adolescents (19 years of age) are diagnosed with type 1 disease each year with a prevalence of 1.7 per 1000. Control of blood sugar and prevention of severe complications, such;as diabetic ketoacidosis (DKA), are important issues nowadays. <br />
* Diabetic ketoacidosis (DKA) and hyperosmolar hyperglycemic state (HHS, also known as nonketotic hyperglycemia) are two of the most serious acute complications of diabetes. They are part of the spectrum of hyperglycemia and each represents an extreme in the spectrum. DKA is characterized by the triad of hyperglycemia, anion gap metabolic acidosis, and ketonemia. Metabolic acidosis is often the major finding. The serum glucose concentration is usually greater than 500 mg/dL (27.8 mmol/L) and less than 800 mg/dL (44.4 mmol/L). Three ketone bodies are produced in DKA: two ketoacids (beta-hydroxybutyric acid and acetoacetic acid), and one neutral ketone (acetone). The treatment of DKA and HHS is similar, including the administration of insulin and correction of the fluid and electrolyte abnormalities that may be present, including hyperglycemia and hyperosmolality, hypovolemia, metabolic acidosis (in DKA), and potassium depletion. <br />
* Existed works (see References below) are focusing on how to take advantages from properties of mammalian cells to express and secrete insulin in a trail-and-error manner. However, from the perspective of synthetic biology, it is possible to engineer a cell works as we want, no matter what type of cells we apply to. <br />
----<br />
==Goal and System Design==<br />
* For the iGEM 2007 project, our team focuses on the development of a prokaryotic system to <br />
** express insulin regulated by external glucose concentration and <br />
** clean the ketone bodies accumulated by mamalian cells due to insufficient intake of glucose <br />
** balance between glucose and insulin to stablize glucose level and prevent low blood glucose condition <br />
* system specification is shown below: <br />
[[Image:GAI-t curve.jpg|center|500px]]<br />
* System diagram (initial state)<br />
[[Image:IGEM2007 insulin circuit diagram.png|center]]<br />
* System under high glucose condition (insulin generating circuit is activated)<br />
[[Image:High_glucose.png|center]]<br />
* System under transition state (insulin increases glucose uptake; whole system shifts from insulin generation to insulin degradation)<br />
[[Image:Trans_glucose.png|center]]<br />
* System under low glucose condition (when glucose is low enough, the insulin generation turns off and insulin degradation turns on)<br />
[[Image:Low_glucose.png|center]]<br />
* System after insulin degradation (both circuits turn off and wait IDE to be naturally degraded; back to initial state)<br />
[[Image:Post_glucose.png|center]]<br />
* Circuit #1: high glucose-triggered insulin generator <br />
** The basic architecture for our insulin secretion design is shown in the figure below: <br />
** External glucose can be sensed by GSD (Glucose Sensing Domain) from gene RcsC and transmits response to ERD (Responsive Domain from EnvZ gene) <br />
** TCS phosphorylation relay activates the expression of insulin (both alpha and beta chains) <br />
** With signal peptide (SP), the assembled insulin (by disulfide bonding) can be exported outside the membrane of the prokaryotic cells <br />
[[Image:Insulin basic2.png|center|500px]] <br />
* Circuit #2: low glucose and insulin triggered insulin inhibitor/destroyer<br />
[[Image:NYMU_Taipei_insulin_inhibitor_2.png|center|500px]] <br />
[[Image:NYMU_Taipei_insulin_inhibitor.png|center|700px]] <br />
----<br />
<br />
==Implementation==<br />
* [[NYMU_Taipei/Part_List | Part List]]<br />
* [[NYMU_Taipei/Lab_Notes | Lab notes]]<br />
* [[NYMU_Taipei/Protocols| Protocols]]<br />
* [[NYMU_Taipei/Primer_List | Primer List]]<br />
----<br />
==Verification==<br />
===pOmpC characterization by testing circuit #1 pOmpC + EYFP===<br />
* first trail: glucose can induce expression pOmpC operon when glucose concentration higher than 1% after 14h culture in the LB medium<br />
[[Image:POmpC-EYFP test1.jpg|center]]<br />
<br /><br />
* second trail: 1% glucose induction will activate the insulin expression after 4hr induction<br />
[[Image:POmpC-EYFP test2.jpg|center]]<br />
<br />
===OmpRBS characterization by testing circuit #2 pLacIQ + OmpRBS + GFP===<br />
===Mammalian Reporting System===<br />
* insulin induces glucose uptake<br />
* cellular growth effect to E.coli-free medium after 15hr culture<br />
[[Image:GWE_15h.jpg|center|500px]]<br />
----<br />
<br />
==References==<br />
* Engineered enteroendocrine cells secrete insulin in response to glucose and reverse hyperglycemia in diabetic mice, 2007 Mol Ther. <br />
* Development of genetically engineered human intestinal cells for regulated insulin secretion using rAAV-mediated gene transfer, 2003 Biochem Biophys Res Commun <br />
* http://www.utdol.com/utd/content/topic.do <br />
* Recombinant DNA Technology in the Synthesis of Human Insulin <br />
* Glucose-Dependent Insulin Release from Genetically Engineered K Cells, 2000 science <br />
* [http://www.nature.com/gt/journal/v7/n21/abs/3301306a.html Glucose-stimulated and self-limiting insulin production by glucose 6-phosphatase promoter driven insulin expression in hepatoma cells, 2000 Gene Therapy]<br />
[https://2007.igem.org/Taipei Back]</div>Blenthttp://2007.igem.org/wiki/index.php/File:GWE_15h.jpgFile:GWE 15h.jpg2007-10-27T04:26:21Z<p>Blent: </p>
<hr />
<div></div>Blenthttp://2007.igem.org/wiki/index.php/NYMU_Taipei/ProjectDescriptionNYMU Taipei/ProjectDescription2007-10-27T04:25:59Z<p>Blent: /* Mammalian Reporting System */</p>
<hr />
<div><p><font size="4"><br />
[[NYMU_Taipei/ProjectDescription| Project Description]] /<br />
[[NYMU_Taipei/Lab_Notes| Lab Notes]] /<br />
[[NYMU_Taipei/Part_List| Part List]] /<br />
[[NYMU_Taipei/Primer_List| Primer List]]<br />
</font><br /><hr></p><br />
<br />
==<font size=5 color=red>GlucOperon</font>==<br />
* A '''Gluc'''ose-based synthetic '''Operon''' system<br />
* An automatic system in E.coli that operates blood sugar wonderfully!<br />
* Insulin secretion system that promotes quality of life for diabetic patients<br />
----<br />
<br />
==Summary==<br />
* Diabetes mellitus is a significant problem especially in developed countries and leads to several severe long-term complications. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Controlling blood sugar in a reasonable level and avoiding severe emergency as diabetes ketoacidosis (DKA) are very important clinical issues. Thus, the NYMU_Taipei iGEM 2007 team is designing a biological system to sense environmental glucose concentration and decrease the level of glucose by releasing insulin. Besides, life-protection functions for removing toxic ketoacids produced during DKA and preventing hypoglycemia status will also be established. This system will be a convenient and safe design for those patients with diabetes mellitus, and further improve their quality of life by avoiding them from diabetes-related morbidity and mortality. <br />
----<br />
==Motivation==<br />
* Over 7 percent of adults in the United States (US) are known to have diabetes mellitus. However, because of the associated microvascular and macrovascular disease, diabetes accounts for almost 14 percent of US health care expenditures, at least one-half of which are related to complications such as myocardial infarction, stroke, end-stage renal disease, retinopathy, and foot ulcers. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus, one of the most common chronic diseases in childhood, is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Data from the Center of Disease Control (CDC) show that more than 13,000 children and adolescents (19 years of age) are diagnosed with type 1 disease each year with a prevalence of 1.7 per 1000. Control of blood sugar and prevention of severe complications, such;as diabetic ketoacidosis (DKA), are important issues nowadays. <br />
* Diabetic ketoacidosis (DKA) and hyperosmolar hyperglycemic state (HHS, also known as nonketotic hyperglycemia) are two of the most serious acute complications of diabetes. They are part of the spectrum of hyperglycemia and each represents an extreme in the spectrum. DKA is characterized by the triad of hyperglycemia, anion gap metabolic acidosis, and ketonemia. Metabolic acidosis is often the major finding. The serum glucose concentration is usually greater than 500 mg/dL (27.8 mmol/L) and less than 800 mg/dL (44.4 mmol/L). Three ketone bodies are produced in DKA: two ketoacids (beta-hydroxybutyric acid and acetoacetic acid), and one neutral ketone (acetone). The treatment of DKA and HHS is similar, including the administration of insulin and correction of the fluid and electrolyte abnormalities that may be present, including hyperglycemia and hyperosmolality, hypovolemia, metabolic acidosis (in DKA), and potassium depletion. <br />
* Existed works (see References below) are focusing on how to take advantages from properties of mammalian cells to express and secrete insulin in a trail-and-error manner. However, from the perspective of synthetic biology, it is possible to engineer a cell works as we want, no matter what type of cells we apply to. <br />
----<br />
==Goal and System Design==<br />
* For the iGEM 2007 project, our team focuses on the development of a prokaryotic system to <br />
** express insulin regulated by external glucose concentration and <br />
** clean the ketone bodies accumulated by mamalian cells due to insufficient intake of glucose <br />
** balance between glucose and insulin to stablize glucose level and prevent low blood glucose condition <br />
* system specification is shown below: <br />
[[Image:GAI-t curve.jpg|center|500px]]<br />
* System diagram (initial state)<br />
[[Image:IGEM2007 insulin circuit diagram.png|center]]<br />
* System under high glucose condition (insulin generating circuit is activated)<br />
[[Image:High_glucose.png|center]]<br />
* System under transition state (insulin increases glucose uptake; whole system shifts from insulin generation to insulin degradation)<br />
[[Image:Trans_glucose.png|center]]<br />
* System under low glucose condition (when glucose is low enough, the insulin generation turns off and insulin degradation turns on)<br />
[[Image:Low_glucose.png|center]]<br />
* System after insulin degradation (both circuits turn off and wait IDE to be naturally degraded; back to initial state)<br />
[[Image:Post_glucose.png|center]]<br />
* Circuit #1: high glucose-triggered insulin generator <br />
** The basic architecture for our insulin secretion design is shown in the figure below: <br />
** External glucose can be sensed by GSD (Glucose Sensing Domain) from gene RcsC and transmits response to ERD (Responsive Domain from EnvZ gene) <br />
** TCS phosphorylation relay activates the expression of insulin (both alpha and beta chains) <br />
** With signal peptide (SP), the assembled insulin (by disulfide bonding) can be exported outside the membrane of the prokaryotic cells <br />
[[Image:Insulin basic2.png|center|500px]] <br />
* Circuit #2: low glucose and insulin triggered insulin inhibitor/destroyer<br />
[[Image:NYMU_Taipei_insulin_inhibitor_2.png|center|500px]] <br />
[[Image:NYMU_Taipei_insulin_inhibitor.png|center|700px]] <br />
----<br />
<br />
==Implementation==<br />
* [[NYMU_Taipei/Part_List | Part List]]<br />
* [[NYMU_Taipei/Lab_Notes | Lab notes]]<br />
* [[NYMU_Taipei/Protocols| Protocols]]<br />
* [[NYMU_Taipei/Primer_List | Primer List]]<br />
----<br />
==Verification==<br />
===pOmpC characterization by testing circuit #1 pOmpC + EYFP===<br />
* first trail: glucose can induce expression pOmpC operon when glucose concentration higher than 1% after 14h culture in the LB medium<br />
[[Image:POmpC-EYFP test1.jpg|center]]<br />
<br /><br />
* second trail: 1% glucose induction will activate the insulin expression after 4hr induction<br />
[[Image:POmpC-EYFP test2.jpg|center]]<br />
<br />
===OmpRBS characterization by testing circuit #2 pLacIQ + OmpRBS + GFP===<br />
===Mammalian Reporting System===<br />
* insulin induces glucose uptake<br />
* cellular growth effect to E.coli-free medium after 15hr culture<br />
[[Image:GWE_15h.jpg]]<br />
----<br />
<br />
==References==<br />
* Engineered enteroendocrine cells secrete insulin in response to glucose and reverse hyperglycemia in diabetic mice, 2007 Mol Ther. <br />
* Development of genetically engineered human intestinal cells for regulated insulin secretion using rAAV-mediated gene transfer, 2003 Biochem Biophys Res Commun <br />
* http://www.utdol.com/utd/content/topic.do <br />
* Recombinant DNA Technology in the Synthesis of Human Insulin <br />
* Glucose-Dependent Insulin Release from Genetically Engineered K Cells, 2000 science <br />
* [http://www.nature.com/gt/journal/v7/n21/abs/3301306a.html Glucose-stimulated and self-limiting insulin production by glucose 6-phosphatase promoter driven insulin expression in hepatoma cells, 2000 Gene Therapy]<br />
[https://2007.igem.org/Taipei Back]</div>Blenthttp://2007.igem.org/wiki/index.php/NYMU_Taipei/ProjectDescriptionNYMU Taipei/ProjectDescription2007-10-27T03:43:05Z<p>Blent: /* Verification */</p>
<hr />
<div><p><font size="4"><br />
[[NYMU_Taipei/ProjectDescription| Project Description]] /<br />
[[NYMU_Taipei/Lab_Notes| Lab Notes]] /<br />
[[NYMU_Taipei/Part_List| Part List]] /<br />
[[NYMU_Taipei/Primer_List| Primer List]]<br />
</font><br /><hr></p><br />
<br />
==<font size=5 color=red>GlucOperon</font>==<br />
* A '''Gluc'''ose-based synthetic '''Operon''' system<br />
* An automatic system in E.coli that operates blood sugar wonderfully!<br />
* Insulin secretion system that promotes quality of life for diabetic patients<br />
----<br />
<br />
==Summary==<br />
* Diabetes mellitus is a significant problem especially in developed countries and leads to several severe long-term complications. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Controlling blood sugar in a reasonable level and avoiding severe emergency as diabetes ketoacidosis (DKA) are very important clinical issues. Thus, the NYMU_Taipei iGEM 2007 team is designing a biological system to sense environmental glucose concentration and decrease the level of glucose by releasing insulin. Besides, life-protection functions for removing toxic ketoacids produced during DKA and preventing hypoglycemia status will also be established. This system will be a convenient and safe design for those patients with diabetes mellitus, and further improve their quality of life by avoiding them from diabetes-related morbidity and mortality. <br />
----<br />
==Motivation==<br />
* Over 7 percent of adults in the United States (US) are known to have diabetes mellitus. However, because of the associated microvascular and macrovascular disease, diabetes accounts for almost 14 percent of US health care expenditures, at least one-half of which are related to complications such as myocardial infarction, stroke, end-stage renal disease, retinopathy, and foot ulcers. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus, one of the most common chronic diseases in childhood, is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Data from the Center of Disease Control (CDC) show that more than 13,000 children and adolescents (19 years of age) are diagnosed with type 1 disease each year with a prevalence of 1.7 per 1000. Control of blood sugar and prevention of severe complications, such;as diabetic ketoacidosis (DKA), are important issues nowadays. <br />
* Diabetic ketoacidosis (DKA) and hyperosmolar hyperglycemic state (HHS, also known as nonketotic hyperglycemia) are two of the most serious acute complications of diabetes. They are part of the spectrum of hyperglycemia and each represents an extreme in the spectrum. DKA is characterized by the triad of hyperglycemia, anion gap metabolic acidosis, and ketonemia. Metabolic acidosis is often the major finding. The serum glucose concentration is usually greater than 500 mg/dL (27.8 mmol/L) and less than 800 mg/dL (44.4 mmol/L). Three ketone bodies are produced in DKA: two ketoacids (beta-hydroxybutyric acid and acetoacetic acid), and one neutral ketone (acetone). The treatment of DKA and HHS is similar, including the administration of insulin and correction of the fluid and electrolyte abnormalities that may be present, including hyperglycemia and hyperosmolality, hypovolemia, metabolic acidosis (in DKA), and potassium depletion. <br />
* Existed works (see References below) are focusing on how to take advantages from properties of mammalian cells to express and secrete insulin in a trail-and-error manner. However, from the perspective of synthetic biology, it is possible to engineer a cell works as we want, no matter what type of cells we apply to. <br />
----<br />
==Goal and System Design==<br />
* For the iGEM 2007 project, our team focuses on the development of a prokaryotic system to <br />
** express insulin regulated by external glucose concentration and <br />
** clean the ketone bodies accumulated by mamalian cells due to insufficient intake of glucose <br />
** balance between glucose and insulin to stablize glucose level and prevent low blood glucose condition <br />
* system specification is shown below: <br />
[[Image:GAI-t curve.jpg|center|500px]]<br />
* System diagram (initial state)<br />
[[Image:IGEM2007 insulin circuit diagram.png|center]]<br />
* System under high glucose condition (insulin generating circuit is activated)<br />
[[Image:High_glucose.png|center]]<br />
* System under transition state (insulin increases glucose uptake; whole system shifts from insulin generation to insulin degradation)<br />
[[Image:Trans_glucose.png|center]]<br />
* System under low glucose condition (when glucose is low enough, the insulin generation turns off and insulin degradation turns on)<br />
[[Image:Low_glucose.png|center]]<br />
* System after insulin degradation (both circuits turn off and wait IDE to be naturally degraded; back to initial state)<br />
[[Image:Post_glucose.png|center]]<br />
* Circuit #1: high glucose-triggered insulin generator <br />
** The basic architecture for our insulin secretion design is shown in the figure below: <br />
** External glucose can be sensed by GSD (Glucose Sensing Domain) from gene RcsC and transmits response to ERD (Responsive Domain from EnvZ gene) <br />
** TCS phosphorylation relay activates the expression of insulin (both alpha and beta chains) <br />
** With signal peptide (SP), the assembled insulin (by disulfide bonding) can be exported outside the membrane of the prokaryotic cells <br />
[[Image:Insulin basic2.png|center|500px]] <br />
* Circuit #2: low glucose and insulin triggered insulin inhibitor/destroyer<br />
[[Image:NYMU_Taipei_insulin_inhibitor_2.png|center|500px]] <br />
[[Image:NYMU_Taipei_insulin_inhibitor.png|center|700px]] <br />
----<br />
<br />
==Implementation==<br />
* [[NYMU_Taipei/Part_List | Part List]]<br />
* [[NYMU_Taipei/Lab_Notes | Lab notes]]<br />
* [[NYMU_Taipei/Protocols| Protocols]]<br />
* [[NYMU_Taipei/Primer_List | Primer List]]<br />
----<br />
==Verification==<br />
===pOmpC characterization by testing circuit #1 pOmpC + EYFP===<br />
* first trail: glucose can induce expression pOmpC operon when glucose concentration higher than 1% after 14h culture in the LB medium<br />
[[Image:POmpC-EYFP test1.jpg|center]]<br />
<br /><br />
* second trail: 1% glucose induction will activate the insulin expression after 4hr induction<br />
[[Image:POmpC-EYFP test2.jpg|center]]<br />
<br />
===OmpRBS characterization by testing circuit #2 pLacIQ + OmpRBS + GFP===<br />
===Mammalian Reporting System===<br />
* insulin induces glucose uptake<br />
* cellular growth effect to E.coli-free medium after 8hr and 14hr<br />
----<br />
<br />
==References==<br />
* Engineered enteroendocrine cells secrete insulin in response to glucose and reverse hyperglycemia in diabetic mice, 2007 Mol Ther. <br />
* Development of genetically engineered human intestinal cells for regulated insulin secretion using rAAV-mediated gene transfer, 2003 Biochem Biophys Res Commun <br />
