http://2007.igem.org/wiki/index.php?title=Special:Contributions/KonniamChan&feed=atom&limit=50&target=KonniamChan&year=&month=2007.igem.org - User contributions [en]2024-03-28T23:58:34ZFrom 2007.igem.orgMediaWiki 1.16.5http://2007.igem.org/wiki/index.php/Berkeley_LBL/MethodsBerkeley LBL/Methods2007-10-27T06:51:20Z<p>KonniamChan: /* Constructs */</p>
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<br />
<br />
==Experimental==<br />
<br />
=== Subcloning ===<br />
<br />
The genes of interest from ''Rhodobacter sphaeroides'', ''Synechocystis sp'', and ''Heliobacillus mobilis'' were amplified using PCR (the details of which can be found in Protocols). The PCR fragments were then digested and ligated with the T7 expression vector pET3a in various ways, resulting in the constructs shown below. They were transformed into ''E.Coli'' (either DH10B or NovaBlue) and glycerol stocks for these cells were saved.<br />
<br />
=== Constructs ===<br />
The T7 expression vector pET3a was used for the subcloning of the genes for magnesium-chelatase. The following constructs were built by inserting various fragments genes from the three organisms into pET3a.<br />
<br />
'''Magnesium-Chelatase'''<br><br />
''Rhodobacter sphaeroides''<br />
{| border="1"<br />
|-<br />
!Construct Name<br />
!Construct Details<br />
|-<br />
|pET3a-R-bchH<br />
|T7p-rbs-bchH-T7Term<br />
|-<br />
|pET3a-R-bchI<br />
|T7p-rbs-bchI-T7Term<br />
|-<br />
|pET3a-R-bchD<br />
|T7p-rbs-bchD-T7Term<br />
|-<br />
|pET3a-R-bchHID<br />
|T7p-rbs-bchH-rbs-bchI-rbs-bchD-T7Term<br />
|}<br />
<br />
<br />
''Synechocystis sp.''<br />
{| border="1"<br />
|-<br />
!Construct Name<br />
!Construct Details<br />
|-<br />
|pET3a-S-chlH<br />
|T7p-rbs-chlH-T7Term<br />
|-<br />
|pET3a-S-chlI<br />
|T7p-rbs-chlI-T7Term<br />
|-<br />
|pET3a-S-chlD<br />
|T7p-rbs-chlD-T7Term<br />
|-<br />
|pET3a-S-chlHID<br />
|T7p-rbs-chlH-rbs-chlI-rbs-chlD-T7Term<br />
|}<br />
<br />
<br />
'''Enzymes for reactions after Mg-insertion step'''<br><br />
<br />
''Heliobacillus mobilis'' (T7 expression vector pET29bEBBX was used for subcloning of heliobacteria)<br />
{| border="1"<br />
|-<br />
!Construct Name<br />
!Construct Details<br />
|-<br />
|pET29-bchB<br />
|T7p-rbs-bchB-T7Term<br />
|-<br />
|pET29-bchE<br />
|T7p-rbs-bchE-T7Term<br />
|-<br />
|pET29-bchM<br />
|T7p-rbs-bchM-T7Term<br />
|-<br />
|pET29-bchN<br />
|T7p-rbs-bchN-T7Term<br />
|-<br />
|pET29-bchI<br />
|T7p-rbs-bchI-T7Term<br />
|-<br />
|pET29-bchL<br />
|T7p-rbs-bchL-T7Term<br />
|}<br />
<br />
=== Expression ===<br />
<br />
Once the constructs are inside DH10B or NovaBlue, they were minipreped and transformed in BL21 (DE3). These cells were then induced to overexpress the proteins of interest. After protein expression, the cell cultures were centrifuged at 10,000rpm for 20 minutes. The resulting cell pellets were dissolved in TRIS buffer (pH 7.8) and sonicated. The sonicated cell extracts were then centrifuged again, with the supernatant containing the soluble proteins. The soluble protein extract was used for enzyme activity assays. Along with the soluble protein extract, the total sonicated cell extract and the insoluble cell extract were analyzed on a SDS-PAGE gel. (all expressions were done by Joyce, Mimi, and Konniam; gel was done on 10/24/07)<br />
<br />
=== Enzyme Activity Assays ===<br />
<br />
The ''E.Coli'' cells with the constructs pET3a-R-bchHID and pET3a-S-bchHID have all the peptides necessary for the enzyme Mg-chelatase. The activity of these enzymes can be measured by the concentration of the reaction product, Mg-protoporphyrin IX. The concentrations were measured by the UV-vis spectroscopy and fluorescence emission spectroscopy, utilizing the Beer-Lambert law. By assuming that the concentration of the product is proportional to the activity of the enzymes, we can determine which enzyme (either from ''Rhodobacter sphaeroides'' or ''Synechocystis sp'') is more efficient.<br />
<br />
== '''Subcloning Protocols''' ==<br />
<br />
'''''Polymerase Chain Reaction (PCR) Protocols:'''''<br />
<br />
[[Berkeley_LBL/PCRphusion|PCR (Using Phusion Polymerase)]]<br />
<br />
[[Berkeley_LBL/PCRextaq|PCR (Using TaKaRa Ex Taq Polymerase)]]<br />
<br />
'''''Clean Up/ Purification Protocols:'''''<br />
<br />
[[Berkeley_LBL/PCRcleanup|PCR Clean Up/Purification]]<br />
<br />
[[Berkeley_LBL/GelExtraction|Gel Extraction]]<br />
<br />
'''''DNA Gel Eletrophoresis Protocol:''''' <br />
<br />
[[Berkeley_LBL/DNAGelElectrophoresis|DNA Gel Electrophoresis]]<br />
<br />
'''''Restriction Digestion Protocols:'''''<br />
<br />
[[Berkeley_LBL/Digestion|Digestion for PCR Product or Miniprepped DNA]]<br />
<br />
[[Berkeley_LBL/Digestion2|Analytic Digestion]]<br />
<br />
'''''Ligation Protocol:'''''<br />
<br />
[[Berkeley_LBL/Ligation|Ligation]]<br />
<br />
'''''Transformation Protocols:'''''<br />
<br />
[[Berkeley_LBL/CompetentCell|KCM Competent Cell Production]]<br />
<br />
[[Berkeley_LBL/CompetentCellTransformation|KCM Competent Cell Transformation]]<br />
<br />
[[Berkeley_LBL/Electroporation|Electroporation Transformation]]<br />
<br />
'''''Miniprep Protocol:'''''<br />
<br />
[[Berkeley_LBL/Miniprep|Miniprep]]<br />
<br />
== '''Expression Protocols''' ==<br />
<br />
'''''Protein Expression Protocols:'''''<br />
<br />
[[Berkeley_LBL/Overexpression|Overexpression]]<br />
<br />
'''''Protein Analysis Protocols:'''''<br />
<br />
[[Berkeley_LBL/Sonication|Sonication]]<br />
<br />
[[Berkeley_LBL/SDS-PAGE|SDS-PAGE]]</div>KonniamChanhttp://2007.igem.org/wiki/index.php/Berkeley_LBL/ResultsBerkeley LBL/Results2007-10-27T06:44:54Z<p>KonniamChan: </p>
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|[[Berkeley_LBL/Resources|Resources]]<br />
|}<br />
<br />
[[Berkeley_LBL/Results/ProteinGel|SDS-PAGE Gel of Soluble Proteins]]<br><br />
[[Berkeley_LBL/Results/Assay|Enzyme Activities of Soluble Proteins]]</div>KonniamChanhttp://2007.igem.org/wiki/index.php/Berkeley_LBL/ResultsBerkeley LBL/Results2007-10-27T06:44:43Z<p>KonniamChan: </p>
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|[[Berkeley_LBL/Resources|Resources]]<br />
|}<br />
<br />
[[Berkeley_LBL/Results/ProteinGel|SDS-PAGE Gel of Soluble Proteins]]<br />
[[Berkeley_LBL/Results/Assay|Enzyme Activities of Soluble Proteins]]</div>KonniamChanhttp://2007.igem.org/wiki/index.php/Berkeley_LBL/KonniamNotebookBerkeley LBL/KonniamNotebook2007-10-27T06:37:22Z<p>KonniamChan: /* Construction of pET3a-R-bchHI */</p>
<hr />
<div>==Construction of pET3A Derivatives Containing R-bchHID==<br />
===Construction of pET3a-R-bchH===<br />
1.)PCR the gene bchH from Rhodobacter sphaeroides<br><br />
Materials:<br />
*1uL R-genomic DNA<br />
*10uL 5x GC buffer<br />
*5uL primer<br />
*0.5uL Phusion<br />
*5uL DMSO<br />
*1uL dNTP<br />
*27.5uL H2O<br />
Conditions:<br />
*98C 30s<br />
*98C 10s*<br />
*63C 30s*<br />
*72C 1:50 min*<br />
*Repeat cycles with * 29x<br />
*72C 10 min<br />
*4C forever<br />
<br />
2.)Run PCR fragments on gel to make sure they are the correct size, then PCR purify<br><br />
3.)Digest R-bchH (8/21/07)<br><br />
*42uL PCR purified fragment<br />
*5uL NEB3<br />
*1.8uL NdeI<br />
*1.2uL BglII<br />
4.)Run the digested PCR fragment on gel, checking that the size is correct (8/21/07)<br><br />
5.)Ligate R-bchH with pET3a that is already digested with NdeI and BamHI (8/21/07)<br><br />
*4.5uL pET3a<br />
*12.5uL R-bchH fragment<br />
*2uL T4 ligase buffer<br />
*1uL T4 ligase<br />
6.)Transform into NovaBlue (8/21/07)<br><br />
7.)Inoculate to LB media (pick 10 colonies) (8/22/07)<br><br />
8.)Miniprep cells with pET3a-R-bchH (8/23/07)<br><br />
9.)Sequence<br />
<br />
===Construction of pET3a-R-bchHI===<br />
1.)PCR the gene bchI from Rhodobacter sphaeroides (7/19/07)<br><br />
Materials:<br />
*1uL R-genomic DNA<br />
*10uL 5x HF buffer<br />
*5uL primer<br />
*0.5uL Phusion<br />
*2.5uL DMSO<br />
*1uL dNTP<br />
*30uL H2O<br />
Conditions:<br />
*98C 30s<br />
*98C 10s*<br />
*62C 30s*<br />
*72C 32s min*<br />
*Repeat cycles with * 29x<br />
*72C 10 min<br />
*4C forever<br />
<br />
2.)Run PCR fragments on gel to make sure they are the correct size, then PCR purify<br><br />
3.)Digest R-bchI with KpnI and BglII(7/20/07)<br><br />
4.)Run the digested PCR fragment on gel, checking that the size is correct (7/21/07)<br><br />
5.)Ligate R-bchH with pET3a-R-bchH that is already digested with KpnI and NsiI(8/28/07)<br><br />
*4.5uL pET3a-R-bchH<br />
*12.5uL R-bchI fragment<br />
*2uL T4 ligase buffer<br />
*1uL T4 ligase<br />
6.)Transform into NovaBlue (9/3/07)<br><br />
7.)Inoculate to LB media (pick 10 colonies) (9/4/07)<br><br />
8.)Miniprep cells with pET3a-R-bchHI (9/14/07)<br><br />
9.)Sequence<br />
<br />
===Construction of pET3a-R-bchHID===<br />
1.)PCR the gene bchD from Rhodobacter sphaeroides (7/20/07)<br><br />
Materials:<br />
*1uL R-genomic DNA<br />
*10uL 5x GC buffer<br />
*5uL primer<br />
*0.5uL Phusion<br />
*5uL DMSO<br />
*1uL dNTP<br />
*27.5uL H2O<br />
Conditions:<br />
*98C 30s<br />
*98C 10s*<br />
*62C 30s*<br />
*72C 1 min*<br />
*Repeat cycles with * 29x<br />
*72C 10 min<br />
*4C forever<br />
<br />
2.)Run PCR fragments on gel to make sure they are the correct size, then PCR purify (7/20/07)<br><br />
3.)Digest R-bchD with SpeI and NsiI (7/23/07)<br><br />
4.)Run the digested PCR fragment on gel, checking that the size is correct (7/23/07)<br><br />
5.)Ligate R-bchD with pET3a-R-bchHI that is already digested with SpeI and NsiI(9/17/07)<br><br />
*4.5uL pET3a-R-bchHI<br />
*12.5uL R-bchD fragment<br />
*2uL T4 ligase buffer<br />
*1uL T4 ligase<br />
6.)Transform into NovaBlue (9/17/07)<br><br />
7.)Inoculate to LB media (pick 10 colonies) (9/18/07)<br><br />
8.)Miniprep cells with pET3a-R-bchHID (9/19/07)<br><br />
9.)Sequence</div>KonniamChanhttp://2007.igem.org/wiki/index.php/Berkeley_LBL/KonniamNotebookBerkeley LBL/KonniamNotebook2007-10-27T06:36:19Z<p>KonniamChan: /* Construction of pET3a-R-bchHID */</p>
<hr />
<div>==Construction of pET3A Derivatives Containing R-bchHID==<br />
===Construction of pET3a-R-bchH===<br />
1.)PCR the gene bchH from Rhodobacter sphaeroides<br><br />
Materials:<br />
*1uL R-genomic DNA<br />
*10uL 5x GC buffer<br />
*5uL primer<br />
*0.5uL Phusion<br />
*5uL DMSO<br />
*1uL dNTP<br />
*27.5uL H2O<br />
Conditions:<br />
*98C 30s<br />
*98C 10s*<br />
*63C 30s*<br />
*72C 1:50 min*<br />
*Repeat cycles with * 29x<br />
*72C 10 min<br />
*4C forever<br />
<br />
2.)Run PCR fragments on gel to make sure they are the correct size, then PCR purify<br><br />
3.)Digest R-bchH (8/21/07)<br><br />
*42uL PCR purified fragment<br />
*5uL NEB3<br />
*1.8uL NdeI<br />
*1.2uL BglII<br />
4.)Run the digested PCR fragment on gel, checking that the size is correct (8/21/07)<br><br />
5.)Ligate R-bchH with pET3a that is already digested with NdeI and BamHI (8/21/07)<br><br />
*4.5uL pET3a<br />
*12.5uL R-bchH fragment<br />
*2uL T4 ligase buffer<br />
*1uL T4 ligase<br />
6.)Transform into NovaBlue (8/21/07)<br><br />
7.)Inoculate to LB media (pick 10 colonies) (8/22/07)<br><br />
8.)Miniprep cells with pET3a-R-bchH (8/23/07)<br><br />
9.)Sequence<br />
<br />
===Construction of pET3a-R-bchHI===<br />
1.)PCR the gene bchI from Rhodobacter sphaeroides (7/19/07)<br><br />
Materials:<br />
*1uL R-genomic DNA<br />
*10uL 5x HF buffer<br />
*5uL primer<br />
*0.5uL Phusion<br />