* http://www.utdol.com/utd/content/topic.do <br />
* Recombinant DNA Technology in the Synthesis of Human Insulin <br />
* Glucose-Dependent Insulin Release from Genetically Engineered K Cells, 2000 science <br />
* [http://www.nature.com/gt/journal/v7/n21/abs/3301306a.html Glucose-stimulated and self-limiting insulin production by glucose 6-phosphatase promoter driven insulin expression in hepatoma cells, 2000 Gene Therapy]<br />
[https://2007.igem.org/Taipei Back]</div>Blenthttp://2007.igem.org/wiki/index.php/NYMU_Taipei/ProjectDescriptionNYMU Taipei/ProjectDescription2007-10-27T03:17:07Z<p>Blent: /* pOmpC characterization by testing circuit #1 pOmpC + EYFP */</p>
<hr />
<div><p><font size="4"><br />
[[NYMU_Taipei/ProjectDescription| Project Description]] /<br />
[[NYMU_Taipei/Lab_Notes| Lab Notes]] /<br />
[[NYMU_Taipei/Part_List| Part List]] /<br />
[[NYMU_Taipei/Primer_List| Primer List]]<br />
</font><br /><hr></p><br />
<br />
==<font size=5 color=red>GlucOperon</font>==<br />
* A '''Gluc'''ose-based synthetic '''Operon''' system<br />
* An automatic system in E.coli that operates blood sugar wonderfully!<br />
* Insulin secretion system that promotes quality of life for diabetic patients<br />
----<br />
<br />
==Summary==<br />
* Diabetes mellitus is a significant problem especially in developed countries and leads to several severe long-term complications. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Controlling blood sugar in a reasonable level and avoiding severe emergency as diabetes ketoacidosis (DKA) are very important clinical issues. Thus, the NYMU_Taipei iGEM 2007 team is designing a biological system to sense environmental glucose concentration and decrease the level of glucose by releasing insulin. Besides, life-protection functions for removing toxic ketoacids produced during DKA and preventing hypoglycemia status will also be established. This system will be a convenient and safe design for those patients with diabetes mellitus, and further improve their quality of life by avoiding them from diabetes-related morbidity and mortality. <br />
----<br />
==Motivation==<br />
* Over 7 percent of adults in the United States (US) are known to have diabetes mellitus. However, because of the associated microvascular and macrovascular disease, diabetes accounts for almost 14 percent of US health care expenditures, at least one-half of which are related to complications such as myocardial infarction, stroke, end-stage renal disease, retinopathy, and foot ulcers. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus, one of the most common chronic diseases in childhood, is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Data from the Center of Disease Control (CDC) show that more than 13,000 children and adolescents (19 years of age) are diagnosed with type 1 disease each year with a prevalence of 1.7 per 1000. Control of blood sugar and prevention of severe complications, such;as diabetic ketoacidosis (DKA), are important issues nowadays. <br />
* Diabetic ketoacidosis (DKA) and hyperosmolar hyperglycemic state (HHS, also known as nonketotic hyperglycemia) are two of the most serious acute complications of diabetes. They are part of the spectrum of hyperglycemia and each represents an extreme in the spectrum. DKA is characterized by the triad of hyperglycemia, anion gap metabolic acidosis, and ketonemia. Metabolic acidosis is often the major finding. The serum glucose concentration is usually greater than 500 mg/dL (27.8 mmol/L) and less than 800 mg/dL (44.4 mmol/L). Three ketone bodies are produced in DKA: two ketoacids (beta-hydroxybutyric acid and acetoacetic acid), and one neutral ketone (acetone). The treatment of DKA and HHS is similar, including the administration of insulin and correction of the fluid and electrolyte abnormalities that may be present, including hyperglycemia and hyperosmolality, hypovolemia, metabolic acidosis (in DKA), and potassium depletion. <br />
* Existed works (see References below) are focusing on how to take advantages from properties of mammalian cells to express and secrete insulin in a trail-and-error manner. However, from the perspective of synthetic biology, it is possible to engineer a cell works as we want, no matter what type of cells we apply to. <br />
----<br />
==Goal and System Design==<br />
* For the iGEM 2007 project, our team focuses on the development of a prokaryotic system to <br />
** express insulin regulated by external glucose concentration and <br />
** clean the ketone bodies accumulated by mamalian cells due to insufficient intake of glucose <br />
** balance between glucose and insulin to stablize glucose level and prevent low blood glucose condition <br />
* system specification is shown below: <br />
[[Image:GAI-t curve.jpg|center|500px]]<br />
* System diagram (initial state)<br />
[[Image:IGEM2007 insulin circuit diagram.png|center]]<br />
* System under high glucose condition (insulin generating circuit is activated)<br />
[[Image:High_glucose.png|center]]<br />
* System under transition state (insulin increases glucose uptake; whole system shifts from insulin generation to insulin degradation)<br />
[[Image:Trans_glucose.png|center]]<br />
* System under low glucose condition (when glucose is low enough, the insulin generation turns off and insulin degradation turns on)<br />
[[Image:Low_glucose.png|center]]<br />
* System after insulin degradation (both circuits turn off and wait IDE to be naturally degraded; back to initial state)<br />
[[Image:Post_glucose.png|center]]<br />
* Circuit #1: high glucose-triggered insulin generator <br />
** The basic architecture for our insulin secretion design is shown in the figure below: <br />
** External glucose can be sensed by GSD (Glucose Sensing Domain) from gene RcsC and transmits response to ERD (Responsive Domain from EnvZ gene) <br />
** TCS phosphorylation relay activates the expression of insulin (both alpha and beta chains) <br />
** With signal peptide (SP), the assembled insulin (by disulfide bonding) can be exported outside the membrane of the prokaryotic cells <br />
[[Image:Insulin basic2.png|center|500px]] <br />
* Circuit #2: low glucose and insulin triggered insulin inhibitor/destroyer<br />
[[Image:NYMU_Taipei_insulin_inhibitor_2.png|center|500px]] <br />
[[Image:NYMU_Taipei_insulin_inhibitor.png|center|700px]] <br />
----<br />
<br />
==Implementation==<br />
* [[NYMU_Taipei/Part_List | Part List]]<br />
* [[NYMU_Taipei/Lab_Notes | Lab notes]]<br />
* [[NYMU_Taipei/Protocols| Protocols]]<br />
* [[NYMU_Taipei/Primer_List | Primer List]]<br />
----<br />
==Verification==<br />
===pOmpC characterization by testing circuit #1 pOmpC + EYFP===<br />
* first trail: glucose can induce expression pOmpC operon when glucose concentration higher than 1% after 14h culture in the LB medium<br />
[[Image:POmpC-EYFP test1.jpg|center]]<br />
<br /><br />
* second trail: 1% glucose induction will activate the insulin expression after 4hr induction<br />
[[Image:POmpC-EYFP test2.jpg|center]]<br />
<br />
===OmpRBS characterization by testing circuit #2 pLacIQ + OmpRBS + GFP===<br />
----<br />
<br />
==References==<br />
* Engineered enteroendocrine cells secrete insulin in response to glucose and reverse hyperglycemia in diabetic mice, 2007 Mol Ther. <br />
* Development of genetically engineered human intestinal cells for regulated insulin secretion using rAAV-mediated gene transfer, 2003 Biochem Biophys Res Commun <br />
* http://www.utdol.com/utd/content/topic.do <br />
* Recombinant DNA Technology in the Synthesis of Human Insulin <br />
* Glucose-Dependent Insulin Release from Genetically Engineered K Cells, 2000 science <br />
* [http://www.nature.com/gt/journal/v7/n21/abs/3301306a.html Glucose-stimulated and self-limiting insulin production by glucose 6-phosphatase promoter driven insulin expression in hepatoma cells, 2000 Gene Therapy]<br />
[https://2007.igem.org/Taipei Back]</div>Blenthttp://2007.igem.org/wiki/index.php/NYMU_Taipei/ProjectDescriptionNYMU Taipei/ProjectDescription2007-10-27T03:15:57Z<p>Blent: /* pOmpC characterization by testing circuit #1 pOmpC + EYFP */</p>
<hr />
<div><p><font size="4"><br />
[[NYMU_Taipei/ProjectDescription| Project Description]] /<br />
[[NYMU_Taipei/Lab_Notes| Lab Notes]] /<br />
[[NYMU_Taipei/Part_List| Part List]] /<br />
[[NYMU_Taipei/Primer_List| Primer List]]<br />
</font><br /><hr></p><br />
<br />
==<font size=5 color=red>GlucOperon</font>==<br />
* A '''Gluc'''ose-based synthetic '''Operon''' system<br />
* An automatic system in E.coli that operates blood sugar wonderfully!<br />
* Insulin secretion system that promotes quality of life for diabetic patients<br />
----<br />
<br />
==Summary==<br />
* Diabetes mellitus is a significant problem especially in developed countries and leads to several severe long-term complications. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Controlling blood sugar in a reasonable level and avoiding severe emergency as diabetes ketoacidosis (DKA) are very important clinical issues. Thus, the NYMU_Taipei iGEM 2007 team is designing a biological system to sense environmental glucose concentration and decrease the level of glucose by releasing insulin. Besides, life-protection functions for removing toxic ketoacids produced during DKA and preventing hypoglycemia status will also be established. This system will be a convenient and safe design for those patients with diabetes mellitus, and further improve their quality of life by avoiding them from diabetes-related morbidity and mortality. <br />
----<br />
==Motivation==<br />
* Over 7 percent of adults in the United States (US) are known to have diabetes mellitus. However, because of the associated microvascular and macrovascular disease, diabetes accounts for almost 14 percent of US health care expenditures, at least one-half of which are related to complications such as myocardial infarction, stroke, end-stage renal disease, retinopathy, and foot ulcers. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus, one of the most common chronic diseases in childhood, is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Data from the Center of Disease Control (CDC) show that more than 13,000 children and adolescents (19 years of age) are diagnosed with type 1 disease each year with a prevalence of 1.7 per 1000. Control of blood sugar and prevention of severe complications, such;as diabetic ketoacidosis (DKA), are important issues nowadays. <br />
* Diabetic ketoacidosis (DKA) and hyperosmolar hyperglycemic state (HHS, also known as nonketotic hyperglycemia) are two of the most serious acute complications of diabetes. They are part of the spectrum of hyperglycemia and each represents an extreme in the spectrum. DKA is characterized by the triad of hyperglycemia, anion gap metabolic acidosis, and ketonemia. Metabolic acidosis is often the major finding. The serum glucose concentration is usually greater than 500 mg/dL (27.8 mmol/L) and less than 800 mg/dL (44.4 mmol/L). Three ketone bodies are produced in DKA: two ketoacids (beta-hydroxybutyric acid and acetoacetic acid), and one neutral ketone (acetone). The treatment of DKA and HHS is similar, including the administration of insulin and correction of the fluid and electrolyte abnormalities that may be present, including hyperglycemia and hyperosmolality, hypovolemia, metabolic acidosis (in DKA), and potassium depletion. <br />
* Existed works (see References below) are focusing on how to take advantages from properties of mammalian cells to express and secrete insulin in a trail-and-error manner. However, from the perspective of synthetic biology, it is possible to engineer a cell works as we want, no matter what type of cells we apply to. <br />
----<br />
==Goal and System Design==<br />
* For the iGEM 2007 project, our team focuses on the development of a prokaryotic system to <br />
** express insulin regulated by external glucose concentration and <br />
** clean the ketone bodies accumulated by mamalian cells due to insufficient intake of glucose <br />
** balance between glucose and insulin to stablize glucose level and prevent low blood glucose condition <br />
* system specification is shown below: <br />
[[Image:GAI-t curve.jpg|center|500px]]<br />
* System diagram (initial state)<br />
[[Image:IGEM2007 insulin circuit diagram.png|center]]<br />
* System under high glucose condition (insulin generating circuit is activated)<br />
[[Image:High_glucose.png|center]]<br />
* System under transition state (insulin increases glucose uptake; whole system shifts from insulin generation to insulin degradation)<br />
[[Image:Trans_glucose.png|center]]<br />
* System under low glucose condition (when glucose is low enough, the insulin generation turns off and insulin degradation turns on)<br />
[[Image:Low_glucose.png|center]]<br />
* System after insulin degradation (both circuits turn off and wait IDE to be naturally degraded; back to initial state)<br />
[[Image:Post_glucose.png|center]]<br />
* Circuit #1: high glucose-triggered insulin generator <br />
** The basic architecture for our insulin secretion design is shown in the figure below: <br />
** External glucose can be sensed by GSD (Glucose Sensing Domain) from gene RcsC and transmits response to ERD (Responsive Domain from EnvZ gene) <br />
** TCS phosphorylation relay activates the expression of insulin (both alpha and beta chains) <br />
** With signal peptide (SP), the assembled insulin (by disulfide bonding) can be exported outside the membrane of the prokaryotic cells <br />
[[Image:Insulin basic2.png|center|500px]] <br />
* Circuit #2: low glucose and insulin triggered insulin inhibitor/destroyer<br />
[[Image:NYMU_Taipei_insulin_inhibitor_2.png|center|500px]] <br />
[[Image:NYMU_Taipei_insulin_inhibitor.png|center|700px]] <br />
----<br />
<br />
==Implementation==<br />
* [[NYMU_Taipei/Part_List | Part List]]<br />
* [[NYMU_Taipei/Lab_Notes | Lab notes]]<br />
* [[NYMU_Taipei/Protocols| Protocols]]<br />
* [[NYMU_Taipei/Primer_List | Primer List]]<br />
----<br />
==Verification==<br />
===pOmpC characterization by testing circuit #1 pOmpC + EYFP===<br />
* first trail: glucose can induce expression pOmpC operon when glucose concentration higher than 1% after 14h culture in the LB medium<br />
[[Image:POmpC-EYFP test1.jpg|center]]<br />
<br /><br />
* second trail: time course for 1% glucose induction<br />
[[Image:POmpC-EYFP test2.jpg|center]]<br />
<br />
===OmpRBS characterization by testing circuit #2 pLacIQ + OmpRBS + GFP===<br />
----<br />
<br />
==References==<br />
* Engineered enteroendocrine cells secrete insulin in response to glucose and reverse hyperglycemia in diabetic mice, 2007 Mol Ther. <br />
* Development of genetically engineered human intestinal cells for regulated insulin secretion using rAAV-mediated gene transfer, 2003 Biochem Biophys Res Commun <br />
* http://www.utdol.com/utd/content/topic.do <br />
* Recombinant DNA Technology in the Synthesis of Human Insulin <br />
* Glucose-Dependent Insulin Release from Genetically Engineered K Cells, 2000 science <br />
* [http://www.nature.com/gt/journal/v7/n21/abs/3301306a.html Glucose-stimulated and self-limiting insulin production by glucose 6-phosphatase promoter driven insulin expression in hepatoma cells, 2000 Gene Therapy]<br />
[https://2007.igem.org/Taipei Back]</div>Blenthttp://2007.igem.org/wiki/index.php/NYMU_Taipei/ProjectDescriptionNYMU Taipei/ProjectDescription2007-10-27T03:15:10Z<p>Blent: /* pOmpC characterization by testing circuit #1 pOmpC + EYFP */</p>
<hr />
<div><p><font size="4"><br />
[[NYMU_Taipei/ProjectDescription| Project Description]] /<br />
[[NYMU_Taipei/Lab_Notes| Lab Notes]] /<br />
[[NYMU_Taipei/Part_List| Part List]] /<br />
[[NYMU_Taipei/Primer_List| Primer List]]<br />
</font><br /><hr></p><br />
<br />
==<font size=5 color=red>GlucOperon</font>==<br />
* A '''Gluc'''ose-based synthetic '''Operon''' system<br />
* An automatic system in E.coli that operates blood sugar wonderfully!<br />
* Insulin secretion system that promotes quality of life for diabetic patients<br />
----<br />
<br />
==Summary==<br />
* Diabetes mellitus is a significant problem especially in developed countries and leads to several severe long-term complications. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Controlling blood sugar in a reasonable level and avoiding severe emergency as diabetes ketoacidosis (DKA) are very important clinical issues. Thus, the NYMU_Taipei iGEM 2007 team is designing a biological system to sense environmental glucose concentration and decrease the level of glucose by releasing insulin. Besides, life-protection functions for removing toxic ketoacids produced during DKA and preventing hypoglycemia status will also be established. This system will be a convenient and safe design for those patients with diabetes mellitus, and further improve their quality of life by avoiding them from diabetes-related morbidity and mortality. <br />
----<br />
==Motivation==<br />
* Over 7 percent of adults in the United States (US) are known to have diabetes mellitus. However, because of the associated microvascular and macrovascular disease, diabetes accounts for almost 14 percent of US health care expenditures, at least one-half of which are related to complications such as myocardial infarction, stroke, end-stage renal disease, retinopathy, and foot ulcers. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus, one of the most common chronic diseases in childhood, is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Data from the Center of Disease Control (CDC) show that more than 13,000 children and adolescents (19 years of age) are diagnosed with type 1 disease each year with a prevalence of 1.7 per 1000. Control of blood sugar and prevention of severe complications, such;as diabetic ketoacidosis (DKA), are important issues nowadays. <br />
* Diabetic ketoacidosis (DKA) and hyperosmolar hyperglycemic state (HHS, also known as nonketotic hyperglycemia) are two of the most serious acute complications of diabetes. They are part of the spectrum of hyperglycemia and each represents an extreme in the spectrum. DKA is characterized by the triad of hyperglycemia, anion gap metabolic acidosis, and ketonemia. Metabolic acidosis is often the major finding. The serum glucose concentration is usually greater than 500 mg/dL (27.8 mmol/L) and less than 800 mg/dL (44.4 mmol/L). Three ketone bodies are produced in DKA: two ketoacids (beta-hydroxybutyric acid and acetoacetic acid), and one neutral ketone (acetone). The treatment of DKA and HHS is similar, including the administration of insulin and correction of the fluid and electrolyte abnormalities that may be present, including hyperglycemia and hyperosmolality, hypovolemia, metabolic acidosis (in DKA), and potassium depletion. <br />
* Existed works (see References below) are focusing on how to take advantages from properties of mammalian cells to express and secrete insulin in a trail-and-error manner. However, from the perspective of synthetic biology, it is possible to engineer a cell works as we want, no matter what type of cells we apply to. <br />
----<br />
==Goal and System Design==<br />
* For the iGEM 2007 project, our team focuses on the development of a prokaryotic system to <br />
** express insulin regulated by external glucose concentration and <br />
** clean the ketone bodies accumulated by mamalian cells due to insufficient intake of glucose <br />
** balance between glucose and insulin to stablize glucose level and prevent low blood glucose condition <br />
* system specification is shown below: <br />
[[Image:GAI-t curve.jpg|center|500px]]<br />
* System diagram (initial state)<br />
[[Image:IGEM2007 insulin circuit diagram.png|center]]<br />
* System under high glucose condition (insulin generating circuit is activated)<br />
[[Image:High_glucose.png|center]]<br />
* System under transition state (insulin increases glucose uptake; whole system shifts from insulin generation to insulin degradation)<br />