*25uL DMSO<br />
*1uL dNTP<br />
*30uL H2O<br />
Conditions:<br />
*98C 30s<br />
*98C 10s*<br />
*62C 30s*<br />
*72C 32s min*<br />
*Repeat cycles with * 29x<br />
*72C 10 min<br />
*4C forever<br />
<br />
2.)Run PCR fragments on gel to make sure they are the correct size, then PCR purify<br><br />
3.)Digest R-bchI with KpnI and BglII(7/20/07)<br><br />
4.)Run the digested PCR fragment on gel, checking that the size is correct (7/21/07)<br><br />
5.)Ligate R-bchH with pET3a-R-bchH that is already digested with KpnI and NsiI(8/28/07)<br><br />
*4.5uL pET3a-R-bchH<br />
*12.5uL R-bchI fragment<br />
*2uL T4 ligase buffer<br />
*1uL T4 ligase<br />
6.)Transform into NovaBlue (9/3/07)<br><br />
7.)Inoculate to LB media (pick 10 colonies) (9/4/07)<br><br />
8.)Miniprep cells with pET3a-R-bchHI (9/14/07)<br><br />
9.)Sequence<br />
<br />
===Construction of pET3a-R-bchHID===<br />
1.)PCR the gene bchD from Rhodobacter sphaeroides (7/20/07)<br><br />
Materials:<br />
*1uL R-genomic DNA<br />
*10uL 5x GC buffer<br />
*5uL primer<br />
*0.5uL Phusion<br />
*5uL DMSO<br />
*1uL dNTP<br />
*27.5uL H2O<br />
Conditions:<br />
*98C 30s<br />
*98C 10s*<br />
*62C 30s*<br />
*72C 1 min*<br />
*Repeat cycles with * 29x<br />
*72C 10 min<br />
*4C forever<br />
<br />
2.)Run PCR fragments on gel to make sure they are the correct size, then PCR purify (7/20/07)<br><br />
3.)Digest R-bchD with SpeI and NsiI (7/23/07)<br><br />
4.)Run the digested PCR fragment on gel, checking that the size is correct (7/23/07)<br><br />
5.)Ligate R-bchD with pET3a-R-bchHI that is already digested with SpeI and NsiI(9/17/07)<br><br />
*4.5uL pET3a-R-bchHI<br />
*12.5uL R-bchD fragment<br />
*2uL T4 ligase buffer<br />
*1uL T4 ligase<br />
6.)Transform into NovaBlue (9/17/07)<br><br />
7.)Inoculate to LB media (pick 10 colonies) (9/18/07)<br><br />
8.)Miniprep cells with pET3a-R-bchHID (9/19/07)<br><br />
9.)Sequence</div>KonniamChanhttp://2007.igem.org/wiki/index.php/Berkeley_LBL/KonniamNotebookBerkeley LBL/KonniamNotebook2007-10-27T06:36:06Z<p>KonniamChan: /* Construction of pET3a-R-bchHI */</p>
<hr />
<div>==Construction of pET3A Derivatives Containing R-bchHID==<br />
===Construction of pET3a-R-bchH===<br />
1.)PCR the gene bchH from Rhodobacter sphaeroides<br><br />
Materials:<br />
*1uL R-genomic DNA<br />
*10uL 5x GC buffer<br />
*5uL primer<br />
*0.5uL Phusion<br />
*5uL DMSO<br />
*1uL dNTP<br />
*27.5uL H2O<br />
Conditions:<br />
*98C 30s<br />
*98C 10s*<br />
*63C 30s*<br />
*72C 1:50 min*<br />
*Repeat cycles with * 29x<br />
*72C 10 min<br />
*4C forever<br />
<br />
2.)Run PCR fragments on gel to make sure they are the correct size, then PCR purify<br><br />
3.)Digest R-bchH (8/21/07)<br><br />
*42uL PCR purified fragment<br />
*5uL NEB3<br />
*1.8uL NdeI<br />
*1.2uL BglII<br />
4.)Run the digested PCR fragment on gel, checking that the size is correct (8/21/07)<br><br />
5.)Ligate R-bchH with pET3a that is already digested with NdeI and BamHI (8/21/07)<br><br />
*4.5uL pET3a<br />
*12.5uL R-bchH fragment<br />
*2uL T4 ligase buffer<br />
*1uL T4 ligase<br />
6.)Transform into NovaBlue (8/21/07)<br><br />
7.)Inoculate to LB media (pick 10 colonies) (8/22/07)<br><br />
8.)Miniprep cells with pET3a-R-bchH (8/23/07)<br><br />
9.)Sequence<br />
<br />
===Construction of pET3a-R-bchHI===<br />
1.)PCR the gene bchI from Rhodobacter sphaeroides (7/19/07)<br><br />
Materials:<br />
*1uL R-genomic DNA<br />
*10uL 5x HF buffer<br />
*5uL primer<br />
*0.5uL Phusion<br />
*25uL DMSO<br />
*1uL dNTP<br />
*30uL H2O<br />
Conditions:<br />
*98C 30s<br />
*98C 10s*<br />
*62C 30s*<br />
*72C 32s min*<br />
*Repeat cycles with * 29x<br />
*72C 10 min<br />
*4C forever<br />
<br />
2.)Run PCR fragments on gel to make sure they are the correct size, then PCR purify<br><br />
3.)Digest R-bchI with KpnI and BglII(7/20/07)<br><br />
4.)Run the digested PCR fragment on gel, checking that the size is correct (7/21/07)<br><br />
5.)Ligate R-bchH with pET3a-R-bchH that is already digested with KpnI and NsiI(8/28/07)<br><br />
*4.5uL pET3a-R-bchH<br />
*12.5uL R-bchI fragment<br />
*2uL T4 ligase buffer<br />
*1uL T4 ligase<br />
6.)Transform into NovaBlue (9/3/07)<br><br />
7.)Inoculate to LB media (pick 10 colonies) (9/4/07)<br><br />
8.)Miniprep cells with pET3a-R-bchHI (9/14/07)<br><br />
9.)Sequence<br />
<br />
===Construction of pET3a-R-bchHID===<br />
1.)PCR the gene bchD from Rhodobacter sphaeroides (7/20/07)<br><br />
Materials:<br />
*1uL R-genomic DNA<br />
*10uL 5x GC buffer<br />
*5uL primer<br />
*0.5uL Phusion<br />
*5uL DMSO<br />
*1uL dNTP<br />
*27.5uL H2O<br />
Conditions:<br />
*98C 30s<br />
*98C 10s*<br />
*62C 30s*<br />
*72C 1 min*<br />
*Repeat cycles with * 29x<br />
*72C 10 min<br />
*4C forever<br />
<br />
2.)Run PCR fragments on gel to make sure they are the correct size, then PCR purify (7/20/07)<br><br />
3.)Digest R-bchD with SpeI and NsiI (7/23/07)<br><br />
4.)Run the digested PCR fragment on gel, checking that the size is correct (7/23/07)<br><br />
5.)Ligate R-bchD with pET3a-R-bchHI that is already digested with SpeI and NsiI(9/17/07)<br><br />
*4.5uL pET3a-R-bchHI<br />
*12.5uL R-bchD fragment<br />
*2uL T4 ligase buffer<br />
*1uL T4 ligase<br />
6.)Transform into NovaBlue (9/17/07)<br><br />
7.)Inoculate to LB media (pick 10 colonies) (9/18/07)<br><br />
8.)Miniprep cells with pET3a-R-bchH (9/19/07)<br><br />
9.)Sequence</div>KonniamChanhttp://2007.igem.org/wiki/index.php/Berkeley_LBL/Results/ProteinGelBerkeley LBL/Results/ProteinGel2007-10-27T03:22:35Z<p>KonniamChan: /* '''Discussion''' */</p>
<hr />
<div>== Results of SDS-PAGE Gel of Soluble Proteins ==<br />
<br />
[[Berkeley_LBL/Ladder|Invitrogen BenchMark Protein Ladder]]<br />