[[Image:Trans_glucose.png|center]]<br />
* System under low glucose condition (when glucose is low enough, the insulin generation turns off and insulin degradation turns on)<br />
[[Image:Low_glucose.png|center]]<br />
* System after insulin degradation (both circuits turn off and wait IDE to be naturally degraded; back to initial state)<br />
[[Image:Post_glucose.png|center]]<br />
* Circuit #1: high glucose-triggered insulin generator <br />
** The basic architecture for our insulin secretion design is shown in the figure below: <br />
** External glucose can be sensed by GSD (Glucose Sensing Domain) from gene RcsC and transmits response to ERD (Responsive Domain from EnvZ gene) <br />
** TCS phosphorylation relay activates the expression of insulin (both alpha and beta chains) <br />
** With signal peptide (SP), the assembled insulin (by disulfide bonding) can be exported outside the membrane of the prokaryotic cells <br />
[[Image:Insulin basic2.png|center|500px]] <br />
* Circuit #2: low glucose and insulin triggered insulin inhibitor/destroyer<br />
[[Image:NYMU_Taipei_insulin_inhibitor_2.png|center|500px]] <br />
[[Image:NYMU_Taipei_insulin_inhibitor.png|center|700px]] <br />
----<br />
<br />
==Implementation==<br />
* [[NYMU_Taipei/Part_List | Part List]]<br />
* [[NYMU_Taipei/Lab_Notes | Lab notes]]<br />
* [[NYMU_Taipei/Protocols| Protocols]]<br />
* [[NYMU_Taipei/Primer_List | Primer List]]<br />
----<br />
==Verification==<br />
===pOmpC characterization by testing circuit #1 pOmpC + EYFP===<br />
* first trail: glucose can induce expression pOmpC operon when glucose concentration higher than 1% after 14h culture in the LB medium<br />
[[Image:POmpC-EYFP test1.jpg|center]]<br />
* second trail: time course for 1% glucose induction<br />
[[Image:POmpC-EYFP test2.jpg|center]]<br />
<br />
===OmpRBS characterization by testing circuit #2 pLacIQ + OmpRBS + GFP===<br />
----<br />
<br />
==References==<br />
* Engineered enteroendocrine cells secrete insulin in response to glucose and reverse hyperglycemia in diabetic mice, 2007 Mol Ther. <br />
* Development of genetically engineered human intestinal cells for regulated insulin secretion using rAAV-mediated gene transfer, 2003 Biochem Biophys Res Commun <br />
* http://www.utdol.com/utd/content/topic.do <br />
* Recombinant DNA Technology in the Synthesis of Human Insulin <br />
* Glucose-Dependent Insulin Release from Genetically Engineered K Cells, 2000 science <br />
* [http://www.nature.com/gt/journal/v7/n21/abs/3301306a.html Glucose-stimulated and self-limiting insulin production by glucose 6-phosphatase promoter driven insulin expression in hepatoma cells, 2000 Gene Therapy]<br />
[https://2007.igem.org/Taipei Back]</div>Blenthttp://2007.igem.org/wiki/index.php/File:POmpC-EYFP_test2.jpgFile:POmpC-EYFP test2.jpg2007-10-27T03:14:45Z<p>Blent: </p>
<hr />
<div></div>Blenthttp://2007.igem.org/wiki/index.php/NYMU_Taipei/ProjectDescriptionNYMU Taipei/ProjectDescription2007-10-27T03:14:30Z<p>Blent: /* pOmpC characterization by testing circuit #1 pOmpC + EYFP */</p>
<hr />
<div><p><font size="4"><br />
[[NYMU_Taipei/ProjectDescription| Project Description]] /<br />
[[NYMU_Taipei/Lab_Notes| Lab Notes]] /<br />
[[NYMU_Taipei/Part_List| Part List]] /<br />
[[NYMU_Taipei/Primer_List| Primer List]]<br />
</font><br /><hr></p><br />
<br />
==<font size=5 color=red>GlucOperon</font>==<br />
* A '''Gluc'''ose-based synthetic '''Operon''' system<br />
* An automatic system in E.coli that operates blood sugar wonderfully!<br />
* Insulin secretion system that promotes quality of life for diabetic patients<br />
----<br />
<br />
==Summary==<br />
* Diabetes mellitus is a significant problem especially in developed countries and leads to several severe long-term complications. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Controlling blood sugar in a reasonable level and avoiding severe emergency as diabetes ketoacidosis (DKA) are very important clinical issues. Thus, the NYMU_Taipei iGEM 2007 team is designing a biological system to sense environmental glucose concentration and decrease the level of glucose by releasing insulin. Besides, life-protection functions for removing toxic ketoacids produced during DKA and preventing hypoglycemia status will also be established. This system will be a convenient and safe design for those patients with diabetes mellitus, and further improve their quality of life by avoiding them from diabetes-related morbidity and mortality. <br />
----<br />
==Motivation==<br />
* Over 7 percent of adults in the United States (US) are known to have diabetes mellitus. However, because of the associated microvascular and macrovascular disease, diabetes accounts for almost 14 percent of US health care expenditures, at least one-half of which are related to complications such as myocardial infarction, stroke, end-stage renal disease, retinopathy, and foot ulcers. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus, one of the most common chronic diseases in childhood, is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Data from the Center of Disease Control (CDC) show that more than 13,000 children and adolescents (19 years of age) are diagnosed with type 1 disease each year with a prevalence of 1.7 per 1000. Control of blood sugar and prevention of severe complications, such;as diabetic ketoacidosis (DKA), are important issues nowadays. <br />
* Diabetic ketoacidosis (DKA) and hyperosmolar hyperglycemic state (HHS, also known as nonketotic hyperglycemia) are two of the most serious acute complications of diabetes. They are part of the spectrum of hyperglycemia and each represents an extreme in the spectrum. DKA is characterized by the triad of hyperglycemia, anion gap metabolic acidosis, and ketonemia. Metabolic acidosis is often the major finding. The serum glucose concentration is usually greater than 500 mg/dL (27.8 mmol/L) and less than 800 mg/dL (44.4 mmol/L). Three ketone bodies are produced in DKA: two ketoacids (beta-hydroxybutyric acid and acetoacetic acid), and one neutral ketone (acetone). The treatment of DKA and HHS is similar, including the administration of insulin and correction of the fluid and electrolyte abnormalities that may be present, including hyperglycemia and hyperosmolality, hypovolemia, metabolic acidosis (in DKA), and potassium depletion. <br />
* Existed works (see References below) are focusing on how to take advantages from properties of mammalian cells to express and secrete insulin in a trail-and-error manner. However, from the perspective of synthetic biology, it is possible to engineer a cell works as we want, no matter what type of cells we apply to. <br />
----<br />
==Goal and System Design==<br />
* For the iGEM 2007 project, our team focuses on the development of a prokaryotic system to <br />
** express insulin regulated by external glucose concentration and <br />
** clean the ketone bodies accumulated by mamalian cells due to insufficient intake of glucose <br />
** balance between glucose and insulin to stablize glucose level and prevent low blood glucose condition <br />
* system specification is shown below: <br />
[[Image:GAI-t curve.jpg|center|500px]]<br />
* System diagram (initial state)<br />
[[Image:IGEM2007 insulin circuit diagram.png|center]]<br />
* System under high glucose condition (insulin generating circuit is activated)<br />
[[Image:High_glucose.png|center]]<br />
* System under transition state (insulin increases glucose uptake; whole system shifts from insulin generation to insulin degradation)<br />
[[Image:Trans_glucose.png|center]]<br />
* System under low glucose condition (when glucose is low enough, the insulin generation turns off and insulin degradation turns on)<br />
[[Image:Low_glucose.png|center]]<br />
* System after insulin degradation (both circuits turn off and wait IDE to be naturally degraded; back to initial state)<br />
[[Image:Post_glucose.png|center]]<br />
* Circuit #1: high glucose-triggered insulin generator <br />
** The basic architecture for our insulin secretion design is shown in the figure below: <br />
** External glucose can be sensed by GSD (Glucose Sensing Domain) from gene RcsC and transmits response to ERD (Responsive Domain from EnvZ gene) <br />
** TCS phosphorylation relay activates the expression of insulin (both alpha and beta chains) <br />
** With signal peptide (SP), the assembled insulin (by disulfide bonding) can be exported outside the membrane of the prokaryotic cells <br />
[[Image:Insulin basic2.png|center|500px]] <br />
* Circuit #2: low glucose and insulin triggered insulin inhibitor/destroyer<br />
[[Image:NYMU_Taipei_insulin_inhibitor_2.png|center|500px]] <br />
[[Image:NYMU_Taipei_insulin_inhibitor.png|center|700px]] <br />
----<br />
<br />
==Implementation==<br />
* [[NYMU_Taipei/Part_List | Part List]]<br />
* [[NYMU_Taipei/Lab_Notes | Lab notes]]<br />
* [[NYMU_Taipei/Protocols| Protocols]]<br />
* [[NYMU_Taipei/Primer_List | Primer List]]<br />
----<br />
==Verification==<br />
===pOmpC characterization by testing circuit #1 pOmpC + EYFP===<br />
* first trail: glucose can induce expression pOmpC operon when glucose concentration higher than 1% after 14h culture in the LB medium<br />
[[Image:POmpC-EYFP test1.jpg]]<br />
* second trail: time course for 1% glucose induction<br />
[[Image:POmpC-EYFP test2.jpg]]<br />
<br />
===OmpRBS characterization by testing circuit #2 pLacIQ + OmpRBS + GFP===<br />
----<br />
<br />
==References==<br />
* Engineered enteroendocrine cells secrete insulin in response to glucose and reverse hyperglycemia in diabetic mice, 2007 Mol Ther. <br />
* Development of genetically engineered human intestinal cells for regulated insulin secretion using rAAV-mediated gene transfer, 2003 Biochem Biophys Res Commun <br />
* http://www.utdol.com/utd/content/topic.do <br />
* Recombinant DNA Technology in the Synthesis of Human Insulin <br />
* Glucose-Dependent Insulin Release from Genetically Engineered K Cells, 2000 science <br />
* [http://www.nature.com/gt/journal/v7/n21/abs/3301306a.html Glucose-stimulated and self-limiting insulin production by glucose 6-phosphatase promoter driven insulin expression in hepatoma cells, 2000 Gene Therapy]<br />
[https://2007.igem.org/Taipei Back]</div>Blenthttp://2007.igem.org/wiki/index.php/NYMU_Taipei/ProjectDescriptionNYMU Taipei/ProjectDescription2007-10-26T23:18:07Z<p>Blent: /* pOmpC characterization by testing circuit #1 pOmpC + EYFP */</p>
<hr />
<div><p><font size="4"><br />
[[NYMU_Taipei/ProjectDescription| Project Description]] /<br />
[[NYMU_Taipei/Lab_Notes| Lab Notes]] /<br />
[[NYMU_Taipei/Part_List| Part List]] /<br />
[[NYMU_Taipei/Primer_List| Primer List]]<br />
</font><br /><hr></p><br />
<br />
==<font size=5 color=red>GlucOperon</font>==<br />
* A '''Gluc'''ose-based synthetic '''Operon''' system<br />
* An automatic system in E.coli that operates blood sugar wonderfully!<br />
* Insulin secretion system that promotes quality of life for diabetic patients<br />
----<br />
<br />
==Summary==<br />
* Diabetes mellitus is a significant problem especially in developed countries and leads to several severe long-term complications. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Controlling blood sugar in a reasonable level and avoiding severe emergency as diabetes ketoacidosis (DKA) are very important clinical issues. Thus, the NYMU_Taipei iGEM 2007 team is designing a biological system to sense environmental glucose concentration and decrease the level of glucose by releasing insulin. Besides, life-protection functions for removing toxic ketoacids produced during DKA and preventing hypoglycemia status will also be established. This system will be a convenient and safe design for those patients with diabetes mellitus, and further improve their quality of life by avoiding them from diabetes-related morbidity and mortality. <br />
----<br />
==Motivation==<br />
* Over 7 percent of adults in the United States (US) are known to have diabetes mellitus. However, because of the associated microvascular and macrovascular disease, diabetes accounts for almost 14 percent of US health care expenditures, at least one-half of which are related to complications such as myocardial infarction, stroke, end-stage renal disease, retinopathy, and foot ulcers. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus, one of the most common chronic diseases in childhood, is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Data from the Center of Disease Control (CDC) show that more than 13,000 children and adolescents (19 years of age) are diagnosed with type 1 disease each year with a prevalence of 1.7 per 1000. Control of blood sugar and prevention of severe complications, such;as diabetic ketoacidosis (DKA), are important issues nowadays. <br />
* Diabetic ketoacidosis (DKA) and hyperosmolar hyperglycemic state (HHS, also known as nonketotic hyperglycemia) are two of the most serious acute complications of diabetes. They are part of the spectrum of hyperglycemia and each represents an extreme in the spectrum. DKA is characterized by the triad of hyperglycemia, anion gap metabolic acidosis, and ketonemia. Metabolic acidosis is often the major finding. The serum glucose concentration is usually greater than 500 mg/dL (27.8 mmol/L) and less than 800 mg/dL (44.4 mmol/L). Three ketone bodies are produced in DKA: two ketoacids (beta-hydroxybutyric acid and acetoacetic acid), and one neutral ketone (acetone). The treatment of DKA and HHS is similar, including the administration of insulin and correction of the fluid and electrolyte abnormalities that may be present, including hyperglycemia and hyperosmolality, hypovolemia, metabolic acidosis (in DKA), and potassium depletion. <br />
* Existed works (see References below) are focusing on how to take advantages from properties of mammalian cells to express and secrete insulin in a trail-and-error manner. However, from the perspective of synthetic biology, it is possible to engineer a cell works as we want, no matter what type of cells we apply to. <br />
----<br />
==Goal and System Design==<br />
* For the iGEM 2007 project, our team focuses on the development of a prokaryotic system to <br />
** express insulin regulated by external glucose concentration and <br />
** clean the ketone bodies accumulated by mamalian cells due to insufficient intake of glucose <br />
** balance between glucose and insulin to stablize glucose level and prevent low blood glucose condition <br />
* system specification is shown below: <br />
[[Image:GAI-t curve.jpg|center|500px]]<br />
* System diagram (initial state)<br />
[[Image:IGEM2007 insulin circuit diagram.png|center]]<br />
* System under high glucose condition (insulin generating circuit is activated)<br />
[[Image:High_glucose.png|center]]<br />
* System under transition state (insulin increases glucose uptake; whole system shifts from insulin generation to insulin degradation)<br />
[[Image:Trans_glucose.png|center]]<br />
* System under low glucose condition (when glucose is low enough, the insulin generation turns off and insulin degradation turns on)<br />
[[Image:Low_glucose.png|center]]<br />
* System after insulin degradation (both circuits turn off and wait IDE to be naturally degraded; back to initial state)<br />
[[Image:Post_glucose.png|center]]<br />
* Circuit #1: high glucose-triggered insulin generator <br />
** The basic architecture for our insulin secretion design is shown in the figure below: <br />
** External glucose can be sensed by GSD (Glucose Sensing Domain) from gene RcsC and transmits response to ERD (Responsive Domain from EnvZ gene) <br />
** TCS phosphorylation relay activates the expression of insulin (both alpha and beta chains) <br />
** With signal peptide (SP), the assembled insulin (by disulfide bonding) can be exported outside the membrane of the prokaryotic cells <br />
[[Image:Insulin basic2.png|center|500px]] <br />
* Circuit #2: low glucose and insulin triggered insulin inhibitor/destroyer<br />
[[Image:NYMU_Taipei_insulin_inhibitor_2.png|center|500px]] <br />
[[Image:NYMU_Taipei_insulin_inhibitor.png|center|700px]] <br />
----<br />
<br />
==Implementation==<br />
* [[NYMU_Taipei/Part_List | Part List]]<br />
* [[NYMU_Taipei/Lab_Notes | Lab notes]]<br />
* [[NYMU_Taipei/Protocols| Protocols]]<br />
* [[NYMU_Taipei/Primer_List | Primer List]]<br />
----<br />
==Verification==<br />
===pOmpC characterization by testing circuit #1 pOmpC + EYFP===<br />
* first trail: glucose can induce expression pOmpC operon when glucose concentration higher than 1% after 14h culture in the LB medium<br />
[[Image:POmpC-EYFP test1.jpg]]<br />
* second trail: time course for 1% glucose induction<br />
<br />
===OmpRBS characterization by testing circuit #2 pLacIQ + OmpRBS + GFP===<br />
----<br />
<br />
==References==<br />
* Engineered enteroendocrine cells secrete insulin in response to glucose and reverse hyperglycemia in diabetic mice, 2007 Mol Ther. <br />
* Development of genetically engineered human intestinal cells for regulated insulin secretion using rAAV-mediated gene transfer, 2003 Biochem Biophys Res Commun <br />
* http://www.utdol.com/utd/content/topic.do <br />
* Recombinant DNA Technology in the Synthesis of Human Insulin <br />
* Glucose-Dependent Insulin Release from Genetically Engineered K Cells, 2000 science <br />
* [http://www.nature.com/gt/journal/v7/n21/abs/3301306a.html Glucose-stimulated and self-limiting insulin production by glucose 6-phosphatase promoter driven insulin expression in hepatoma cells, 2000 Gene Therapy]<br />
[https://2007.igem.org/Taipei Back]</div>Blenthttp://2007.igem.org/wiki/index.php/NYMU_Taipei/ProjectDescriptionNYMU Taipei/ProjectDescription2007-10-26T23:16:27Z<p>Blent: /* pOmpC characterization by testing circuit #1 pOmpC + EYFP */</p>
<hr />
<div><p><font size="4"><br />
[[NYMU_Taipei/ProjectDescription| Project Description]] /<br />
[[NYMU_Taipei/Lab_Notes| Lab Notes]] /<br />
[[NYMU_Taipei/Part_List| Part List]] /<br />
[[NYMU_Taipei/Primer_List| Primer List]]<br />
</font><br /><hr></p><br />
<br />
==<font size=5 color=red>GlucOperon</font>==<br />
* A '''Gluc'''ose-based synthetic '''Operon''' system<br />
* An automatic system in E.coli that operates blood sugar wonderfully!<br />
* Insulin secretion system that promotes quality of life for diabetic patients<br />
----<br />
<br />
==Summary==<br />
* Diabetes mellitus is a significant problem especially in developed countries and leads to several severe long-term complications. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Controlling blood sugar in a reasonable level and avoiding severe emergency as diabetes ketoacidosis (DKA) are very important clinical issues. Thus, the NYMU_Taipei iGEM 2007 team is designing a biological system to sense environmental glucose concentration and decrease the level of glucose by releasing insulin. Besides, life-protection functions for removing toxic ketoacids produced during DKA and preventing hypoglycemia status will also be established. This system will be a convenient and safe design for those patients with diabetes mellitus, and further improve their quality of life by avoiding them from diabetes-related morbidity and mortality. <br />
----<br />
==Motivation==<br />
* Over 7 percent of adults in the United States (US) are known to have diabetes mellitus. However, because of the associated microvascular and macrovascular disease, diabetes accounts for almost 14 percent of US health care expenditures, at least one-half of which are related to complications such as myocardial infarction, stroke, end-stage renal disease, retinopathy, and foot ulcers. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus, one of the most common chronic diseases in childhood, is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Data from the Center of Disease Control (CDC) show that more than 13,000 children and adolescents (19 years of age) are diagnosed with type 1 disease each year with a prevalence of 1.7 per 1000. Control of blood sugar and prevention of severe complications, such;as diabetic ketoacidosis (DKA), are important issues nowadays. <br />