<br />
[[Berkeley_LBL/Key|Plasmid Key for Protein Gels]]<br />
<br />
[[Image:ProteinGel1.jpg]]<br />
<br />
[[Image:Gel1Key.jpg]]<br />
<br />
[[Image:ProteinGel2.jpg]]<br />
<br />
[[Image:Gel2Key.jpg]]<br />
<br />
[[Image:ProteinGel3.jpg]]<br />
<br />
[[Image:Gel3Key.jpg]]<br />
<br />
[[Image:ProteinGel4.jpg]]<br />
<br />
[[Image:Gel4Key.jpg]]<br />
<br />
<br />
== '''Discussion''' ==<br />
<br />
'''''Prior to running the protein gels:'''''<br />
<br />
''*We expected to see protein bands for the constructs that contained:''<br />
<br />
* 1. -H gene (~140kDa)that were induced with IPTG<br />
<br />
* 2. -I gene(~38kDa) that were induced with IPTG<br />
<br />
''*We did not expect to see bands that coded for:''<br />
<br />
* 1. -D gene (~70kDa) <br />
<br />
* 2. - All constructs that were not induced with IPTG<br />
<br />
'''''After running the protein gels:'''''<br />
<br />
*Although the induced constructs that contained the -H gene had strong bands at ~140kDa, the uninduced constructs also showed strong bands in the same area. The -H gene was able to be expressed with or without induction of IPTG. <br />
<br />
*In addition, protein bands that coded for the -I gene showed a strong band, when alone. <br />
<br />
*However, when the -I gene was expressed with the -H gene, it did not clearly show a strong band at ~38 kDa in comparison to the benchmark ladder. <br />
<br />
*As expected, protein bands for the -D gene also did not show, alone nor in conjunction with the -H and -I gene. <br />
<br />
'''''Conclusion:''''' <br />
<br />
'''* Although our protein gels did not convey expression of neither the -I nor -D genes, our genes may still be expressed and may have enough activity to catalyze the Mg-chelatase enzyme to produce Mg-protoporphyrin IX.'''</div>KonniamChanhttp://2007.igem.org/wiki/index.php/Berkeley_LBL/Results/ProteinGelBerkeley LBL/Results/ProteinGel2007-10-27T03:21:56Z<p>KonniamChan: /* '''Discussion''' */</p>
<hr />
<div>== Results of SDS-PAGE Gel of Soluble Proteins ==<br />
<br />
[[Berkeley_LBL/Ladder|Invitrogen BenchMark Protein Ladder]]<br />
<br />
[[Berkeley_LBL/Key|Plasmid Key for Protein Gels]]<br />
<br />
[[Image:ProteinGel1.jpg]]<br />
<br />
[[Image:Gel1Key.jpg]]<br />
<br />
[[Image:ProteinGel2.jpg]]<br />
<br />
[[Image:Gel2Key.jpg]]<br />
<br />
[[Image:ProteinGel3.jpg]]<br />
<br />
[[Image:Gel3Key.jpg]]<br />
<br />
[[Image:ProteinGel4.jpg]]<br />
<br />
[[Image:Gel4Key.jpg]]<br />
<br />
<br />
== '''Discussion''' ==<br />
<br />
'''''Prior to running the protein gels:'''''<br />
<br />
''*We expected to see protein bands for the constructs that contained:''<br />
<br />
* 1. -H gene (~140kDa)that were induced with IPTG<br />
<br />
* 2. -I gene(~38kDa) that were induced with IPTG<br />
<br />
''*We did not expect to see bands that coded for:''<br />
<br />
* 1. -D gene (~70kDa) <br />
<br />
* 2. - All constructs that were not induced with IPTG<br />
<br />
'''''After running the protein gels:'''''<br />
<br />
*Although the induced constructs that contained the -H gene had strong bands at ~140kDa, the uninduced constructs also showed strong bands in the same area. The -H gene was able to be expressed with or without induction of IPTG. <br />
<br />
*In addition, protein bands that coded for the -I gene showed a strong band, when alone. <br />
<br />
*However, when the -I gene was expressed with the -H gene, it did not clearly show a strong band at ~38 kDa in comparison to the benchmark ladder. <br />
<br />
*As expected, protein bands for the -D gene also did not show, alone nor in conjunction with the -H and -I gene. <br />
''<br />
'''Conclusion:''''' <br />
<br />
'''* Although our protein gels did not convey expression of neither the -I nor -D genes, our genes may still be expressed and may have enough activity to catalyze the Mg-chelatase enzyme to produce Mg-protoporphyrin IX.'''</div>KonniamChanhttp://2007.igem.org/wiki/index.php/Berkeley_LBL/Results/ProteinGelBerkeley LBL/Results/ProteinGel2007-10-27T03:20:39Z<p>KonniamChan: /* '''Discussion''' */</p>
<hr />
<div>== Results of SDS-PAGE Gel of Soluble Proteins ==<br />
<br />
[[Berkeley_LBL/Ladder|Invitrogen BenchMark Protein Ladder]]<br />
<br />
[[Berkeley_LBL/Key|Plasmid Key for Protein Gels]]<br />
<br />
[[Image:ProteinGel1.jpg]]<br />
<br />
[[Image:Gel1Key.jpg]]<br />
<br />
[[Image:ProteinGel2.jpg]]<br />
<br />
[[Image:Gel2Key.jpg]]<br />
<br />
[[Image:ProteinGel3.jpg]]<br />
<br />
[[Image:Gel3Key.jpg]]<br />
<br />
[[Image:ProteinGel4.jpg]]<br />
<br />
[[Image:Gel4Key.jpg]]<br />
<br />
<br />
== '''Discussion''' ==<br />
<br />
'''''Prior to running the protein gels:'''''<br />
<br />
''*We expected to see protein bands for the constructs that contained:''<br />
<br />
* 1. -H gene (~140kDa)that were induced with IPTG<br />
<br />
* 2. -I gene(~38kDa) that were induced with IPTG<br />
<br />
''*We did not expect to see bands that coded for:''<br />
<br />
1. -D gene (~70kDa) <br />
<br />
2. - All constructs that were not induced with IPTG<br />
<br />
*Although the induced constructs that contained the -H gene had strong bands at ~140kDa, the uninduced constructs also showed strong bands in the same area. The -H gene was able to be expressed with or without induction of IPTG. <br />
<br />
*In addition, protein bands that coded for the -I gene showed a strong band, when alone. <br />
<br />
*However, when the -I gene was expressed with the -H gene, it did not clearly show a strong band at ~38 kDa in comparison to the benchmark ladder. <br />
<br />
*As expected, protein bands for the -D gene also did not show, alone nor in conjunction with the -H and -I gene. <br />