* Diabetic ketoacidosis (DKA) and hyperosmolar hyperglycemic state (HHS, also known as nonketotic hyperglycemia) are two of the most serious acute complications of diabetes. They are part of the spectrum of hyperglycemia and each represents an extreme in the spectrum. DKA is characterized by the triad of hyperglycemia, anion gap metabolic acidosis, and ketonemia. Metabolic acidosis is often the major finding. The serum glucose concentration is usually greater than 500 mg/dL (27.8 mmol/L) and less than 800 mg/dL (44.4 mmol/L). Three ketone bodies are produced in DKA: two ketoacids (beta-hydroxybutyric acid and acetoacetic acid), and one neutral ketone (acetone). The treatment of DKA and HHS is similar, including the administration of insulin and correction of the fluid and electrolyte abnormalities that may be present, including hyperglycemia and hyperosmolality, hypovolemia, metabolic acidosis (in DKA), and potassium depletion. <br />
* Existed works (see References below) are focusing on how to take advantages from properties of mammalian cells to express and secrete insulin in a trail-and-error manner. However, from the perspective of synthetic biology, it is possible to engineer a cell works as we want, no matter what type of cells we apply to. <br />
----<br />
==Goal and System Design==<br />
* For the iGEM 2007 project, our team focuses on the development of a prokaryotic system to <br />
** express insulin regulated by external glucose concentration and <br />
** clean the ketone bodies accumulated by mamalian cells due to insufficient intake of glucose <br />
** balance between glucose and insulin to stablize glucose level and prevent low blood glucose condition <br />
* system specification is shown below: <br />
[[Image:GAI-t curve.jpg|center|500px]]<br />
* System diagram (initial state)<br />
[[Image:IGEM2007 insulin circuit diagram.png|center]]<br />
* System under high glucose condition (insulin generating circuit is activated)<br />
[[Image:High_glucose.png|center]]<br />
* System under transition state (insulin increases glucose uptake; whole system shifts from insulin generation to insulin degradation)<br />
[[Image:Trans_glucose.png|center]]<br />
* System under low glucose condition (when glucose is low enough, the insulin generation turns off and insulin degradation turns on)<br />
[[Image:Low_glucose.png|center]]<br />
* System after insulin degradation (both circuits turn off and wait IDE to be naturally degraded; back to initial state)<br />
[[Image:Post_glucose.png|center]]<br />
* Circuit #1: high glucose-triggered insulin generator <br />
** The basic architecture for our insulin secretion design is shown in the figure below: <br />
** External glucose can be sensed by GSD (Glucose Sensing Domain) from gene RcsC and transmits response to ERD (Responsive Domain from EnvZ gene) <br />
** TCS phosphorylation relay activates the expression of insulin (both alpha and beta chains) <br />
** With signal peptide (SP), the assembled insulin (by disulfide bonding) can be exported outside the membrane of the prokaryotic cells <br />
[[Image:Insulin basic2.png|center|500px]] <br />
* Circuit #2: low glucose and insulin triggered insulin inhibitor/destroyer<br />
[[Image:NYMU_Taipei_insulin_inhibitor_2.png|center|500px]] <br />
[[Image:NYMU_Taipei_insulin_inhibitor.png|center|700px]] <br />
----<br />
<br />
==Implementation==<br />
* [[NYMU_Taipei/Part_List | Part List]]<br />
* [[NYMU_Taipei/Lab_Notes | Lab notes]]<br />
* [[NYMU_Taipei/Protocols| Protocols]]<br />
* [[NYMU_Taipei/Primer_List | Primer List]]<br />
----<br />
==Verification==<br />
===pOmpC characterization by testing circuit #1 pOmpC + EYFP===<br />
* first trial: glucose can induce expression pOmpC operon when 1% glucose concentration in the LB medium after 14h culture<br />
[[Image:POmpC-EYFP test1.jpg]]<br />
* second trail: time course for 1% glucose induction<br />
<br />
===OmpRBS characterization by testing circuit #2 pLacIQ + OmpRBS + GFP===<br />
----<br />
<br />
==References==<br />
* Engineered enteroendocrine cells secrete insulin in response to glucose and reverse hyperglycemia in diabetic mice, 2007 Mol Ther. <br />
* Development of genetically engineered human intestinal cells for regulated insulin secretion using rAAV-mediated gene transfer, 2003 Biochem Biophys Res Commun <br />
* http://www.utdol.com/utd/content/topic.do <br />
* Recombinant DNA Technology in the Synthesis of Human Insulin <br />
* Glucose-Dependent Insulin Release from Genetically Engineered K Cells, 2000 science <br />
* [http://www.nature.com/gt/journal/v7/n21/abs/3301306a.html Glucose-stimulated and self-limiting insulin production by glucose 6-phosphatase promoter driven insulin expression in hepatoma cells, 2000 Gene Therapy]<br />
[https://2007.igem.org/Taipei Back]</div>Blenthttp://2007.igem.org/wiki/index.php/NYMU_Taipei/ProjectDescriptionNYMU Taipei/ProjectDescription2007-10-26T22:56:46Z<p>Blent: /* Goal and system design */</p>
<hr />
<div><p><font size="4"><br />
[[NYMU_Taipei/ProjectDescription| Project Description]] /<br />
[[NYMU_Taipei/Lab_Notes| Lab Notes]] /<br />
[[NYMU_Taipei/Part_List| Part List]] /<br />
[[NYMU_Taipei/Primer_List| Primer List]]<br />
</font><br /><hr></p><br />
<br />
==<font size=5 color=red>GlucOperon</font>==<br />
* A '''Gluc'''ose-based synthetic '''Operon''' system<br />
* An automatic system in E.coli that operates blood sugar wonderfully!<br />
* Insulin secretion system that promotes quality of life for diabetic patients<br />
----<br />
<br />
==Summary==<br />
* Diabetes mellitus is a significant problem especially in developed countries and leads to several severe long-term complications. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Controlling blood sugar in a reasonable level and avoiding severe emergency as diabetes ketoacidosis (DKA) are very important clinical issues. Thus, the NYMU_Taipei iGEM 2007 team is designing a biological system to sense environmental glucose concentration and decrease the level of glucose by releasing insulin. Besides, life-protection functions for removing toxic ketoacids produced during DKA and preventing hypoglycemia status will also be established. This system will be a convenient and safe design for those patients with diabetes mellitus, and further improve their quality of life by avoiding them from diabetes-related morbidity and mortality. <br />
----<br />
==Motivation==<br />
* Over 7 percent of adults in the United States (US) are known to have diabetes mellitus. However, because of the associated microvascular and macrovascular disease, diabetes accounts for almost 14 percent of US health care expenditures, at least one-half of which are related to complications such as myocardial infarction, stroke, end-stage renal disease, retinopathy, and foot ulcers. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus, one of the most common chronic diseases in childhood, is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Data from the Center of Disease Control (CDC) show that more than 13,000 children and adolescents (19 years of age) are diagnosed with type 1 disease each year with a prevalence of 1.7 per 1000. Control of blood sugar and prevention of severe complications, such;as diabetic ketoacidosis (DKA), are important issues nowadays. <br />
* Diabetic ketoacidosis (DKA) and hyperosmolar hyperglycemic state (HHS, also known as nonketotic hyperglycemia) are two of the most serious acute complications of diabetes. They are part of the spectrum of hyperglycemia and each represents an extreme in the spectrum. DKA is characterized by the triad of hyperglycemia, anion gap metabolic acidosis, and ketonemia. Metabolic acidosis is often the major finding. The serum glucose concentration is usually greater than 500 mg/dL (27.8 mmol/L) and less than 800 mg/dL (44.4 mmol/L). Three ketone bodies are produced in DKA: two ketoacids (beta-hydroxybutyric acid and acetoacetic acid), and one neutral ketone (acetone). The treatment of DKA and HHS is similar, including the administration of insulin and correction of the fluid and electrolyte abnormalities that may be present, including hyperglycemia and hyperosmolality, hypovolemia, metabolic acidosis (in DKA), and potassium depletion. <br />
* Existed works (see References below) are focusing on how to take advantages from properties of mammalian cells to express and secrete insulin in a trail-and-error manner. However, from the perspective of synthetic biology, it is possible to engineer a cell works as we want, no matter what type of cells we apply to. <br />
----<br />
==Goal and System Design==<br />
* For the iGEM 2007 project, our team focuses on the development of a prokaryotic system to <br />
** express insulin regulated by external glucose concentration and <br />
** clean the ketone bodies accumulated by mamalian cells due to insufficient intake of glucose <br />
** balance between glucose and insulin to stablize glucose level and prevent low blood glucose condition <br />
* system specification is shown below: <br />
[[Image:GAI-t curve.jpg|center|500px]]<br />
* System diagram (initial state)<br />
[[Image:IGEM2007 insulin circuit diagram.png|center]]<br />
* System under high glucose condition (insulin generating circuit is activated)<br />
[[Image:High_glucose.png|center]]<br />
* System under transition state (insulin increases glucose uptake; whole system shifts from insulin generation to insulin degradation)<br />
[[Image:Trans_glucose.png|center]]<br />
* System under low glucose condition (when glucose is low enough, the insulin generation turns off and insulin degradation turns on)<br />
[[Image:Low_glucose.png|center]]<br />
* System after insulin degradation (both circuits turn off and wait IDE to be naturally degraded; back to initial state)<br />
[[Image:Post_glucose.png|center]]<br />
* Circuit #1: high glucose-triggered insulin generator <br />
** The basic architecture for our insulin secretion design is shown in the figure below: <br />
** External glucose can be sensed by GSD (Glucose Sensing Domain) from gene RcsC and transmits response to ERD (Responsive Domain from EnvZ gene) <br />
** TCS phosphorylation relay activates the expression of insulin (both alpha and beta chains) <br />
** With signal peptide (SP), the assembled insulin (by disulfide bonding) can be exported outside the membrane of the prokaryotic cells <br />
[[Image:Insulin basic2.png|center|500px]] <br />
* Circuit #2: low glucose and insulin triggered insulin inhibitor/destroyer<br />
[[Image:NYMU_Taipei_insulin_inhibitor_2.png|center|500px]] <br />
[[Image:NYMU_Taipei_insulin_inhibitor.png|center|700px]] <br />
----<br />
<br />
==Implementation==<br />
* [[NYMU_Taipei/Part_List | Part List]]<br />
* [[NYMU_Taipei/Lab_Notes | Lab notes]]<br />
* [[NYMU_Taipei/Protocols| Protocols]]<br />
* [[NYMU_Taipei/Primer_List | Primer List]]<br />
----<br />
==Verification==<br />
===pOmpC characterization by testing circuit #1 pOmpC + EYFP===<br />
* glucose can induce expression pOmpC operon when 1% glucose concentration in the LB medium after 14h culture<br />
[[Image:POmpC-EYFP test1.jpg]]<br />
===OmpRBS characterization by testing circuit #2 pLacIQ + OmpRBS + GFP===<br />
----<br />
<br />
==References==<br />
* Engineered enteroendocrine cells secrete insulin in response to glucose and reverse hyperglycemia in diabetic mice, 2007 Mol Ther. <br />
* Development of genetically engineered human intestinal cells for regulated insulin secretion using rAAV-mediated gene transfer, 2003 Biochem Biophys Res Commun <br />
* http://www.utdol.com/utd/content/topic.do <br />
* Recombinant DNA Technology in the Synthesis of Human Insulin <br />
* Glucose-Dependent Insulin Release from Genetically Engineered K Cells, 2000 science <br />
* [http://www.nature.com/gt/journal/v7/n21/abs/3301306a.html Glucose-stimulated and self-limiting insulin production by glucose 6-phosphatase promoter driven insulin expression in hepatoma cells, 2000 Gene Therapy]<br />
[https://2007.igem.org/Taipei Back]</div>Blenthttp://2007.igem.org/wiki/index.php/NYMU_Taipei/ProjectDescriptionNYMU Taipei/ProjectDescription2007-10-26T22:49:54Z<p>Blent: /* pOmpC characterization by testing circuit pOmpC+EYFP */</p>
<hr />
<div><p><font size="4"><br />
[[NYMU_Taipei/ProjectDescription| Project Description]] /<br />
[[NYMU_Taipei/Lab_Notes| Lab Notes]] /<br />
[[NYMU_Taipei/Part_List| Part List]] /<br />
[[NYMU_Taipei/Primer_List| Primer List]]<br />
</font><br /><hr></p><br />
<br />
==<font size=5 color=red>GlucOperon</font>==<br />
* A '''Gluc'''ose-based synthetic '''Operon''' system<br />
* An automatic system in E.coli that operates blood sugar wonderfully!<br />
* Insulin secretion system that promotes quality of life for diabetic patients<br />
----<br />
<br />
==Summary==<br />
* Diabetes mellitus is a significant problem especially in developed countries and leads to several severe long-term complications. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Controlling blood sugar in a reasonable level and avoiding severe emergency as diabetes ketoacidosis (DKA) are very important clinical issues. Thus, the NYMU_Taipei iGEM 2007 team is designing a biological system to sense environmental glucose concentration and decrease the level of glucose by releasing insulin. Besides, life-protection functions for removing toxic ketoacids produced during DKA and preventing hypoglycemia status will also be established. This system will be a convenient and safe design for those patients with diabetes mellitus, and further improve their quality of life by avoiding them from diabetes-related morbidity and mortality. <br />
----<br />
==Motivation==<br />
* Over 7 percent of adults in the United States (US) are known to have diabetes mellitus. However, because of the associated microvascular and macrovascular disease, diabetes accounts for almost 14 percent of US health care expenditures, at least one-half of which are related to complications such as myocardial infarction, stroke, end-stage renal disease, retinopathy, and foot ulcers. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus, one of the most common chronic diseases in childhood, is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Data from the Center of Disease Control (CDC) show that more than 13,000 children and adolescents (19 years of age) are diagnosed with type 1 disease each year with a prevalence of 1.7 per 1000. Control of blood sugar and prevention of severe complications, such;as diabetic ketoacidosis (DKA), are important issues nowadays. <br />
* Diabetic ketoacidosis (DKA) and hyperosmolar hyperglycemic state (HHS, also known as nonketotic hyperglycemia) are two of the most serious acute complications of diabetes. They are part of the spectrum of hyperglycemia and each represents an extreme in the spectrum. DKA is characterized by the triad of hyperglycemia, anion gap metabolic acidosis, and ketonemia. Metabolic acidosis is often the major finding. The serum glucose concentration is usually greater than 500 mg/dL (27.8 mmol/L) and less than 800 mg/dL (44.4 mmol/L). Three ketone bodies are produced in DKA: two ketoacids (beta-hydroxybutyric acid and acetoacetic acid), and one neutral ketone (acetone). The treatment of DKA and HHS is similar, including the administration of insulin and correction of the fluid and electrolyte abnormalities that may be present, including hyperglycemia and hyperosmolality, hypovolemia, metabolic acidosis (in DKA), and potassium depletion. <br />
* Existed works (see References below) are focusing on how to take advantages from properties of mammalian cells to express and secrete insulin in a trail-and-error manner. However, from the perspective of synthetic biology, it is possible to engineer a cell works as we want, no matter what type of cells we apply to. <br />
----<br />
==Goal and system design==<br />
* For the iGEM 2007 project, our team focuses on the development of a prokaryotic system to <br />
** express insulin regulated by external glucose concentration and <br />
** clean the ketone bodies accumulated by mamalian cells due to insufficient intake of glucose <br />
** balance between glucose and insulin to stablize glucose level and prevent low blood glucose condition <br />
* system specification is shown below: <br />
[[Image:GAI-t curve.jpg|center|500px]]<br />
* System diagram (initial state)<br />
[[Image:IGEM2007 insulin circuit diagram.png|center]]<br />
* System under high glucose condition (insulin generating circuit is activated)<br />
[[Image:High_glucose.png|center]]<br />
* System under transition state (insulin increases glucose uptake; whole system shifts from insulin generation to insulin degradation)<br />
[[Image:Trans_glucose.png|center]]<br />
* System under low glucose condition (when glucose is low enough, the insulin generation turns off and insulin degradation turns on)<br />
[[Image:Low_glucose.png|center]]<br />
* System after insulin degradation (both circuits turn off and wait IDE to be naturally degraded; back to initial state)<br />
[[Image:Post_glucose.png|center]]<br />
* Circuit #1: high glucose-triggered insulin generator <br />
** The basic architecture for our insulin secretion design is shown in the figure below: <br />
** External glucose can be sensed by GSD (Glucose Sensing Domain) from gene RcsC and transmits response to ERD (Responsive Domain from EnvZ gene) <br />
** TCS phosphorylation relay activates the expression of insulin (both alpha and beta chains) <br />
** With signal peptide (SP), the assembled insulin (by disulfide bonding) can be exported outside the membrane of the prokaryotic cells <br />
[[Image:Insulin basic2.png|center|500px]] <br />
* Circuit #2: low glucose and insulin triggered insulin inhibitor/destroyer<br />
[[Image:NYMU_Taipei_insulin_inhibitor_2.png|center|500px]] <br />
[[Image:NYMU_Taipei_insulin_inhibitor.png|center|700px]] <br />
----<br />
<br />
==Implementation==<br />
* [[NYMU_Taipei/Part_List | Part List]]<br />
* [[NYMU_Taipei/Lab_Notes | Lab notes]]<br />
* [[NYMU_Taipei/Protocols| Protocols]]<br />
* [[NYMU_Taipei/Primer_List | Primer List]]<br />
----<br />
==Verification==<br />
===pOmpC characterization by testing circuit #1 pOmpC + EYFP===<br />
* glucose can induce expression pOmpC operon when 1% glucose concentration in the LB medium after 14h culture<br />
[[Image:POmpC-EYFP test1.jpg]]<br />
===OmpRBS characterization by testing circuit #2 pLacIQ + OmpRBS + GFP===<br />
----<br />
<br />
==References==<br />
* Engineered enteroendocrine cells secrete insulin in response to glucose and reverse hyperglycemia in diabetic mice, 2007 Mol Ther. <br />
* Development of genetically engineered human intestinal cells for regulated insulin secretion using rAAV-mediated gene transfer, 2003 Biochem Biophys Res Commun <br />
* http://www.utdol.com/utd/content/topic.do <br />
* Recombinant DNA Technology in the Synthesis of Human Insulin <br />
* Glucose-Dependent Insulin Release from Genetically Engineered K Cells, 2000 science <br />
* [http://www.nature.com/gt/journal/v7/n21/abs/3301306a.html Glucose-stimulated and self-limiting insulin production by glucose 6-phosphatase promoter driven insulin expression in hepatoma cells, 2000 Gene Therapy]<br />
[https://2007.igem.org/Taipei Back]</div>Blenthttp://2007.igem.org/wiki/index.php/NYMU_Taipei/ProjectDescriptionNYMU Taipei/ProjectDescription2007-10-26T22:48:44Z<p>Blent: /* Verification */</p>
<hr />
<div><p><font size="4"><br />
[[NYMU_Taipei/ProjectDescription| Project Description]] /<br />
[[NYMU_Taipei/Lab_Notes| Lab Notes]] /<br />
[[NYMU_Taipei/Part_List| Part List]] /<br />
[[NYMU_Taipei/Primer_List| Primer List]]<br />
</font><br /><hr></p><br />
<br />
==<font size=5 color=red>GlucOperon</font>==<br />
* A '''Gluc'''ose-based synthetic '''Operon''' system<br />
* An automatic system in E.coli that operates blood sugar wonderfully!<br />
* Insulin secretion system that promotes quality of life for diabetic patients<br />
----<br />
<br />
==Summary==<br />
* Diabetes mellitus is a significant problem especially in developed countries and leads to several severe long-term complications. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Controlling blood sugar in a reasonable level and avoiding severe emergency as diabetes ketoacidosis (DKA) are very important clinical issues. Thus, the NYMU_Taipei iGEM 2007 team is designing a biological system to sense environmental glucose concentration and decrease the level of glucose by releasing insulin. Besides, life-protection functions for removing toxic ketoacids produced during DKA and preventing hypoglycemia status will also be established. This system will be a convenient and safe design for those patients with diabetes mellitus, and further improve their quality of life by avoiding them from diabetes-related morbidity and mortality. <br />
----<br />
==Motivation==<br />
* Over 7 percent of adults in the United States (US) are known to have diabetes mellitus. However, because of the associated microvascular and macrovascular disease, diabetes accounts for almost 14 percent of US health care expenditures, at least one-half of which are related to complications such as myocardial infarction, stroke, end-stage renal disease, retinopathy, and foot ulcers. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus, one of the most common chronic diseases in childhood, is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Data from the Center of Disease Control (CDC) show that more than 13,000 children and adolescents (19 years of age) are diagnosed with type 1 disease each year with a prevalence of 1.7 per 1000. Control of blood sugar and prevention of severe complications, such;as diabetic ketoacidosis (DKA), are important issues nowadays. <br />
* Diabetic ketoacidosis (DKA) and hyperosmolar hyperglycemic state (HHS, also known as nonketotic hyperglycemia) are two of the most serious acute complications of diabetes. They are part of the spectrum of hyperglycemia and each represents an extreme in the spectrum. DKA is characterized by the triad of hyperglycemia, anion gap metabolic acidosis, and ketonemia. Metabolic acidosis is often the major finding. The serum glucose concentration is usually greater than 500 mg/dL (27.8 mmol/L) and less than 800 mg/dL (44.4 mmol/L). Three ketone bodies are produced in DKA: two ketoacids (beta-hydroxybutyric acid and acetoacetic acid), and one neutral ketone (acetone). The treatment of DKA and HHS is similar, including the administration of insulin and correction of the fluid and electrolyte abnormalities that may be present, including hyperglycemia and hyperosmolality, hypovolemia, metabolic acidosis (in DKA), and potassium depletion. <br />
* Existed works (see References below) are focusing on how to take advantages from properties of mammalian cells to express and secrete insulin in a trail-and-error manner. However, from the perspective of synthetic biology, it is possible to engineer a cell works as we want, no matter what type of cells we apply to. <br />
----<br />
==Goal and system design==<br />
* For the iGEM 2007 project, our team focuses on the development of a prokaryotic system to <br />
** express insulin regulated by external glucose concentration and <br />
** clean the ketone bodies accumulated by mamalian cells due to insufficient intake of glucose <br />
** balance between glucose and insulin to stablize glucose level and prevent low blood glucose condition <br />
* system specification is shown below: <br />
[[Image:GAI-t curve.jpg|center|500px]]<br />
* System diagram (initial state)<br />
[[Image:IGEM2007 insulin circuit diagram.png|center]]<br />
* System under high glucose condition (insulin generating circuit is activated)<br />
[[Image:High_glucose.png|center]]<br />
* System under transition state (insulin increases glucose uptake; whole system shifts from insulin generation to insulin degradation)<br />
[[Image:Trans_glucose.png|center]]<br />
* System under low glucose condition (when glucose is low enough, the insulin generation turns off and insulin degradation turns on)<br />
[[Image:Low_glucose.png|center]]<br />
* System after insulin degradation (both circuits turn off and wait IDE to be naturally degraded; back to initial state)<br />
[[Image:Post_glucose.png|center]]<br />
* Circuit #1: high glucose-triggered insulin generator <br />
** The basic architecture for our insulin secretion design is shown in the figure below: <br />
** External glucose can be sensed by GSD (Glucose Sensing Domain) from gene RcsC and transmits response to ERD (Responsive Domain from EnvZ gene) <br />
** TCS phosphorylation relay activates the expression of insulin (both alpha and beta chains) <br />
** With signal peptide (SP), the assembled insulin (by disulfide bonding) can be exported outside the membrane of the prokaryotic cells <br />
[[Image:Insulin basic2.png|center|500px]] <br />
* Circuit #2: low glucose and insulin triggered insulin inhibitor/destroyer<br />
[[Image:NYMU_Taipei_insulin_inhibitor_2.png|center|500px]] <br />
[[Image:NYMU_Taipei_insulin_inhibitor.png|center|700px]] <br />
----<br />
<br />
==Implementation==<br />
* [[NYMU_Taipei/Part_List | Part List]]<br />
* [[NYMU_Taipei/Lab_Notes | Lab notes]]<br />
* [[NYMU_Taipei/Protocols| Protocols]]<br />
* [[NYMU_Taipei/Primer_List | Primer List]]<br />
----<br />
==Verification==<br />
===pOmpC characterization by testing circuit pOmpC+EYFP===<br />
* glucose can induce expression pOmpC operon when 1% glucose concentration in the LB medium after 14h culture<br />
[[Image:POmpC-EYFP test1.jpg]]<br />
----<br />
<br />
==References==<br />
* Engineered enteroendocrine cells secrete insulin in response to glucose and reverse hyperglycemia in diabetic mice, 2007 Mol Ther. <br />
* Development of genetically engineered human intestinal cells for regulated insulin secretion using rAAV-mediated gene transfer, 2003 Biochem Biophys Res Commun <br />
* http://www.utdol.com/utd/content/topic.do <br />
* Recombinant DNA Technology in the Synthesis of Human Insulin <br />
* Glucose-Dependent Insulin Release from Genetically Engineered K Cells, 2000 science <br />
* [http://www.nature.com/gt/journal/v7/n21/abs/3301306a.html Glucose-stimulated and self-limiting insulin production by glucose 6-phosphatase promoter driven insulin expression in hepatoma cells, 2000 Gene Therapy]<br />
[https://2007.igem.org/Taipei Back]</div>Blenthttp://2007.igem.org/wiki/index.php/File:POmpC-EYFP_test1.jpgFile:POmpC-EYFP test1.jpg2007-10-26T22:46:50Z<p>Blent: </p>
<hr />
<div></div>Blenthttp://2007.igem.org/wiki/index.php/NYMU_Taipei/ProjectDescriptionNYMU Taipei/ProjectDescription2007-10-26T22:44:58Z<p>Blent: /* Verification */</p>
<hr />
<div><p><font size="4"><br />
[[NYMU_Taipei/ProjectDescription| Project Description]] /<br />
[[NYMU_Taipei/Lab_Notes| Lab Notes]] /<br />
[[NYMU_Taipei/Part_List| Part List]] /<br />
[[NYMU_Taipei/Primer_List| Primer List]]<br />
</font><br /><hr></p><br />
<br />
==<font size=5 color=red>GlucOperon</font>==<br />
* A '''Gluc'''ose-based synthetic '''Operon''' system<br />
* An automatic system in E.coli that operates blood sugar wonderfully!<br />
* Insulin secretion system that promotes quality of life for diabetic patients<br />
----<br />
<br />
==Summary==<br />
* Diabetes mellitus is a significant problem especially in developed countries and leads to several severe long-term complications. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Controlling blood sugar in a reasonable level and avoiding severe emergency as diabetes ketoacidosis (DKA) are very important clinical issues. Thus, the NYMU_Taipei iGEM 2007 team is designing a biological system to sense environmental glucose concentration and decrease the level of glucose by releasing insulin. Besides, life-protection functions for removing toxic ketoacids produced during DKA and preventing hypoglycemia status will also be established. This system will be a convenient and safe design for those patients with diabetes mellitus, and further improve their quality of life by avoiding them from diabetes-related morbidity and mortality. <br />
----<br />
==Motivation==<br />
* Over 7 percent of adults in the United States (US) are known to have diabetes mellitus. However, because of the associated microvascular and macrovascular disease, diabetes accounts for almost 14 percent of US health care expenditures, at least one-half of which are related to complications such as myocardial infarction, stroke, end-stage renal disease, retinopathy, and foot ulcers. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus, one of the most common chronic diseases in childhood, is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Data from the Center of Disease Control (CDC) show that more than 13,000 children and adolescents (19 years of age) are diagnosed with type 1 disease each year with a prevalence of 1.7 per 1000. Control of blood sugar and prevention of severe complications, such;as diabetic ketoacidosis (DKA), are important issues nowadays. <br />
* Diabetic ketoacidosis (DKA) and hyperosmolar hyperglycemic state (HHS, also known as nonketotic hyperglycemia) are two of the most serious acute complications of diabetes. They are part of the spectrum of hyperglycemia and each represents an extreme in the spectrum. DKA is characterized by the triad of hyperglycemia, anion gap metabolic acidosis, and ketonemia. Metabolic acidosis is often the major finding. The serum glucose concentration is usually greater than 500 mg/dL (27.8 mmol/L) and less than 800 mg/dL (44.4 mmol/L). Three ketone bodies are produced in DKA: two ketoacids (beta-hydroxybutyric acid and acetoacetic acid), and one neutral ketone (acetone). The treatment of DKA and HHS is similar, including the administration of insulin and correction of the fluid and electrolyte abnormalities that may be present, including hyperglycemia and hyperosmolality, hypovolemia, metabolic acidosis (in DKA), and potassium depletion. <br />
* Existed works (see References below) are focusing on how to take advantages from properties of mammalian cells to express and secrete insulin in a trail-and-error manner. However, from the perspective of synthetic biology, it is possible to engineer a cell works as we want, no matter what type of cells we apply to. <br />
----<br />
==Goal and system design==<br />
* For the iGEM 2007 project, our team focuses on the development of a prokaryotic system to <br />
** express insulin regulated by external glucose concentration and <br />
** clean the ketone bodies accumulated by mamalian cells due to insufficient intake of glucose <br />
** balance between glucose and insulin to stablize glucose level and prevent low blood glucose condition <br />
* system specification is shown below: <br />
[[Image:GAI-t curve.jpg|center|500px]]<br />
* System diagram (initial state)<br />
[[Image:IGEM2007 insulin circuit diagram.png|center]]<br />
* System under high glucose condition (insulin generating circuit is activated)<br />
[[Image:High_glucose.png|center]]<br />
* System under transition state (insulin increases glucose uptake; whole system shifts from insulin generation to insulin degradation)<br />
[[Image:Trans_glucose.png|center]]<br />
* System under low glucose condition (when glucose is low enough, the insulin generation turns off and insulin degradation turns on)<br />
[[Image:Low_glucose.png|center]]<br />
* System after insulin degradation (both circuits turn off and wait IDE to be naturally degraded; back to initial state)<br />
[[Image:Post_glucose.png|center]]<br />
* Circuit #1: high glucose-triggered insulin generator <br />
** The basic architecture for our insulin secretion design is shown in the figure below: <br />
** External glucose can be sensed by GSD (Glucose Sensing Domain) from gene RcsC and transmits response to ERD (Responsive Domain from EnvZ gene) <br />
** TCS phosphorylation relay activates the expression of insulin (both alpha and beta chains) <br />
** With signal peptide (SP), the assembled insulin (by disulfide bonding) can be exported outside the membrane of the prokaryotic cells <br />
[[Image:Insulin basic2.png|center|500px]] <br />
* Circuit #2: low glucose and insulin triggered insulin inhibitor/destroyer<br />
[[Image:NYMU_Taipei_insulin_inhibitor_2.png|center|500px]] <br />
[[Image:NYMU_Taipei_insulin_inhibitor.png|center|700px]] <br />
----<br />
<br />
==Implementation==<br />
* [[NYMU_Taipei/Part_List | Part List]]<br />
* [[NYMU_Taipei/Lab_Notes | Lab notes]]<br />
* [[NYMU_Taipei/Protocols| Protocols]]<br />
* [[NYMU_Taipei/Primer_List | Primer List]]<br />
----<br />
==Verification==<br />
* pOmpC characterization by testing circuit pOmpC+EYFP<br />
----<br />
<br />
==References==<br />
* Engineered enteroendocrine cells secrete insulin in response to glucose and reverse hyperglycemia in diabetic mice, 2007 Mol Ther. <br />
* Development of genetically engineered human intestinal cells for regulated insulin secretion using rAAV-mediated gene transfer, 2003 Biochem Biophys Res Commun <br />
* http://www.utdol.com/utd/content/topic.do <br />
* Recombinant DNA Technology in the Synthesis of Human Insulin <br />
* Glucose-Dependent Insulin Release from Genetically Engineered K Cells, 2000 science <br />
* [http://www.nature.com/gt/journal/v7/n21/abs/3301306a.html Glucose-stimulated and self-limiting insulin production by glucose 6-phosphatase promoter driven insulin expression in hepatoma cells, 2000 Gene Therapy]<br />
[https://2007.igem.org/Taipei Back]</div>Blenthttp://2007.igem.org/wiki/index.php/NYMU_Taipei/ProjectDescriptionNYMU Taipei/ProjectDescription2007-10-26T22:41:44Z<p>Blent: /* Implementation */</p>
<hr />
<div><p><font size="4"><br />
[[NYMU_Taipei/ProjectDescription| Project Description]] /<br />
[[NYMU_Taipei/Lab_Notes| Lab Notes]] /<br />
[[NYMU_Taipei/Part_List| Part List]] /<br />
[[NYMU_Taipei/Primer_List| Primer List]]<br />
</font><br /><hr></p><br />
<br />
==<font size=5 color=red>GlucOperon</font>==<br />
* A '''Gluc'''ose-based synthetic '''Operon''' system<br />
* An automatic system in E.coli that operates blood sugar wonderfully!<br />
* Insulin secretion system that promotes quality of life for diabetic patients<br />
----<br />
<br />
==Summary==<br />
* Diabetes mellitus is a significant problem especially in developed countries and leads to several severe long-term complications. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Controlling blood sugar in a reasonable level and avoiding severe emergency as diabetes ketoacidosis (DKA) are very important clinical issues. Thus, the NYMU_Taipei iGEM 2007 team is designing a biological system to sense environmental glucose concentration and decrease the level of glucose by releasing insulin. Besides, life-protection functions for removing toxic ketoacids produced during DKA and preventing hypoglycemia status will also be established. This system will be a convenient and safe design for those patients with diabetes mellitus, and further improve their quality of life by avoiding them from diabetes-related morbidity and mortality. <br />
----<br />
==Motivation==<br />
* Over 7 percent of adults in the United States (US) are known to have diabetes mellitus. However, because of the associated microvascular and macrovascular disease, diabetes accounts for almost 14 percent of US health care expenditures, at least one-half of which are related to complications such as myocardial infarction, stroke, end-stage renal disease, retinopathy, and foot ulcers. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus, one of the most common chronic diseases in childhood, is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Data from the Center of Disease Control (CDC) show that more than 13,000 children and adolescents (19 years of age) are diagnosed with type 1 disease each year with a prevalence of 1.7 per 1000. Control of blood sugar and prevention of severe complications, such;as diabetic ketoacidosis (DKA), are important issues nowadays. <br />
* Diabetic ketoacidosis (DKA) and hyperosmolar hyperglycemic state (HHS, also known as nonketotic hyperglycemia) are two of the most serious acute complications of diabetes. They are part of the spectrum of hyperglycemia and each represents an extreme in the spectrum. DKA is characterized by the triad of hyperglycemia, anion gap metabolic acidosis, and ketonemia. Metabolic acidosis is often the major finding. The serum glucose concentration is usually greater than 500 mg/dL (27.8 mmol/L) and less than 800 mg/dL (44.4 mmol/L). Three ketone bodies are produced in DKA: two ketoacids (beta-hydroxybutyric acid and acetoacetic acid), and one neutral ketone (acetone). The treatment of DKA and HHS is similar, including the administration of insulin and correction of the fluid and electrolyte abnormalities that may be present, including hyperglycemia and hyperosmolality, hypovolemia, metabolic acidosis (in DKA), and potassium depletion. <br />
* Existed works (see References below) are focusing on how to take advantages from properties of mammalian cells to express and secrete insulin in a trail-and-error manner. However, from the perspective of synthetic biology, it is possible to engineer a cell works as we want, no matter what type of cells we apply to. <br />
----<br />
==Goal and system design==<br />
* For the iGEM 2007 project, our team focuses on the development of a prokaryotic system to <br />
** express insulin regulated by external glucose concentration and <br />
** clean the ketone bodies accumulated by mamalian cells due to insufficient intake of glucose <br />
** balance between glucose and insulin to stablize glucose level and prevent low blood glucose condition <br />
* system specification is shown below: <br />
[[Image:GAI-t curve.jpg|center|500px]]<br />
* System diagram (initial state)<br />
[[Image:IGEM2007 insulin circuit diagram.png|center]]<br />
* System under high glucose condition (insulin generating circuit is activated)<br />
[[Image:High_glucose.png|center]]<br />
* System under transition state (insulin increases glucose uptake; whole system shifts from insulin generation to insulin degradation)<br />
[[Image:Trans_glucose.png|center]]<br />
* System under low glucose condition (when glucose is low enough, the insulin generation turns off and insulin degradation turns on)<br />
[[Image:Low_glucose.png|center]]<br />
* System after insulin degradation (both circuits turn off and wait IDE to be naturally degraded; back to initial state)<br />
[[Image:Post_glucose.png|center]]<br />
* Circuit #1: high glucose-triggered insulin generator <br />
** The basic architecture for our insulin secretion design is shown in the figure below: <br />
** External glucose can be sensed by GSD (Glucose Sensing Domain) from gene RcsC and transmits response to ERD (Responsive Domain from EnvZ gene) <br />
** TCS phosphorylation relay activates the expression of insulin (both alpha and beta chains) <br />
** With signal peptide (SP), the assembled insulin (by disulfide bonding) can be exported outside the membrane of the prokaryotic cells <br />
[[Image:Insulin basic2.png|center|500px]] <br />
* Circuit #2: low glucose and insulin triggered insulin inhibitor/destroyer<br />
[[Image:NYMU_Taipei_insulin_inhibitor_2.png|center|500px]] <br />
[[Image:NYMU_Taipei_insulin_inhibitor.png|center|700px]] <br />
----<br />
<br />
==Implementation==<br />
* [[NYMU_Taipei/Part_List | Part List]]<br />
* [[NYMU_Taipei/Lab_Notes | Lab notes]]<br />
* [[NYMU_Taipei/Protocols| Protocols]]<br />
* [[NYMU_Taipei/Primer_List | Primer List]]<br />
----<br />
==Verification==<br />
----<br />
<br />
==References==<br />
* Engineered enteroendocrine cells secrete insulin in response to glucose and reverse hyperglycemia in diabetic mice, 2007 Mol Ther. <br />
* Development of genetically engineered human intestinal cells for regulated insulin secretion using rAAV-mediated gene transfer, 2003 Biochem Biophys Res Commun <br />
* http://www.utdol.com/utd/content/topic.do <br />
* Recombinant DNA Technology in the Synthesis of Human Insulin <br />
* Glucose-Dependent Insulin Release from Genetically Engineered K Cells, 2000 science <br />