''<br />
'''Conclusion:''''' <br />
<br />
'''* Although our protein gels did not convey expression of neither the -I nor -D genes, our genes may still be expressed and may have enough activity to catalyze the Mg-chelatase enzyme to produce Mg-protoporphyrin IX.'''</div>KonniamChanhttp://2007.igem.org/wiki/index.php/Berkeley_LBL/Results/ProteinGelBerkeley LBL/Results/ProteinGel2007-10-27T03:19:08Z<p>KonniamChan: /* Discussion */</p>
<hr />
<div>== Results of SDS-PAGE Gel of Soluble Proteins ==<br />
<br />
[[Berkeley_LBL/Ladder|Invitrogen BenchMark Protein Ladder]]<br />
<br />
[[Berkeley_LBL/Key|Plasmid Key for Protein Gels]]<br />
<br />
[[Image:ProteinGel1.jpg]]<br />
<br />
[[Image:Gel1Key.jpg]]<br />
<br />
[[Image:ProteinGel2.jpg]]<br />
<br />
[[Image:Gel2Key.jpg]]<br />
<br />
[[Image:ProteinGel3.jpg]]<br />
<br />
[[Image:Gel3Key.jpg]]<br />
<br />
[[Image:ProteinGel4.jpg]]<br />
<br />
[[Image:Gel4Key.jpg]]<br />
<br />
<br />
== '''Discussion''' ==<br />
<br />
'''''Prior to running the protein gels:'''''<br />
<br />
''*We expected to see protein bands for the constructs that contained:''<br />
<br />
1. -H gene (~140kDa)that were induced with IPTG<br />
<br />
2. -I gene(~38kDa) that were induced with IPTG<br />
<br />
''*We did not expect to see bands that coded for:''<br />
<br />
1. -D gene (~70kDa) <br />
<br />
2. - All constructs that were not induced with IPTG<br />
<br />
* Although the induced constructs that contained the -H gene had strong bands at ~140kDa, the uninduced constructs also showed strong bands in the same area. The -H gene was able to be expressed with or without induction of IPTG. <br />
<br />
* In addition, protein bands that coded for the -I gene showed a strong band, when alone. <br />
<br />
* However, when the -I gene was expressed with the -H gene, it did not clearly show a strong band at ~38 kDa in comparison to the benchmark ladder. <br />
<br />
* As expected, protein bands for the -D gene also did not show, alone nor in conjunction with the -H and -I gene. <br />
<br />
'''* Although our protein gels did not convey expression of neither the -I nor -D genes, our genes may still be expressed and may have enough activity to catalyze the Mg-chelatase enzyme to produce Mg-protoporphyrin IX.'''</div>KonniamChanhttp://2007.igem.org/wiki/index.php/Berkeley_LBL/MethodsBerkeley LBL/Methods2007-10-27T03:11:45Z<p>KonniamChan: /* Expression */</p>
<hr />
<div>{| border="0" cellspacing="8px" cellpadding="15" width="80%"<br />
|-<br />
|[[Berkeley_LBL|Home]]<br />
|[[Berkeley_LBL/Project|Project Description]]<br />
|[[Berkeley_LBL/Methods|Methods]]<br />
|[[Berkeley_LBL/Notebook|Notebook]]<br />
|[[Berkeley_LBL/Results|Results and Discussion]]<br />
|[[Berkeley_LBL/Resources|Resources]]<br />
|}<br />
<br />
<br />
==Experimental==<br />
<br />
=== Subcloning ===<br />
<br />
The genes of interest from ''Rhodobacter sphaeroides'', ''Synechocystis sp'', and ''Heliobacillus mobilis'' were amplified using PCR (the details of which can be found in Protocols). The PCR fragments were then digested and ligated with the T7 expression vector pET3a in various ways, resulting in the constructs shown below. They were transformed into ''E.Coli'' (either DH10B or NovaBlue) and glycerol stocks for these cells were saved.<br />
<br />
=== Constructs ===<br />
The T7 expression vector pET3a was used for the subcloning of the genes for magnesium-chelatase. The following constructs were built by inserting various fragments genes from the three organisms into pET3a.<br />
<br />
'''Magnesium-Chelatase'''<br><br />
''Rhodobacter sphaeroides''<br />
{| border="1"<br />
|-<br />
!Construct Name<br />
!Construct Details<br />
|-<br />
|pET3a-R-bchH<br />
|T7p-rbs-bchH-T7Term<br />
|-<br />
|pET3a-R-bchI<br />
|T7p-rbs-bchI-T7Term<br />
|-<br />
|pET3a-R-bchD<br />
|T7p-rbs-bchD-T7Term<br />
|-<br />
|pET3a-R-bchHID<br />
|T7p-rbs-bchH-rbs-bchI-rbs-bchD-T7Term<br />
|}<br />
<br />
<br />
''Synechocystis sp.''<br />
{| border="1"<br />
|-<br />
!Construct Name<br />
!Construct Details<br />
|-<br />
|pET3a-S-chlH<br />
|T7p-rbs-chlH-T7Term<br />
|-<br />
|pET3a-S-chlI<br />
|T7p-rbs-chlI-T7Term<br />
|-<br />
|pET3a-S-chlD<br />
|T7p-rbs-chlD-T7Term<br />
|-<br />
|pET3a-S-chlHID<br />
|T7p-rbs-chlH-rbs-chlI-rbs-chlD-T7Term<br />
|}<br />
<br />
<br />
'''Enzymes for reactions after Mg-insertion step'''<br><br />
<br />
''Heliobacillus mobilis'' (T7 expression vector pET29eBEBBX was used for subcloning of heliobacteria)<br />
{| border="1"<br />
|-<br />
!Construct Name<br />
!Construct Details<br />
|-<br />
|pET29-bchB<br />
|T7p-rbs-bchB-T7Term<br />
|-<br />
|pET29-bchE<br />
|T7p-rbs-bchE-T7Term<br />
|-<br />
|pET29-bchM<br />
|T7p-rbs-bchM-T7Term<br />
|-<br />
|pET29-bchN<br />
|T7p-rbs-bchN-T7Term<br />
|-<br />
|pET29-bchI<br />
|T7p-rbs-bchI-T7Term<br />
|-<br />
|pET29-bchL<br />
|T7p-rbs-bchL-T7Term<br />
|}<br />
<br />
=== Expression ===<br />
<br />
Once the constructs are inside DH10B or NovaBlue, they were minipreped and transformed in BL21 (DE3). These cells were then induced to overexpress the proteins of interest. After protein expression, the cell cultures were centrifuged at 10,000rpm for 20 minutes. The resulting cell pellets were dissolved in TRIS buffer (pH 7.8) and sonicated. The sonicated cell extracts were then centrifuged again, with the supernatant containing the soluble proteins. The soluble protein extract was used for enzyme activity assays. Along with the soluble protein extract, the total sonicated cell extract and the insoluble cell extract were analyzed on a SDS-PAGE gel. (all expressions were done by Joyce, Mimi, and Konniam; gel was done on 10/24/07)<br />
<br />