* [http://www.nature.com/gt/journal/v7/n21/abs/3301306a.html Glucose-stimulated and self-limiting insulin production by glucose 6-phosphatase promoter driven insulin expression in hepatoma cells, 2000 Gene Therapy]<br />
[https://2007.igem.org/Taipei Back]</div>Blenthttp://2007.igem.org/wiki/index.php/NYMU_Taipei/ProjectDescriptionNYMU Taipei/ProjectDescription2007-10-26T22:41:14Z<p>Blent: /* Goal and system design */</p>
<hr />
<div><p><font size="4"><br />
[[NYMU_Taipei/ProjectDescription| Project Description]] /<br />
[[NYMU_Taipei/Lab_Notes| Lab Notes]] /<br />
[[NYMU_Taipei/Part_List| Part List]] /<br />
[[NYMU_Taipei/Primer_List| Primer List]]<br />
</font><br /><hr></p><br />
<br />
==<font size=5 color=red>GlucOperon</font>==<br />
* A '''Gluc'''ose-based synthetic '''Operon''' system<br />
* An automatic system in E.coli that operates blood sugar wonderfully!<br />
* Insulin secretion system that promotes quality of life for diabetic patients<br />
----<br />
<br />
==Summary==<br />
* Diabetes mellitus is a significant problem especially in developed countries and leads to several severe long-term complications. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Controlling blood sugar in a reasonable level and avoiding severe emergency as diabetes ketoacidosis (DKA) are very important clinical issues. Thus, the NYMU_Taipei iGEM 2007 team is designing a biological system to sense environmental glucose concentration and decrease the level of glucose by releasing insulin. Besides, life-protection functions for removing toxic ketoacids produced during DKA and preventing hypoglycemia status will also be established. This system will be a convenient and safe design for those patients with diabetes mellitus, and further improve their quality of life by avoiding them from diabetes-related morbidity and mortality. <br />
----<br />
==Motivation==<br />
* Over 7 percent of adults in the United States (US) are known to have diabetes mellitus. However, because of the associated microvascular and macrovascular disease, diabetes accounts for almost 14 percent of US health care expenditures, at least one-half of which are related to complications such as myocardial infarction, stroke, end-stage renal disease, retinopathy, and foot ulcers. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus, one of the most common chronic diseases in childhood, is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Data from the Center of Disease Control (CDC) show that more than 13,000 children and adolescents (19 years of age) are diagnosed with type 1 disease each year with a prevalence of 1.7 per 1000. Control of blood sugar and prevention of severe complications, such;as diabetic ketoacidosis (DKA), are important issues nowadays. <br />
* Diabetic ketoacidosis (DKA) and hyperosmolar hyperglycemic state (HHS, also known as nonketotic hyperglycemia) are two of the most serious acute complications of diabetes. They are part of the spectrum of hyperglycemia and each represents an extreme in the spectrum. DKA is characterized by the triad of hyperglycemia, anion gap metabolic acidosis, and ketonemia. Metabolic acidosis is often the major finding. The serum glucose concentration is usually greater than 500 mg/dL (27.8 mmol/L) and less than 800 mg/dL (44.4 mmol/L). Three ketone bodies are produced in DKA: two ketoacids (beta-hydroxybutyric acid and acetoacetic acid), and one neutral ketone (acetone). The treatment of DKA and HHS is similar, including the administration of insulin and correction of the fluid and electrolyte abnormalities that may be present, including hyperglycemia and hyperosmolality, hypovolemia, metabolic acidosis (in DKA), and potassium depletion. <br />
* Existed works (see References below) are focusing on how to take advantages from properties of mammalian cells to express and secrete insulin in a trail-and-error manner. However, from the perspective of synthetic biology, it is possible to engineer a cell works as we want, no matter what type of cells we apply to. <br />
----<br />
==Goal and system design==<br />
* For the iGEM 2007 project, our team focuses on the development of a prokaryotic system to <br />
** express insulin regulated by external glucose concentration and <br />
** clean the ketone bodies accumulated by mamalian cells due to insufficient intake of glucose <br />
** balance between glucose and insulin to stablize glucose level and prevent low blood glucose condition <br />
* system specification is shown below: <br />
[[Image:GAI-t curve.jpg|center|500px]]<br />
* System diagram (initial state)<br />
[[Image:IGEM2007 insulin circuit diagram.png|center]]<br />
* System under high glucose condition (insulin generating circuit is activated)<br />
[[Image:High_glucose.png|center]]<br />
* System under transition state (insulin increases glucose uptake; whole system shifts from insulin generation to insulin degradation)<br />
[[Image:Trans_glucose.png|center]]<br />
* System under low glucose condition (when glucose is low enough, the insulin generation turns off and insulin degradation turns on)<br />
[[Image:Low_glucose.png|center]]<br />
* System after insulin degradation (both circuits turn off and wait IDE to be naturally degraded; back to initial state)<br />
[[Image:Post_glucose.png|center]]<br />
* Circuit #1: high glucose-triggered insulin generator <br />
** The basic architecture for our insulin secretion design is shown in the figure below: <br />
** External glucose can be sensed by GSD (Glucose Sensing Domain) from gene RcsC and transmits response to ERD (Responsive Domain from EnvZ gene) <br />
** TCS phosphorylation relay activates the expression of insulin (both alpha and beta chains) <br />
** With signal peptide (SP), the assembled insulin (by disulfide bonding) can be exported outside the membrane of the prokaryotic cells <br />
[[Image:Insulin basic2.png|center|500px]] <br />
* Circuit #2: low glucose and insulin triggered insulin inhibitor/destroyer<br />
[[Image:NYMU_Taipei_insulin_inhibitor_2.png|center|500px]] <br />
[[Image:NYMU_Taipei_insulin_inhibitor.png|center|700px]] <br />
----<br />
<br />
==Implementation==<br />
* [[NYMU_Taipei/Part_List | Part List]]<br />
* [[NYMU_Taipei/Lab_Notes | Lab notes]]<br />
* [[NYMU_Taipei/Protocols| Protocols]]<br />
* [[NYMU_Taipei/Primer_List | Primer List]]<br />
----<br />
<br />
==References==<br />
* Engineered enteroendocrine cells secrete insulin in response to glucose and reverse hyperglycemia in diabetic mice, 2007 Mol Ther. <br />
* Development of genetically engineered human intestinal cells for regulated insulin secretion using rAAV-mediated gene transfer, 2003 Biochem Biophys Res Commun <br />
* http://www.utdol.com/utd/content/topic.do <br />
* Recombinant DNA Technology in the Synthesis of Human Insulin <br />
* Glucose-Dependent Insulin Release from Genetically Engineered K Cells, 2000 science <br />
* [http://www.nature.com/gt/journal/v7/n21/abs/3301306a.html Glucose-stimulated and self-limiting insulin production by glucose 6-phosphatase promoter driven insulin expression in hepatoma cells, 2000 Gene Therapy]<br />
[https://2007.igem.org/Taipei Back]</div>Blenthttp://2007.igem.org/wiki/index.php/NYMU_Taipei/ProjectDescriptionNYMU Taipei/ProjectDescription2007-10-26T22:39:39Z<p>Blent: /* Goal and system design */</p>
<hr />
<div><p><font size="4"><br />
[[NYMU_Taipei/ProjectDescription| Project Description]] /<br />
[[NYMU_Taipei/Lab_Notes| Lab Notes]] /<br />
[[NYMU_Taipei/Part_List| Part List]] /<br />
[[NYMU_Taipei/Primer_List| Primer List]]<br />
</font><br /><hr></p><br />
<br />
==<font size=5 color=red>GlucOperon</font>==<br />
* A '''Gluc'''ose-based synthetic '''Operon''' system<br />
* An automatic system in E.coli that operates blood sugar wonderfully!<br />
* Insulin secretion system that promotes quality of life for diabetic patients<br />
----<br />
<br />
==Summary==<br />
* Diabetes mellitus is a significant problem especially in developed countries and leads to several severe long-term complications. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Controlling blood sugar in a reasonable level and avoiding severe emergency as diabetes ketoacidosis (DKA) are very important clinical issues. Thus, the NYMU_Taipei iGEM 2007 team is designing a biological system to sense environmental glucose concentration and decrease the level of glucose by releasing insulin. Besides, life-protection functions for removing toxic ketoacids produced during DKA and preventing hypoglycemia status will also be established. This system will be a convenient and safe design for those patients with diabetes mellitus, and further improve their quality of life by avoiding them from diabetes-related morbidity and mortality. <br />
----<br />
==Motivation==<br />
* Over 7 percent of adults in the United States (US) are known to have diabetes mellitus. However, because of the associated microvascular and macrovascular disease, diabetes accounts for almost 14 percent of US health care expenditures, at least one-half of which are related to complications such as myocardial infarction, stroke, end-stage renal disease, retinopathy, and foot ulcers. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus, one of the most common chronic diseases in childhood, is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Data from the Center of Disease Control (CDC) show that more than 13,000 children and adolescents (19 years of age) are diagnosed with type 1 disease each year with a prevalence of 1.7 per 1000. Control of blood sugar and prevention of severe complications, such;as diabetic ketoacidosis (DKA), are important issues nowadays. <br />
* Diabetic ketoacidosis (DKA) and hyperosmolar hyperglycemic state (HHS, also known as nonketotic hyperglycemia) are two of the most serious acute complications of diabetes. They are part of the spectrum of hyperglycemia and each represents an extreme in the spectrum. DKA is characterized by the triad of hyperglycemia, anion gap metabolic acidosis, and ketonemia. Metabolic acidosis is often the major finding. The serum glucose concentration is usually greater than 500 mg/dL (27.8 mmol/L) and less than 800 mg/dL (44.4 mmol/L). Three ketone bodies are produced in DKA: two ketoacids (beta-hydroxybutyric acid and acetoacetic acid), and one neutral ketone (acetone). The treatment of DKA and HHS is similar, including the administration of insulin and correction of the fluid and electrolyte abnormalities that may be present, including hyperglycemia and hyperosmolality, hypovolemia, metabolic acidosis (in DKA), and potassium depletion. <br />
* Existed works (see References below) are focusing on how to take advantages from properties of mammalian cells to express and secrete insulin in a trail-and-error manner. However, from the perspective of synthetic biology, it is possible to engineer a cell works as we want, no matter what type of cells we apply to. <br />
----<br />
==Goal and system design==<br />
* For the iGEM 2007 project, our team focuses on the development of a prokaryotic system to <br />
** express insulin regulated by external glucose concentration and <br />
** clean the ketone bodies accumulated by mamalian cells due to insufficient intake of glucose <br />
** balance between glucose and insulin to stablize glucose level and prevent low blood glucose condition <br />
* system specification is shown below: <br />
[[Image:GAI-t curve.jpg|center|500px]]<br />
* Circuit diagram (initial state)<br />
[[Image:IGEM2007 insulin circuit diagram.png|center]]<br />
* Circuit under high glucose condition (insulin generating device is activated)<br />
[[Image:High_glucose.png|center]]<br />
* Circuit under transition state (insulin increases glucose uptake; whole system shifts from insulin generation to insulin degradation)<br />
[[Image:Trans_glucose.png|center]]<br />
* Circuit under low glucose condition (when glucose is low enough, the insulin generation turns off and insulin degradation turns on)<br />
[[Image:Low_glucose.png|center]]<br />
* Circuit after insulin degradation (both circuits turn off and wait IDE to be naturally degraded; back to initial state)<br />
[[Image:Post_glucose.png|center]]<br />
* Sub-circuit #1: high glucose-triggered insulin generator <br />
** The basic architecture for our insulin secretion design is shown in the figure below: <br />
** External glucose can be sensed by GSD (Glucose Sensing Domain) from gene RcsC and transmits response to ERD (Responsive Domain from EnvZ gene) <br />
** TCS phosphorylation relay activates the expression of insulin (both alpha and beta chains) <br />
** With signal peptide (SP), the assembled insulin (by disulfide bonding) can be exported outside the membrane of the prokaryotic cells <br />
[[Image:Insulin basic2.png|center|500px]] <br />
* Sub-circuit #2: low glucose and insulin triggered insulin inhibitor/destroyer<br />
[[Image:NYMU_Taipei_insulin_inhibitor_2.png|center|500px]] <br />
[[Image:NYMU_Taipei_insulin_inhibitor.png|center|700px]] <br />
----<br />
<br />
==Implementation==<br />
* [[NYMU_Taipei/Part_List | Part List]]<br />
* [[NYMU_Taipei/Lab_Notes | Lab notes]]<br />
* [[NYMU_Taipei/Protocols| Protocols]]<br />
* [[NYMU_Taipei/Primer_List | Primer List]]<br />
----<br />
<br />
==References==<br />
* Engineered enteroendocrine cells secrete insulin in response to glucose and reverse hyperglycemia in diabetic mice, 2007 Mol Ther. <br />
* Development of genetically engineered human intestinal cells for regulated insulin secretion using rAAV-mediated gene transfer, 2003 Biochem Biophys Res Commun <br />
* http://www.utdol.com/utd/content/topic.do <br />
* Recombinant DNA Technology in the Synthesis of Human Insulin <br />
* Glucose-Dependent Insulin Release from Genetically Engineered K Cells, 2000 science <br />
* [http://www.nature.com/gt/journal/v7/n21/abs/3301306a.html Glucose-stimulated and self-limiting insulin production by glucose 6-phosphatase promoter driven insulin expression in hepatoma cells, 2000 Gene Therapy]<br />
[https://2007.igem.org/Taipei Back]</div>Blenthttp://2007.igem.org/wiki/index.php/File:Post_glucose.pngFile:Post glucose.png2007-10-26T22:38:03Z<p>Blent: </p>
<hr />
<div></div>Blenthttp://2007.igem.org/wiki/index.php/NYMU_Taipei/ProjectDescriptionNYMU Taipei/ProjectDescription2007-10-26T22:36:31Z<p>Blent: /* Goal and system design */</p>
<hr />
<div><p><font size="4"><br />
[[NYMU_Taipei/ProjectDescription| Project Description]] /<br />
[[NYMU_Taipei/Lab_Notes| Lab Notes]] /<br />
[[NYMU_Taipei/Part_List| Part List]] /<br />
[[NYMU_Taipei/Primer_List| Primer List]]<br />
</font><br /><hr></p><br />
<br />
==<font size=5 color=red>GlucOperon</font>==<br />
* A '''Gluc'''ose-based synthetic '''Operon''' system<br />
* An automatic system in E.coli that operates blood sugar wonderfully!<br />
* Insulin secretion system that promotes quality of life for diabetic patients<br />
----<br />
<br />
==Summary==<br />
* Diabetes mellitus is a significant problem especially in developed countries and leads to several severe long-term complications. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Controlling blood sugar in a reasonable level and avoiding severe emergency as diabetes ketoacidosis (DKA) are very important clinical issues. Thus, the NYMU_Taipei iGEM 2007 team is designing a biological system to sense environmental glucose concentration and decrease the level of glucose by releasing insulin. Besides, life-protection functions for removing toxic ketoacids produced during DKA and preventing hypoglycemia status will also be established. This system will be a convenient and safe design for those patients with diabetes mellitus, and further improve their quality of life by avoiding them from diabetes-related morbidity and mortality. <br />
----<br />
==Motivation==<br />
* Over 7 percent of adults in the United States (US) are known to have diabetes mellitus. However, because of the associated microvascular and macrovascular disease, diabetes accounts for almost 14 percent of US health care expenditures, at least one-half of which are related to complications such as myocardial infarction, stroke, end-stage renal disease, retinopathy, and foot ulcers. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus, one of the most common chronic diseases in childhood, is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Data from the Center of Disease Control (CDC) show that more than 13,000 children and adolescents (19 years of age) are diagnosed with type 1 disease each year with a prevalence of 1.7 per 1000. Control of blood sugar and prevention of severe complications, such;as diabetic ketoacidosis (DKA), are important issues nowadays. <br />
* Diabetic ketoacidosis (DKA) and hyperosmolar hyperglycemic state (HHS, also known as nonketotic hyperglycemia) are two of the most serious acute complications of diabetes. They are part of the spectrum of hyperglycemia and each represents an extreme in the spectrum. DKA is characterized by the triad of hyperglycemia, anion gap metabolic acidosis, and ketonemia. Metabolic acidosis is often the major finding. The serum glucose concentration is usually greater than 500 mg/dL (27.8 mmol/L) and less than 800 mg/dL (44.4 mmol/L). Three ketone bodies are produced in DKA: two ketoacids (beta-hydroxybutyric acid and acetoacetic acid), and one neutral ketone (acetone). The treatment of DKA and HHS is similar, including the administration of insulin and correction of the fluid and electrolyte abnormalities that may be present, including hyperglycemia and hyperosmolality, hypovolemia, metabolic acidosis (in DKA), and potassium depletion. <br />
* Existed works (see References below) are focusing on how to take advantages from properties of mammalian cells to express and secrete insulin in a trail-and-error manner. However, from the perspective of synthetic biology, it is possible to engineer a cell works as we want, no matter what type of cells we apply to. <br />
----<br />
==Goal and system design==<br />
* For the iGEM 2007 project, our team focuses on the development of a prokaryotic system to <br />
** express insulin regulated by external glucose concentration and <br />
** clean the ketone bodies accumulated by mamalian cells due to insufficient intake of glucose <br />
** balance between glucose and insulin to stablize glucose level and prevent low blood glucose condition <br />
* system specification is shown below: <br />
[[Image:GAI-t curve.jpg|center|500px]]<br />
* Circuit diagram (initial state)<br />
[[Image:IGEM2007 insulin circuit diagram.png|center]]<br />
* Circuit under high glucose condition (insulin generating device is activated)<br />
[[Image:High_glucose.png|center]]<br />
* Circuit under transition state (insulin increases glucose uptake; whole system shifts from insulin generation to insulin degradation)<br />
[[Image:Trans_glucose.png|center]]<br />
* Circuit under low glucose condition (when glucose is low enough, the insulin generation turns off and insulin degradation turns on)<br />
[[Image:Low_glucose.png|center]]<br />
* Circuit after insulin degradation<br />
[[Image:Post_glucose.png|center]]<br />
* Sub-circuit #1: high glucose-triggered insulin generator <br />
** The basic architecture for our insulin secretion design is shown in the figure below: <br />
** External glucose can be sensed by GSD (Glucose Sensing Domain) from gene RcsC and transmits response to ERD (Responsive Domain from EnvZ gene) <br />
** TCS phosphorylation relay activates the expression of insulin (both alpha and beta chains) <br />
** With signal peptide (SP), the assembled insulin (by disulfide bonding) can be exported outside the membrane of the prokaryotic cells <br />
[[Image:Insulin basic2.png|center|500px]] <br />
* Sub-circuit #2: low glucose and insulin triggered insulin inhibitor/destroyer<br />
[[Image:NYMU_Taipei_insulin_inhibitor_2.png|center|500px]] <br />
[[Image:NYMU_Taipei_insulin_inhibitor.png|center|700px]] <br />
----<br />
<br />
==Implementation==<br />
* [[NYMU_Taipei/Part_List | Part List]]<br />
* [[NYMU_Taipei/Lab_Notes | Lab notes]]<br />