=== Enzyme Activity Assays ===<br />
<br />
The E.Coli cells with the constructs pET3a-R-bchHID and pET3a-S-bchHID have all the peptides necessary for the enzyme Mg-chelatase. The activity of these enzymes can be measured by the concentration of the reaction product, Mg-protoporphyrin IX. The concentrations were measured by the UV-vis spectroscopy and fluorescence emission spectroscopy, utilizing the Beer-Lambert law. By assuming that the concentration of the product is proportional to the activity of the enzymes, we can determine which enzyme (either from Rhodobacter sphaeroides or Synechocystis sp) is more efficient.<br />
<br />
<br />
== '''Subcloning Protocols''' ==<br />
<br />
'''''Polymerase Chain Reaction (PCR) Protocols:'''''<br />
<br />
[[Berkeley_LBL/PCRphusion|PCR (Using Phusion Polymerase)]]<br />
<br />
[[Berkeley_LBL/PCRextaq|PCR (Using TaKaRa Ex Taq Polymerase)]]<br />
<br />
'''''Clean Up/ Purification Protocols:'''''<br />
<br />
[[Berkeley_LBL/PCRcleanup|PCR Clean Up/Purification]]<br />
<br />
[[Berkeley_LBL/GelExtraction|Gel Extraction]]<br />
<br />
'''''DNA Gel Eletrophoresis Protocol:''''' <br />
<br />
[[Berkeley_LBL/DNAGelElectrophoresis|DNA Gel Electrophoresis]]<br />
<br />
''''''Restriction Digestion Protocols:''''''<br />
<br />
[[Berkeley_LBL/Digestion|Digestion for PCR Product or Miniprepped DNA]]<br />
<br />
[[Berkeley_LBL/Digestion2|Analytic Digestion]]<br />
<br />
''''''Ligation Protocol:''''''<br />
<br />
[[Berkeley_LBL/Ligation|Ligation]]<br />
<br />
'''''Transformation Protocols:'''''<br />
<br />
[[Berkeley_LBL/CompetentCell|KCM Competent Cell Production]]<br />
<br />
[[Berkeley_LBL/CompetentCellTransformation|KCM Competent Cell Transformation]]<br />
<br />
[[Berkeley_LBL/Electroporation|Electroporation Transformation]]<br />
<br />
'''''Miniprep Protocol:'''''<br />
<br />
[[Berkeley_LBL/Miniprep|Miniprep]]<br />
<br />
<br />
== '''Expression Protocols''' ==<br />
<br />
'''''Protein Expression Protocols:'''''<br />
<br />
[[Berkeley_LBL/Overexpression|Overexpression]]<br />
<br />
'''''Protein Analysis Protocols:'''''<br />
<br />
[[Berkeley_LBL/Sonication|Sonication]]<br />
<br />
[[Berkeley_LBL/SDS-PAGE|SDS-PAGE]]</div>KonniamChanhttp://2007.igem.org/wiki/index.php/Berkeley_LBL/MethodsBerkeley LBL/Methods2007-10-27T03:07:46Z<p>KonniamChan: /* Constructs */</p>
<hr />
<div>{| border="0" cellspacing="8px" cellpadding="15" width="80%"<br />
|-<br />
|[[Berkeley_LBL|Home]]<br />
|[[Berkeley_LBL/Project|Project Description]]<br />
|[[Berkeley_LBL/Methods|Methods]]<br />
|[[Berkeley_LBL/Notebook|Notebook]]<br />
|[[Berkeley_LBL/Results|Results and Discussion]]<br />
|[[Berkeley_LBL/Resources|Resources]]<br />
|}<br />
<br />
<br />
==Experimental==<br />
<br />
=== Subcloning ===<br />
<br />
The genes of interest from ''Rhodobacter sphaeroides'', ''Synechocystis sp'', and ''Heliobacillus mobilis'' were amplified using PCR (the details of which can be found in Protocols). The PCR fragments were then digested and ligated with the T7 expression vector pET3a in various ways, resulting in the constructs shown below. They were transformed into ''E.Coli'' (either DH10B or NovaBlue) and glycerol stocks for these cells were saved.<br />
<br />
=== Constructs ===<br />
The T7 expression vector pET3a was used for the subcloning of the genes for magnesium-chelatase. The following constructs were built by inserting various fragments genes from the three organisms into pET3a.<br />
<br />
'''Magnesium-Chelatase'''<br><br />
''Rhodobacter sphaeroides''<br />
{| border="1"<br />
|-<br />
!Construct Name<br />
!Construct Details<br />
|-<br />
|pET3a-R-bchH<br />
|T7p-rbs-bchH-T7Term<br />
|-<br />
|pET3a-R-bchI<br />
|T7p-rbs-bchI-T7Term<br />
|-<br />
|pET3a-R-bchD<br />
|T7p-rbs-bchD-T7Term<br />
|-<br />
|pET3a-R-bchHID<br />
|T7p-rbs-bchH-rbs-bchI-rbs-bchD-T7Term<br />
|}<br />
<br />
<br />
''Synechocystis sp.''<br />
{| border="1"<br />
|-<br />
!Construct Name<br />
!Construct Details<br />
|-<br />
|pET3a-S-chlH<br />
|T7p-rbs-chlH-T7Term<br />
|-<br />
|pET3a-S-chlI<br />
|T7p-rbs-chlI-T7Term<br />
|-<br />
|pET3a-S-chlD<br />
|T7p-rbs-chlD-T7Term<br />
|-<br />
|pET3a-S-chlHID<br />
|T7p-rbs-chlH-rbs-chlI-rbs-chlD-T7Term<br />
|}<br />
<br />
<br />
'''Enzymes for reactions after Mg-insertion step'''<br><br />
<br />
''Heliobacillus mobilis'' (T7 expression vector pET29eBEBBX was used for subcloning of heliobacteria)<br />
{| border="1"<br />
|-<br />
!Construct Name<br />
!Construct Details<br />
|-<br />
|pET29-bchB<br />
|T7p-rbs-bchB-T7Term<br />
|-<br />
|pET29-bchE<br />
|T7p-rbs-bchE-T7Term<br />
|-<br />
|pET29-bchM<br />
|T7p-rbs-bchM-T7Term<br />
|-<br />
|pET29-bchN<br />
|T7p-rbs-bchN-T7Term<br />
|-<br />
|pET29-bchI<br />
|T7p-rbs-bchI-T7Term<br />
|-<br />
|pET29-bchL<br />
|T7p-rbs-bchL-T7Term<br />
|}<br />
<br />
=== Expression ===<br />
<br />
Once the constructs are inside DH10B or NovaBlue, they were minipreped and transformed in BL21 (DE3). These cells were then induced to overexpress the proteins of interest. After protein expression, the cell cultures were centrifuged at 10,000rpm for 20 minutes. The resulting cell pellets were dissolved in TRIS buffer (pH 7.8) and sonicated. The sonicated cell extracts were then centrifuged again, with the supernatant containing the soluble proteins. The soluble protein extract was used for enzyme activity assays. Along with the soluble protein extract, the total sonicated cell extract and the insoluble cell extract were analyzed on a SDS-PAGE gel.<br />
<br />
=== Enzyme Activity Assays ===<br />
<br />