* [[NYMU_Taipei/Protocols| Protocols]]<br />
* [[NYMU_Taipei/Primer_List | Primer List]]<br />
----<br />
<br />
==References==<br />
* Engineered enteroendocrine cells secrete insulin in response to glucose and reverse hyperglycemia in diabetic mice, 2007 Mol Ther. <br />
* Development of genetically engineered human intestinal cells for regulated insulin secretion using rAAV-mediated gene transfer, 2003 Biochem Biophys Res Commun <br />
* http://www.utdol.com/utd/content/topic.do <br />
* Recombinant DNA Technology in the Synthesis of Human Insulin <br />
* Glucose-Dependent Insulin Release from Genetically Engineered K Cells, 2000 science <br />
* [http://www.nature.com/gt/journal/v7/n21/abs/3301306a.html Glucose-stimulated and self-limiting insulin production by glucose 6-phosphatase promoter driven insulin expression in hepatoma cells, 2000 Gene Therapy]<br />
[https://2007.igem.org/Taipei Back]</div>Blenthttp://2007.igem.org/wiki/index.php/NYMU_Taipei/ProjectDescriptionNYMU Taipei/ProjectDescription2007-10-26T22:29:31Z<p>Blent: /* Goal and system design */</p>
<hr />
<div><p><font size="4"><br />
[[NYMU_Taipei/ProjectDescription| Project Description]] /<br />
[[NYMU_Taipei/Lab_Notes| Lab Notes]] /<br />
[[NYMU_Taipei/Part_List| Part List]] /<br />
[[NYMU_Taipei/Primer_List| Primer List]]<br />
</font><br /><hr></p><br />
<br />
==<font size=5 color=red>GlucOperon</font>==<br />
* A '''Gluc'''ose-based synthetic '''Operon''' system<br />
* An automatic system in E.coli that operates blood sugar wonderfully!<br />
* Insulin secretion system that promotes quality of life for diabetic patients<br />
----<br />
<br />
==Summary==<br />
* Diabetes mellitus is a significant problem especially in developed countries and leads to several severe long-term complications. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Controlling blood sugar in a reasonable level and avoiding severe emergency as diabetes ketoacidosis (DKA) are very important clinical issues. Thus, the NYMU_Taipei iGEM 2007 team is designing a biological system to sense environmental glucose concentration and decrease the level of glucose by releasing insulin. Besides, life-protection functions for removing toxic ketoacids produced during DKA and preventing hypoglycemia status will also be established. This system will be a convenient and safe design for those patients with diabetes mellitus, and further improve their quality of life by avoiding them from diabetes-related morbidity and mortality. <br />
----<br />
==Motivation==<br />
* Over 7 percent of adults in the United States (US) are known to have diabetes mellitus. However, because of the associated microvascular and macrovascular disease, diabetes accounts for almost 14 percent of US health care expenditures, at least one-half of which are related to complications such as myocardial infarction, stroke, end-stage renal disease, retinopathy, and foot ulcers. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus, one of the most common chronic diseases in childhood, is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Data from the Center of Disease Control (CDC) show that more than 13,000 children and adolescents (19 years of age) are diagnosed with type 1 disease each year with a prevalence of 1.7 per 1000. Control of blood sugar and prevention of severe complications, such;as diabetic ketoacidosis (DKA), are important issues nowadays. <br />
* Diabetic ketoacidosis (DKA) and hyperosmolar hyperglycemic state (HHS, also known as nonketotic hyperglycemia) are two of the most serious acute complications of diabetes. They are part of the spectrum of hyperglycemia and each represents an extreme in the spectrum. DKA is characterized by the triad of hyperglycemia, anion gap metabolic acidosis, and ketonemia. Metabolic acidosis is often the major finding. The serum glucose concentration is usually greater than 500 mg/dL (27.8 mmol/L) and less than 800 mg/dL (44.4 mmol/L). Three ketone bodies are produced in DKA: two ketoacids (beta-hydroxybutyric acid and acetoacetic acid), and one neutral ketone (acetone). The treatment of DKA and HHS is similar, including the administration of insulin and correction of the fluid and electrolyte abnormalities that may be present, including hyperglycemia and hyperosmolality, hypovolemia, metabolic acidosis (in DKA), and potassium depletion. <br />
* Existed works (see References below) are focusing on how to take advantages from properties of mammalian cells to express and secrete insulin in a trail-and-error manner. However, from the perspective of synthetic biology, it is possible to engineer a cell works as we want, no matter what type of cells we apply to. <br />
----<br />
==Goal and system design==<br />
* For the iGEM 2007 project, our team focuses on the development of a prokaryotic system to <br />
** express insulin regulated by external glucose concentration and <br />
** clean the ketone bodies accumulated by mamalian cells due to insufficient intake of glucose <br />
** balance between glucose and insulin to stablize glucose level and prevent low blood glucose condition <br />
* system specification is shown below: <br />
[[Image:GAI-t curve.jpg|center|500px]]<br />
* Circuit diagram (initial state)<br />
[[Image:IGEM2007 insulin circuit diagram.png|center]]<br />
* Circuit under high glucose condition (insulin generating device is activated)<br />
[[Image:High_glucose.png|center]]<br />
* Circuit under transition state (insulin increases glucose uptake; whole system shifts from insulin generation to insulin degradation)<br />
[[Image:Trans_glucose.png|center]]<br />
* Circuit under low glucose condition<br />
[[Image:Low_glucose.png|center]]<br />
* Sub-circuit #1: high glucose-triggered insulin generator (insulin generation turns off, and insulin degradation turns on)<br />
** The basic architecture for our insulin secretion design is shown in the figure below: <br />
** External glucose can be sensed by GSD (Glucose Sensing Domain) from gene RcsC and transmits response to ERD (Responsive Domain from EnvZ gene) <br />
** TCS phosphorylation relay activates the expression of insulin (both alpha and beta chains) <br />
** With signal peptide (SP), the assembled insulin (by disulfide bonding) can be exported outside the membrane of the prokaryotic cells <br />
[[Image:Insulin basic2.png|center|500px]] <br />
* Sub-circuit #2: low glucose and insulin triggered insulin inhibitor/destroyer<br />
[[Image:NYMU_Taipei_insulin_inhibitor_2.png|center|500px]] <br />
[[Image:NYMU_Taipei_insulin_inhibitor.png|center|700px]] <br />
----<br />
<br />
==Implementation==<br />
* [[NYMU_Taipei/Part_List | Part List]]<br />
* [[NYMU_Taipei/Lab_Notes | Lab notes]]<br />
* [[NYMU_Taipei/Protocols| Protocols]]<br />
* [[NYMU_Taipei/Primer_List | Primer List]]<br />
----<br />
<br />
==References==<br />
* Engineered enteroendocrine cells secrete insulin in response to glucose and reverse hyperglycemia in diabetic mice, 2007 Mol Ther. <br />
* Development of genetically engineered human intestinal cells for regulated insulin secretion using rAAV-mediated gene transfer, 2003 Biochem Biophys Res Commun <br />
* http://www.utdol.com/utd/content/topic.do <br />
* Recombinant DNA Technology in the Synthesis of Human Insulin <br />
* Glucose-Dependent Insulin Release from Genetically Engineered K Cells, 2000 science <br />
* [http://www.nature.com/gt/journal/v7/n21/abs/3301306a.html Glucose-stimulated and self-limiting insulin production by glucose 6-phosphatase promoter driven insulin expression in hepatoma cells, 2000 Gene Therapy]<br />
[https://2007.igem.org/Taipei Back]</div>Blenthttp://2007.igem.org/wiki/index.php/File:Trans_glucose.pngFile:Trans glucose.png2007-10-26T22:21:59Z<p>Blent: </p>
<hr />
<div></div>Blenthttp://2007.igem.org/wiki/index.php/NYMU_Taipei/ProjectDescriptionNYMU Taipei/ProjectDescription2007-10-26T22:21:36Z<p>Blent: /* Goal and system design */</p>
<hr />
<div><p><font size="4"><br />
[[NYMU_Taipei/ProjectDescription| Project Description]] /<br />
[[NYMU_Taipei/Lab_Notes| Lab Notes]] /<br />
[[NYMU_Taipei/Part_List| Part List]] /<br />
[[NYMU_Taipei/Primer_List| Primer List]]<br />
</font><br /><hr></p><br />
<br />
==<font size=5 color=red>GlucOperon</font>==<br />
* A '''Gluc'''ose-based synthetic '''Operon''' system<br />
* An automatic system in E.coli that operates blood sugar wonderfully!<br />
* Insulin secretion system that promotes quality of life for diabetic patients<br />
----<br />
<br />
==Summary==<br />
* Diabetes mellitus is a significant problem especially in developed countries and leads to several severe long-term complications. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Controlling blood sugar in a reasonable level and avoiding severe emergency as diabetes ketoacidosis (DKA) are very important clinical issues. Thus, the NYMU_Taipei iGEM 2007 team is designing a biological system to sense environmental glucose concentration and decrease the level of glucose by releasing insulin. Besides, life-protection functions for removing toxic ketoacids produced during DKA and preventing hypoglycemia status will also be established. This system will be a convenient and safe design for those patients with diabetes mellitus, and further improve their quality of life by avoiding them from diabetes-related morbidity and mortality. <br />
----<br />
==Motivation==<br />
* Over 7 percent of adults in the United States (US) are known to have diabetes mellitus. However, because of the associated microvascular and macrovascular disease, diabetes accounts for almost 14 percent of US health care expenditures, at least one-half of which are related to complications such as myocardial infarction, stroke, end-stage renal disease, retinopathy, and foot ulcers. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus, one of the most common chronic diseases in childhood, is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Data from the Center of Disease Control (CDC) show that more than 13,000 children and adolescents (19 years of age) are diagnosed with type 1 disease each year with a prevalence of 1.7 per 1000. Control of blood sugar and prevention of severe complications, such;as diabetic ketoacidosis (DKA), are important issues nowadays. <br />
* Diabetic ketoacidosis (DKA) and hyperosmolar hyperglycemic state (HHS, also known as nonketotic hyperglycemia) are two of the most serious acute complications of diabetes. They are part of the spectrum of hyperglycemia and each represents an extreme in the spectrum. DKA is characterized by the triad of hyperglycemia, anion gap metabolic acidosis, and ketonemia. Metabolic acidosis is often the major finding. The serum glucose concentration is usually greater than 500 mg/dL (27.8 mmol/L) and less than 800 mg/dL (44.4 mmol/L). Three ketone bodies are produced in DKA: two ketoacids (beta-hydroxybutyric acid and acetoacetic acid), and one neutral ketone (acetone). The treatment of DKA and HHS is similar, including the administration of insulin and correction of the fluid and electrolyte abnormalities that may be present, including hyperglycemia and hyperosmolality, hypovolemia, metabolic acidosis (in DKA), and potassium depletion. <br />
* Existed works (see References below) are focusing on how to take advantages from properties of mammalian cells to express and secrete insulin in a trail-and-error manner. However, from the perspective of synthetic biology, it is possible to engineer a cell works as we want, no matter what type of cells we apply to. <br />
----<br />
==Goal and system design==<br />
* For the iGEM 2007 project, our team focuses on the development of a prokaryotic system to <br />
** express insulin regulated by external glucose concentration and <br />
** clean the ketone bodies accumulated by mamalian cells due to insufficient intake of glucose <br />
** balance between glucose and insulin to stablize glucose level and prevent low blood glucose condition <br />
* system specification is shown below: <br />
[[Image:GAI-t curve.jpg|center|500px]]<br />
* Circuit diagram (initial state)<br />
[[Image:IGEM2007 insulin circuit diagram.png|center]]<br />
* Circuit under high glucose condition<br />
[[Image:High_glucose.png|center]]<br />
* Circuit under transition state (insulin increases glucose uptake)<br />
[[Image:Trans_glucose.png|center]]<br />
* Circuit under low glucose condition<br />
[[Image:Low_glucose.png|center]]<br />
* Sub-circuit #1: high glucose-triggered insulin generator<br />
** The basic architecture for our insulin secretion design is shown in the figure below: <br />
** External glucose can be sensed by GSD (Glucose Sensing Domain) from gene RcsC and transmits response to ERD (Responsive Domain from EnvZ gene) <br />
** TCS phosphorylation relay activates the expression of insulin (both alpha and beta chains) <br />
** With signal peptide (SP), the assembled insulin (by disulfide bonding) can be exported outside the membrane of the prokaryotic cells <br />
[[Image:Insulin basic2.png|center|500px]] <br />
* Sub-circuit #2: low glucose and insulin triggered insulin inhibitor/destroyer<br />
[[Image:NYMU_Taipei_insulin_inhibitor_2.png|center|500px]] <br />
[[Image:NYMU_Taipei_insulin_inhibitor.png|center|700px]] <br />
----<br />
<br />
==Implementation==<br />
* [[NYMU_Taipei/Part_List | Part List]]<br />
* [[NYMU_Taipei/Lab_Notes | Lab notes]]<br />
* [[NYMU_Taipei/Protocols| Protocols]]<br />
* [[NYMU_Taipei/Primer_List | Primer List]]<br />
----<br />
<br />
==References==<br />
* Engineered enteroendocrine cells secrete insulin in response to glucose and reverse hyperglycemia in diabetic mice, 2007 Mol Ther. <br />
* Development of genetically engineered human intestinal cells for regulated insulin secretion using rAAV-mediated gene transfer, 2003 Biochem Biophys Res Commun <br />
* http://www.utdol.com/utd/content/topic.do <br />
* Recombinant DNA Technology in the Synthesis of Human Insulin <br />
* Glucose-Dependent Insulin Release from Genetically Engineered K Cells, 2000 science <br />
* [http://www.nature.com/gt/journal/v7/n21/abs/3301306a.html Glucose-stimulated and self-limiting insulin production by glucose 6-phosphatase promoter driven insulin expression in hepatoma cells, 2000 Gene Therapy]<br />
[https://2007.igem.org/Taipei Back]</div>Blenthttp://2007.igem.org/wiki/index.php/NYMU_Taipei/ProjectDescriptionNYMU Taipei/ProjectDescription2007-10-26T22:20:48Z<p>Blent: /* Goal and system design */</p>
<hr />
<div><p><font size="4"><br />
[[NYMU_Taipei/ProjectDescription| Project Description]] /<br />
[[NYMU_Taipei/Lab_Notes| Lab Notes]] /<br />
[[NYMU_Taipei/Part_List| Part List]] /<br />
[[NYMU_Taipei/Primer_List| Primer List]]<br />
</font><br /><hr></p><br />
<br />
==<font size=5 color=red>GlucOperon</font>==<br />
* A '''Gluc'''ose-based synthetic '''Operon''' system<br />
* An automatic system in E.coli that operates blood sugar wonderfully!<br />
* Insulin secretion system that promotes quality of life for diabetic patients<br />
----<br />
<br />
==Summary==<br />
* Diabetes mellitus is a significant problem especially in developed countries and leads to several severe long-term complications. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Controlling blood sugar in a reasonable level and avoiding severe emergency as diabetes ketoacidosis (DKA) are very important clinical issues. Thus, the NYMU_Taipei iGEM 2007 team is designing a biological system to sense environmental glucose concentration and decrease the level of glucose by releasing insulin. Besides, life-protection functions for removing toxic ketoacids produced during DKA and preventing hypoglycemia status will also be established. This system will be a convenient and safe design for those patients with diabetes mellitus, and further improve their quality of life by avoiding them from diabetes-related morbidity and mortality. <br />
----<br />
==Motivation==<br />
* Over 7 percent of adults in the United States (US) are known to have diabetes mellitus. However, because of the associated microvascular and macrovascular disease, diabetes accounts for almost 14 percent of US health care expenditures, at least one-half of which are related to complications such as myocardial infarction, stroke, end-stage renal disease, retinopathy, and foot ulcers. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus, one of the most common chronic diseases in childhood, is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Data from the Center of Disease Control (CDC) show that more than 13,000 children and adolescents (19 years of age) are diagnosed with type 1 disease each year with a prevalence of 1.7 per 1000. Control of blood sugar and prevention of severe complications, such;as diabetic ketoacidosis (DKA), are important issues nowadays. <br />
* Diabetic ketoacidosis (DKA) and hyperosmolar hyperglycemic state (HHS, also known as nonketotic hyperglycemia) are two of the most serious acute complications of diabetes. They are part of the spectrum of hyperglycemia and each represents an extreme in the spectrum. DKA is characterized by the triad of hyperglycemia, anion gap metabolic acidosis, and ketonemia. Metabolic acidosis is often the major finding. The serum glucose concentration is usually greater than 500 mg/dL (27.8 mmol/L) and less than 800 mg/dL (44.4 mmol/L). Three ketone bodies are produced in DKA: two ketoacids (beta-hydroxybutyric acid and acetoacetic acid), and one neutral ketone (acetone). The treatment of DKA and HHS is similar, including the administration of insulin and correction of the fluid and electrolyte abnormalities that may be present, including hyperglycemia and hyperosmolality, hypovolemia, metabolic acidosis (in DKA), and potassium depletion. <br />
* Existed works (see References below) are focusing on how to take advantages from properties of mammalian cells to express and secrete insulin in a trail-and-error manner. However, from the perspective of synthetic biology, it is possible to engineer a cell works as we want, no matter what type of cells we apply to. <br />
----<br />
==Goal and system design==<br />
* For the iGEM 2007 project, our team focuses on the development of a prokaryotic system to <br />
** express insulin regulated by external glucose concentration and <br />
** clean the ketone bodies accumulated by mamalian cells due to insufficient intake of glucose <br />
** balance between glucose and insulin to stablize glucose level and prevent low blood glucose condition <br />
* system specification is shown below: <br />
[[Image:GAI-t curve.jpg|center|500px]]<br />
* Circuit diagram (initial state)<br />
[[Image:IGEM2007 insulin circuit diagram.png|center]]<br />
* Circuit under high glucose condition<br />
[[Image:High_glucose.png|center]]<br />
* Circuit under transition state (insulin increases glucose uptake)<br />
[[Image:bm2.jpg|center]]<br />
* Circuit under low glucose condition<br />
[[Image:Low_glucose.png|center]]<br />
* Sub-circuit #1: high glucose-triggered insulin generator<br />
** The basic architecture for our insulin secretion design is shown in the figure below: <br />
** External glucose can be sensed by GSD (Glucose Sensing Domain) from gene RcsC and transmits response to ERD (Responsive Domain from EnvZ gene) <br />
** TCS phosphorylation relay activates the expression of insulin (both alpha and beta chains) <br />
** With signal peptide (SP), the assembled insulin (by disulfide bonding) can be exported outside the membrane of the prokaryotic cells <br />
[[Image:Insulin basic2.png|center|500px]] <br />
* Sub-circuit #2: low glucose and insulin triggered insulin inhibitor/destroyer<br />
[[Image:NYMU_Taipei_insulin_inhibitor_2.png|center|500px]] <br />
[[Image:NYMU_Taipei_insulin_inhibitor.png|center|700px]] <br />
----<br />
<br />
==Implementation==<br />
* [[NYMU_Taipei/Part_List | Part List]]<br />
* [[NYMU_Taipei/Lab_Notes | Lab notes]]<br />
* [[NYMU_Taipei/Protocols| Protocols]]<br />
* [[NYMU_Taipei/Primer_List | Primer List]]<br />
----<br />
<br />
==References==<br />
* Engineered enteroendocrine cells secrete insulin in response to glucose and reverse hyperglycemia in diabetic mice, 2007 Mol Ther. <br />