The E.Coli cells with the constructs pET3a-R-bchHID and pET3a-S-bchHID have all the peptides necessary for the enzyme Mg-chelatase. The activity of these enzymes can be measured by the concentration of the reaction product, Mg-protoporphyrin IX. The concentrations were measured by the UV-vis spectroscopy and fluorescence emission spectroscopy, utilizing the Beer-Lambert law. By assuming that the concentration of the product is proportional to the activity of the enzymes, we can determine which enzyme (either from Rhodobacter sphaeroides or Synechocystis sp) is more efficient.<br />
<br />
<br />
== '''Subcloning Protocols''' ==<br />
<br />
'''''Polymerase Chain Reaction (PCR) Protocols:'''''<br />
<br />
[[Berkeley_LBL/PCRphusion|PCR (Using Phusion Polymerase)]]<br />
<br />
[[Berkeley_LBL/PCRextaq|PCR (Using TaKaRa Ex Taq Polymerase)]]<br />
<br />
'''''Clean Up/ Purification Protocols:'''''<br />
<br />
[[Berkeley_LBL/PCRcleanup|PCR Clean Up/Purification]]<br />
<br />
[[Berkeley_LBL/GelExtraction|Gel Extraction]]<br />
<br />
'''''DNA Gel Eletrophoresis Protocol:''''' <br />
<br />
[[Berkeley_LBL/DNAGelElectrophoresis|DNA Gel Electrophoresis]]<br />
<br />
''''''Restriction Digestion Protocols:''''''<br />
<br />
[[Berkeley_LBL/Digestion|Digestion for PCR Product or Miniprepped DNA]]<br />
<br />
[[Berkeley_LBL/Digestion2|Analytic Digestion]]<br />
<br />
''''''Ligation Protocol:''''''<br />
<br />
[[Berkeley_LBL/Ligation|Ligation]]<br />
<br />
'''''Transformation Protocols:'''''<br />
<br />
[[Berkeley_LBL/CompetentCell|KCM Competent Cell Production]]<br />
<br />
[[Berkeley_LBL/CompetentCellTransformation|KCM Competent Cell Transformation]]<br />
<br />
[[Berkeley_LBL/Electroporation|Electroporation Transformation]]<br />
<br />
'''''Miniprep Protocol:'''''<br />
<br />
[[Berkeley_LBL/Miniprep|Miniprep]]<br />
<br />
<br />
== '''Expression Protocols''' ==<br />
<br />
'''''Protein Expression Protocols:'''''<br />
<br />
[[Berkeley_LBL/Overexpression|Overexpression]]<br />
<br />
'''''Protein Analysis Protocols:'''''<br />
<br />
[[Berkeley_LBL/Sonication|Sonication]]<br />
<br />
[[Berkeley_LBL/SDS-PAGE|SDS-PAGE]]</div>KonniamChanhttp://2007.igem.org/wiki/index.php/Berkeley_LBL/Results/ProteinGelBerkeley LBL/Results/ProteinGel2007-10-27T02:57:06Z<p>KonniamChan: </p>
<hr />
<div>== Results of SDS-PAGE Gel of Soluble Proteins ==<br />
<br />
[[Berkeley_LBL/Ladder|Invitrogen BenchMark Protein Ladder]]<br />
<br />
[[Berkeley_LBL/Key|Plasmid Key for Protein Gels]]<br />
<br />
[[Image:ProteinGel1.jpg]]<br />
<br />
[[Image:Gel1Key.jpg]]<br />
<br />
[[Image:ProteinGel2.jpg]]<br />
<br />
[[Image:Gel2Key.jpg]]<br />
<br />
[[Image:ProteinGel3.jpg]]<br />
<br />
[[Image:Gel3Key.jpg]]<br />
<br />
[[Image:ProteinGel4.jpg]]<br />
<br />
[[Image:Gel4Key.jpg]]<br />
<br />
<br />
== Discussion ==<br />
<br />
Prior to running the protein gels, we expected to see protein bands for the constructs that contained the -H and -I gene(~140kDa and ~38kDa, respectively) and were also induced with IPTG . We did not expect to see bands that coded for the -D gene (~70kDa) and for all constructs that were not induced with IPTG. Although the induced constructs that contained the -H gene had strong bands at ~140kDa, the uninduced constructs also showed strong bands in the same area. This gene was able to be expressed with or without induction of IPTG. In addition, protein bands that coded for the -I gene showed a strong band; however, when expressed with the -H gene, it did not clearly show a strong band at ~38 kDa in comparison to the benchmark ladder. As expected, protein bands for the -D gene also did not show, alone nor in conjunction with the -H and -I gene. Although our protein gels did not convey expression of neither the -I nor -D genes, our genes may still be expressed and may have enough activity to catalyze the Mg-chelatase enzyme to produce Mg-protoporphyrin IX.</div>KonniamChanhttp://2007.igem.org/wiki/index.php/Berkeley_LBL/ResultsBerkeley LBL/Results2007-10-27T02:56:40Z<p>KonniamChan: </p>
<hr />
<div>{| border="0" cellspacing="8px" cellpadding="15" width="80%"<br />
|-<br />
|[[Berkeley_LBL|Home]]<br />
|[[Berkeley_LBL/Project|Project Description]]<br />
|[[Berkeley_LBL/Methods|Methods]]<br />
|[[Berkeley_LBL/Notebook|Notebook]]<br />
|[[Berkeley_LBL/Results|Results and Discussion]]<br />
|[[Berkeley_LBL/Resources|Resources]]<br />
|}<br />
<br />
[[Berkeley_LBL/Results/ProteinGel|SDS-PAGE Gel of Soluble Proteins]]</div>KonniamChanhttp://2007.igem.org/wiki/index.php/Berkeley_LBL/Results/ProteinGelBerkeley LBL/Results/ProteinGel2007-10-27T02:56:01Z<p>KonniamChan: </p>
<hr />
<div>== Results ==<br />
<br />
[[Berkeley_LBL/Ladder|Invitrogen BenchMark Protein Ladder]]<br />
<br />
[[Berkeley_LBL/Key|Plasmid Key for Protein Gels]]<br />
<br />
[[Image:ProteinGel1.jpg]]<br />
<br />
[[Image:Gel1Key.jpg]]<br />
<br />
[[Image:ProteinGel2.jpg]]<br />
<br />
[[Image:Gel2Key.jpg]]<br />
<br />
[[Image:ProteinGel3.jpg]]<br />
<br />
[[Image:Gel3Key.jpg]]<br />
<br />
[[Image:ProteinGel4.jpg]]<br />
<br />
[[Image:Gel4Key.jpg]]<br />
<br />
<br />
== Discussion ==<br />
<br />
Prior to running the protein gels, we expected to see protein bands for the constructs that contained the -H and -I gene(~140kDa and ~38kDa, respectively) and were also induced with IPTG . We did not expect to see bands that coded for the -D gene (~70kDa) and for all constructs that were not induced with IPTG. Although the induced constructs that contained the -H gene had strong bands at ~140kDa, the uninduced constructs also showed strong bands in the same area. This gene was able to be expressed with or without induction of IPTG. In addition, protein bands that coded for the -I gene showed a strong band; however, when expressed with the -H gene, it did not clearly show a strong band at ~38 kDa in comparison to the benchmark ladder. As expected, protein bands for the -D gene also did not show, alone nor in conjunction with the -H and -I gene. Although our protein gels did not convey expression of neither the -I nor -D genes, our genes may still be expressed and may have enough activity to catalyze the Mg-chelatase enzyme to produce Mg-protoporphyrin IX.</div>KonniamChanhttp://2007.igem.org/wiki/index.php/Berkeley_LBL/ResultsBerkeley LBL/Results2007-10-27T02:55:19Z<p>KonniamChan: </p>