* Development of genetically engineered human intestinal cells for regulated insulin secretion using rAAV-mediated gene transfer, 2003 Biochem Biophys Res Commun <br />
* http://www.utdol.com/utd/content/topic.do <br />
* Recombinant DNA Technology in the Synthesis of Human Insulin <br />
* Glucose-Dependent Insulin Release from Genetically Engineered K Cells, 2000 science <br />
* [http://www.nature.com/gt/journal/v7/n21/abs/3301306a.html Glucose-stimulated and self-limiting insulin production by glucose 6-phosphatase promoter driven insulin expression in hepatoma cells, 2000 Gene Therapy]<br />
[https://2007.igem.org/Taipei Back]</div>Blenthttp://2007.igem.org/wiki/index.php/NYMU_Taipei/ProjectDescriptionNYMU Taipei/ProjectDescription2007-10-26T22:16:28Z<p>Blent: /* Goal and system design */</p>
<hr />
<div><p><font size="4"><br />
[[NYMU_Taipei/ProjectDescription| Project Description]] /<br />
[[NYMU_Taipei/Lab_Notes| Lab Notes]] /<br />
[[NYMU_Taipei/Part_List| Part List]] /<br />
[[NYMU_Taipei/Primer_List| Primer List]]<br />
</font><br /><hr></p><br />
<br />
==<font size=5 color=red>GlucOperon</font>==<br />
* A '''Gluc'''ose-based synthetic '''Operon''' system<br />
* An automatic system in E.coli that operates blood sugar wonderfully!<br />
* Insulin secretion system that promotes quality of life for diabetic patients<br />
----<br />
<br />
==Summary==<br />
* Diabetes mellitus is a significant problem especially in developed countries and leads to several severe long-term complications. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Controlling blood sugar in a reasonable level and avoiding severe emergency as diabetes ketoacidosis (DKA) are very important clinical issues. Thus, the NYMU_Taipei iGEM 2007 team is designing a biological system to sense environmental glucose concentration and decrease the level of glucose by releasing insulin. Besides, life-protection functions for removing toxic ketoacids produced during DKA and preventing hypoglycemia status will also be established. This system will be a convenient and safe design for those patients with diabetes mellitus, and further improve their quality of life by avoiding them from diabetes-related morbidity and mortality. <br />
----<br />
==Motivation==<br />
* Over 7 percent of adults in the United States (US) are known to have diabetes mellitus. However, because of the associated microvascular and macrovascular disease, diabetes accounts for almost 14 percent of US health care expenditures, at least one-half of which are related to complications such as myocardial infarction, stroke, end-stage renal disease, retinopathy, and foot ulcers. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus, one of the most common chronic diseases in childhood, is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Data from the Center of Disease Control (CDC) show that more than 13,000 children and adolescents (19 years of age) are diagnosed with type 1 disease each year with a prevalence of 1.7 per 1000. Control of blood sugar and prevention of severe complications, such;as diabetic ketoacidosis (DKA), are important issues nowadays. <br />
* Diabetic ketoacidosis (DKA) and hyperosmolar hyperglycemic state (HHS, also known as nonketotic hyperglycemia) are two of the most serious acute complications of diabetes. They are part of the spectrum of hyperglycemia and each represents an extreme in the spectrum. DKA is characterized by the triad of hyperglycemia, anion gap metabolic acidosis, and ketonemia. Metabolic acidosis is often the major finding. The serum glucose concentration is usually greater than 500 mg/dL (27.8 mmol/L) and less than 800 mg/dL (44.4 mmol/L). Three ketone bodies are produced in DKA: two ketoacids (beta-hydroxybutyric acid and acetoacetic acid), and one neutral ketone (acetone). The treatment of DKA and HHS is similar, including the administration of insulin and correction of the fluid and electrolyte abnormalities that may be present, including hyperglycemia and hyperosmolality, hypovolemia, metabolic acidosis (in DKA), and potassium depletion. <br />
* Existed works (see References below) are focusing on how to take advantages from properties of mammalian cells to express and secrete insulin in a trail-and-error manner. However, from the perspective of synthetic biology, it is possible to engineer a cell works as we want, no matter what type of cells we apply to. <br />
----<br />
==Goal and system design==<br />
* For the iGEM 2007 project, our team focuses on the development of a prokaryotic system to <br />
** express insulin regulated by external glucose concentration and <br />
** clean the ketone bodies accumulated by mamalian cells due to insufficient intake of glucose <br />
** balance between glucose and insulin to stablize glucose level and prevent low blood glucose condition <br />
* system specification is shown below: <br />
[[Image:GAI-t curve.jpg|center|500px]]<br />
* Circuit diagram (initial state)<br />
[[Image:IGEM2007 insulin circuit diagram.png|center]]<br />
* Circuit under high glucose condition<br />
[[Image:High_glucose.png|center]]<br />
* Circuit under low glucose condition<br />
[[Image:Low_glucose.png|center]]<br />
* Sub-circuit #1: high glucose-triggered insulin generator<br />
** The basic architecture for our insulin secretion design is shown in the figure below: <br />
** External glucose can be sensed by GSD (Glucose Sensing Domain) from gene RcsC and transmits response to ERD (Responsive Domain from EnvZ gene) <br />
** TCS phosphorylation relay activates the expression of insulin (both alpha and beta chains) <br />
** With signal peptide (SP), the assembled insulin (by disulfide bonding) can be exported outside the membrane of the prokaryotic cells <br />
[[Image:Insulin basic2.png|center|500px]] <br />
* Sub-circuit #2: low glucose and insulin triggered insulin inhibitor/destroyer<br />
[[Image:NYMU_Taipei_insulin_inhibitor_2.png|center|500px]] <br />
[[Image:NYMU_Taipei_insulin_inhibitor.png|center|700px]] <br />
----<br />
<br />
==Implementation==<br />
* [[NYMU_Taipei/Part_List | Part List]]<br />
* [[NYMU_Taipei/Lab_Notes | Lab notes]]<br />
* [[NYMU_Taipei/Protocols| Protocols]]<br />
* [[NYMU_Taipei/Primer_List | Primer List]]<br />
----<br />
<br />
==References==<br />
* Engineered enteroendocrine cells secrete insulin in response to glucose and reverse hyperglycemia in diabetic mice, 2007 Mol Ther. <br />
* Development of genetically engineered human intestinal cells for regulated insulin secretion using rAAV-mediated gene transfer, 2003 Biochem Biophys Res Commun <br />
* http://www.utdol.com/utd/content/topic.do <br />
* Recombinant DNA Technology in the Synthesis of Human Insulin <br />
* Glucose-Dependent Insulin Release from Genetically Engineered K Cells, 2000 science <br />
* [http://www.nature.com/gt/journal/v7/n21/abs/3301306a.html Glucose-stimulated and self-limiting insulin production by glucose 6-phosphatase promoter driven insulin expression in hepatoma cells, 2000 Gene Therapy]<br />
[https://2007.igem.org/Taipei Back]</div>Blenthttp://2007.igem.org/wiki/index.php/NYMU_Taipei/ProjectDescriptionNYMU Taipei/ProjectDescription2007-10-26T22:13:05Z<p>Blent: /* Goal and system design */</p>
<hr />
<div><p><font size="4"><br />
[[NYMU_Taipei/ProjectDescription| Project Description]] /<br />
[[NYMU_Taipei/Lab_Notes| Lab Notes]] /<br />
[[NYMU_Taipei/Part_List| Part List]] /<br />
[[NYMU_Taipei/Primer_List| Primer List]]<br />
</font><br /><hr></p><br />
<br />
==<font size=5 color=red>GlucOperon</font>==<br />
* A '''Gluc'''ose-based synthetic '''Operon''' system<br />
* An automatic system in E.coli that operates blood sugar wonderfully!<br />
* Insulin secretion system that promotes quality of life for diabetic patients<br />
----<br />
<br />
==Summary==<br />
* Diabetes mellitus is a significant problem especially in developed countries and leads to several severe long-term complications. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Controlling blood sugar in a reasonable level and avoiding severe emergency as diabetes ketoacidosis (DKA) are very important clinical issues. Thus, the NYMU_Taipei iGEM 2007 team is designing a biological system to sense environmental glucose concentration and decrease the level of glucose by releasing insulin. Besides, life-protection functions for removing toxic ketoacids produced during DKA and preventing hypoglycemia status will also be established. This system will be a convenient and safe design for those patients with diabetes mellitus, and further improve their quality of life by avoiding them from diabetes-related morbidity and mortality. <br />
----<br />
==Motivation==<br />
* Over 7 percent of adults in the United States (US) are known to have diabetes mellitus. However, because of the associated microvascular and macrovascular disease, diabetes accounts for almost 14 percent of US health care expenditures, at least one-half of which are related to complications such as myocardial infarction, stroke, end-stage renal disease, retinopathy, and foot ulcers. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus, one of the most common chronic diseases in childhood, is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Data from the Center of Disease Control (CDC) show that more than 13,000 children and adolescents (19 years of age) are diagnosed with type 1 disease each year with a prevalence of 1.7 per 1000. Control of blood sugar and prevention of severe complications, such;as diabetic ketoacidosis (DKA), are important issues nowadays. <br />
* Diabetic ketoacidosis (DKA) and hyperosmolar hyperglycemic state (HHS, also known as nonketotic hyperglycemia) are two of the most serious acute complications of diabetes. They are part of the spectrum of hyperglycemia and each represents an extreme in the spectrum. DKA is characterized by the triad of hyperglycemia, anion gap metabolic acidosis, and ketonemia. Metabolic acidosis is often the major finding. The serum glucose concentration is usually greater than 500 mg/dL (27.8 mmol/L) and less than 800 mg/dL (44.4 mmol/L). Three ketone bodies are produced in DKA: two ketoacids (beta-hydroxybutyric acid and acetoacetic acid), and one neutral ketone (acetone). The treatment of DKA and HHS is similar, including the administration of insulin and correction of the fluid and electrolyte abnormalities that may be present, including hyperglycemia and hyperosmolality, hypovolemia, metabolic acidosis (in DKA), and potassium depletion. <br />
* Existed works (see References below) are focusing on how to take advantages from properties of mammalian cells to express and secrete insulin in a trail-and-error manner. However, from the perspective of synthetic biology, it is possible to engineer a cell works as we want, no matter what type of cells we apply to. <br />
----<br />
==Goal and system design==<br />
* For the iGEM 2007 project, our team focuses on the development of a prokaryotic system to <br />
** express insulin regulated by external glucose concentration and <br />
** clean the ketone bodies accumulated by mamalian cells due to insufficient intake of glucose <br />
** balance between glucose and insulin to stablize glucose level and prevent low blood glucose condition <br />
* system specification is shown below: <br />
[[Image:GAI-t curve.jpg|center|500px]]<br />
* Circuit diagram (initial state)<br />
[[Image:IGEM2007 insulin circuit diagram.png|center|500px]]<br />
* Circuit under high glucose condition<br />
[[Image:High_glucose.png|center]]<br />
* Circuit under low glucose condition<br />
[[Image:Low_glucose.png|center]]<br />
* Sub-circuit #1: high glucose-triggered insulin generator<br />
** The basic architecture for our insulin secretion design is shown in the figure below: <br />
** External glucose can be sensed by GSD (Glucose Sensing Domain) from gene RcsC and transmits response to ERD (Responsive Domain from EnvZ gene) <br />
** TCS phosphorylation relay activates the expression of insulin (both alpha and beta chains) <br />
** With signal peptide (SP), the assembled insulin (by disulfide bonding) can be exported outside the membrane of the prokaryotic cells <br />
[[Image:Insulin basic2.png|center|500px]] <br />
* Sub-circuit #2: low glucose and insulin triggered insulin inhibitor/destroyer<br />
[[Image:NYMU_Taipei_insulin_inhibitor_2.png|center|500px]] <br />
[[Image:NYMU_Taipei_insulin_inhibitor.png|center|700px]] <br />
----<br />
<br />
==Implementation==<br />
* [[NYMU_Taipei/Part_List | Part List]]<br />
* [[NYMU_Taipei/Lab_Notes | Lab notes]]<br />
* [[NYMU_Taipei/Protocols| Protocols]]<br />
* [[NYMU_Taipei/Primer_List | Primer List]]<br />
----<br />
<br />
==References==<br />
* Engineered enteroendocrine cells secrete insulin in response to glucose and reverse hyperglycemia in diabetic mice, 2007 Mol Ther. <br />
* Development of genetically engineered human intestinal cells for regulated insulin secretion using rAAV-mediated gene transfer, 2003 Biochem Biophys Res Commun <br />
* http://www.utdol.com/utd/content/topic.do <br />
* Recombinant DNA Technology in the Synthesis of Human Insulin <br />
* Glucose-Dependent Insulin Release from Genetically Engineered K Cells, 2000 science <br />
* [http://www.nature.com/gt/journal/v7/n21/abs/3301306a.html Glucose-stimulated and self-limiting insulin production by glucose 6-phosphatase promoter driven insulin expression in hepatoma cells, 2000 Gene Therapy]<br />
[https://2007.igem.org/Taipei Back]</div>Blenthttp://2007.igem.org/wiki/index.php/NYMU_Taipei/ProjectDescriptionNYMU Taipei/ProjectDescription2007-10-26T22:12:11Z<p>Blent: /* Goal and system design */</p>
<hr />
<div><p><font size="4"><br />
[[NYMU_Taipei/ProjectDescription| Project Description]] /<br />
[[NYMU_Taipei/Lab_Notes| Lab Notes]] /<br />
[[NYMU_Taipei/Part_List| Part List]] /<br />
[[NYMU_Taipei/Primer_List| Primer List]]<br />
</font><br /><hr></p><br />
<br />
==<font size=5 color=red>GlucOperon</font>==<br />
* A '''Gluc'''ose-based synthetic '''Operon''' system<br />
* An automatic system in E.coli that operates blood sugar wonderfully!<br />
* Insulin secretion system that promotes quality of life for diabetic patients<br />
----<br />
<br />
==Summary==<br />
* Diabetes mellitus is a significant problem especially in developed countries and leads to several severe long-term complications. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Controlling blood sugar in a reasonable level and avoiding severe emergency as diabetes ketoacidosis (DKA) are very important clinical issues. Thus, the NYMU_Taipei iGEM 2007 team is designing a biological system to sense environmental glucose concentration and decrease the level of glucose by releasing insulin. Besides, life-protection functions for removing toxic ketoacids produced during DKA and preventing hypoglycemia status will also be established. This system will be a convenient and safe design for those patients with diabetes mellitus, and further improve their quality of life by avoiding them from diabetes-related morbidity and mortality. <br />
----<br />
==Motivation==<br />
* Over 7 percent of adults in the United States (US) are known to have diabetes mellitus. However, because of the associated microvascular and macrovascular disease, diabetes accounts for almost 14 percent of US health care expenditures, at least one-half of which are related to complications such as myocardial infarction, stroke, end-stage renal disease, retinopathy, and foot ulcers. Compared to well-known type 2 diabetes mellitus, manifested with different degrees of insulin resistance, type 1 diabetes mellitus, one of the most common chronic diseases in childhood, is caused by insulin deficiency following destruction of the insulin-producing pancreatic beta cells. Data from the Center of Disease Control (CDC) show that more than 13,000 children and adolescents (19 years of age) are diagnosed with type 1 disease each year with a prevalence of 1.7 per 1000. Control of blood sugar and prevention of severe complications, such;as diabetic ketoacidosis (DKA), are important issues nowadays. <br />
* Diabetic ketoacidosis (DKA) and hyperosmolar hyperglycemic state (HHS, also known as nonketotic hyperglycemia) are two of the most serious acute complications of diabetes. They are part of the spectrum of hyperglycemia and each represents an extreme in the spectrum. DKA is characterized by the triad of hyperglycemia, anion gap metabolic acidosis, and ketonemia. Metabolic acidosis is often the major finding. The serum glucose concentration is usually greater than 500 mg/dL (27.8 mmol/L) and less than 800 mg/dL (44.4 mmol/L). Three ketone bodies are produced in DKA: two ketoacids (beta-hydroxybutyric acid and acetoacetic acid), and one neutral ketone (acetone). The treatment of DKA and HHS is similar, including the administration of insulin and correction of the fluid and electrolyte abnormalities that may be present, including hyperglycemia and hyperosmolality, hypovolemia, metabolic acidosis (in DKA), and potassium depletion. <br />
* Existed works (see References below) are focusing on how to take advantages from properties of mammalian cells to express and secrete insulin in a trail-and-error manner. However, from the perspective of synthetic biology, it is possible to engineer a cell works as we want, no matter what type of cells we apply to. <br />
----<br />
==Goal and system design==<br />
* For the iGEM 2007 project, our team focuses on the development of a prokaryotic system to <br />
** express insulin regulated by external glucose concentration and <br />
** clean the ketone bodies accumulated by mamalian cells due to insufficient intake of glucose <br />
** balance between glucose and insulin to stablize glucose level and prevent low blood glucose condition <br />
* system specification is shown below: <br />
[[Image:GAI-t curve.jpg|center|500px]]<br />
* Circuit diagram (initial state)<br />
[[Image:IGEM2007 insulin circuit diagram.png|center|500px]]<br />
* Circuit works under high glucose condition<br />
[[Image:High_glucose.png|center]]<br />
* Circuit works under low glucose condition<br />
[[Image:Low_glucose.png|center]]<br />
* Sub-circuit #1: high glucose-triggered insulin generator<br />
** The basic architecture for our insulin secretion design is shown in the figure below: <br />
** External glucose can be sensed by GSD (Glucose Sensing Domain) from gene RcsC and transmits response to ERD (Responsive Domain from EnvZ gene) <br />
** TCS phosphorylation relay activates the expression of insulin (both alpha and beta chains) <br />
** With signal peptide (SP), the assembled insulin (by disulfide bonding) can be exported outside the membrane of the prokaryotic cells <br />
[[Image:Insulin basic2.png|center|500px]] <br />
* Sub-circuit #2: low glucose and insulin triggered insulin inhibitor/destroyer<br />
[[Image:NYMU_Taipei_insulin_inhibitor_2.png|center|500px]] <br />
[[Image:NYMU_Taipei_insulin_inhibitor.png|center|700px]] <br />
----<br />
<br />
==Implementation==<br />
* [[NYMU_Taipei/Part_List | Part List]]<br />
* [[NYMU_Taipei/Lab_Notes | Lab notes]]<br />
* [[NYMU_Taipei/Protocols| Protocols]]<br />
* [[NYMU_Taipei/Primer_List | Primer List]]<br />
----<br />
<br />
==References==<br />
* Engineered enteroendocrine cells secrete insulin in response to glucose and reverse hyperglycemia in diabetic mice, 2007 Mol Ther. <br />
* Development of genetically engineered human intestinal cells for regulated insulin secretion using rAAV-mediated gene transfer, 2003 Biochem Biophys Res Commun <br />
* http://www.utdol.com/utd/content/topic.do <br />
* Recombinant DNA Technology in the Synthesis of Human Insulin <br />
* Glucose-Dependent Insulin Release from Genetically Engineered K Cells, 2000 science <br />
* [http://www.nature.com/gt/journal/v7/n21/abs/3301306a.html Glucose-stimulated and self-limiting insulin production by glucose 6-phosphatase promoter driven insulin expression in hepatoma cells, 2000 Gene Therapy]<br />
[https://2007.igem.org/Taipei Back]</div>Blent