<hr />
<div>{| border="0" cellspacing="8px" cellpadding="15" width="80%"<br />
|-<br />
|[[Berkeley_LBL|Home]]<br />
|[[Berkeley_LBL/Project|Project Description]]<br />
|[[Berkeley_LBL/Methods|Methods]]<br />
|[[Berkeley_LBL/Notebook|Notebook]]<br />
|[[Berkeley_LBL/Results|Results and Discussion]]<br />
|[[Berkeley_LBL/Resources|Resources]]<br />
|}<br />
<br />
[[Berkeley_LBL/Results/ProteinGel|SDS-PAGE Gel of Soluble Proteins]]<br />
== Results ==<br />
<br />
[[Berkeley_LBL/Ladder|Invitrogen BenchMark Protein Ladder]]<br />
<br />
[[Berkeley_LBL/Key|Plasmid Key for Protein Gels]]<br />
<br />
[[Image:ProteinGel1.jpg]]<br />
<br />
[[Image:Gel1Key.jpg]]<br />
<br />
[[Image:ProteinGel2.jpg]]<br />
<br />
[[Image:Gel2Key.jpg]]<br />
<br />
[[Image:ProteinGel3.jpg]]<br />
<br />
[[Image:Gel3Key.jpg]]<br />
<br />
[[Image:ProteinGel4.jpg]]<br />
<br />
[[Image:Gel4Key.jpg]]<br />
<br />
<br />
== Discussion ==<br />
<br />
Prior to running the protein gels, we expected to see protein bands for the constructs that contained the -H and -I gene(~140kDa and ~38kDa, respectively) and were also induced with IPTG . We did not expect to see bands that coded for the -D gene (~70kDa) and for all constructs that were not induced with IPTG. Although the induced constructs that contained the -H gene had strong bands at ~140kDa, the uninduced constructs also showed strong bands in the same area. This gene was able to be expressed with or without induction of IPTG. In addition, protein bands that coded for the -I gene showed a strong band; however, when expressed with the -H gene, it did not clearly show a strong band at ~38 kDa in comparison to the benchmark ladder. As expected, protein bands for the -D gene also did not show, alone nor in conjunction with the -H and -I gene. Although our protein gels did not convey expression of neither the -I nor -D genes, our genes may still be expressed and may have enough activity to catalyze the Mg-chelatase enzyme to produce Mg-protoporphyrin IX.</div>KonniamChanhttp://2007.igem.org/wiki/index.php/Berkeley_LBL/KonniamNotebookBerkeley LBL/KonniamNotebook2007-10-27T02:49:45Z<p>KonniamChan: /* Construction of pET3A Derivatives Containing R-bchHID */</p>
<hr />
<div>==Construction of pET3A Derivatives Containing R-bchHID==<br />
===Construction of pET3a-R-bchH===<br />
1.)PCR the gene bchH from Rhodobacter sphaeroides<br><br />
Materials:<br />
*1uL R-genomic DNA<br />
*10uL 5x GC buffer<br />
*5uL primer<br />
*0.5uL Phusion<br />
*5uL DMSO<br />
*1uL dNTP<br />
*27.5uL H2O<br />
Conditions:<br />
*98C 30s<br />
*98C 10s*<br />
*63C 30s*<br />
*72C 1:50 min*<br />
*Repeat cycles with * 29x<br />
*72C 10 min<br />
*4C forever<br />
<br />
2.)Run PCR fragments on gel to make sure they are the correct size, then PCR purify<br><br />
3.)Digest R-bchH (8/21/07)<br><br />
*42uL PCR purified fragment<br />
*5uL NEB3<br />
*1.8uL NdeI<br />
*1.2uL BglII<br />
4.)Run the digested PCR fragment on gel, checking that the size is correct (8/21/07)<br><br />
5.)Ligate R-bchH with pET3a that is already digested with NdeI and BamHI (8/21/07)<br><br />
*4.5uL pET3a<br />
*12.5uL R-bchH fragment<br />
*2uL T4 ligase buffer<br />
*1uL T4 ligase<br />
6.)Transform into NovaBlue (8/21/07)<br><br />
7.)Inoculate to LB media (pick 10 colonies) (8/22/07)<br><br />
8.)Miniprep cells with pET3a-R-bchH (8/23/07)<br><br />
9.)Sequence<br />
<br />
===Construction of pET3a-R-bchHI===<br />
1.)PCR the gene bchI from Rhodobacter sphaeroides (7/19/07)<br><br />
Materials:<br />
*1uL R-genomic DNA<br />
*10uL 5x HF buffer<br />
*5uL primer<br />
*0.5uL Phusion<br />
*25uL DMSO<br />
*1uL dNTP<br />
*30uL H2O<br />
Conditions:<br />
*98C 30s<br />
*98C 10s*<br />
*62C 30s*<br />
*72C 32s min*<br />
*Repeat cycles with * 29x<br />
*72C 10 min<br />
*4C forever<br />
<br />
2.)Run PCR fragments on gel to make sure they are the correct size, then PCR purify<br><br />
3.)Digest R-bchI with KpnI and BglII(7/20/07)<br><br />
4.)Run the digested PCR fragment on gel, checking that the size is correct (7/21/07)<br><br />
5.)Ligate R-bchH with pET3a-R-bchH that is already digested with KpnI and NsiI(8/28/07)<br><br />
*4.5uL pET3a-R-bchH<br />
*12.5uL R-bchI fragment<br />
*2uL T4 ligase buffer<br />
*1uL T4 ligase<br />
6.)Transform into NovaBlue (9/3/07)<br><br />
7.)Inoculate to LB media (pick 10 colonies) (9/4/07)<br><br />
8.)Miniprep cells with pET3a-R-bchH (9/14/07)<br><br />
9.)Sequence<br />
<br />
===Construction of pET3a-R-bchHID===<br />
1.)PCR the gene bchD from Rhodobacter sphaeroides (7/20/07)<br><br />
Materials:<br />
*1uL R-genomic DNA<br />
*10uL 5x GC buffer<br />
*5uL primer<br />
*0.5uL Phusion<br />
*5uL DMSO<br />
*1uL dNTP<br />
*27.5uL H2O<br />
Conditions:<br />
*98C 30s<br />
*98C 10s*<br />
*62C 30s*<br />
*72C 1 min*<br />
*Repeat cycles with * 29x<br />
*72C 10 min<br />
*4C forever<br />
<br />
2.)Run PCR fragments on gel to make sure they are the correct size, then PCR purify (7/20/07)<br><br />
3.)Digest R-bchD with SpeI and NsiI (7/23/07)<br><br />
4.)Run the digested PCR fragment on gel, checking that the size is correct (7/23/07)<br><br />
5.)Ligate R-bchD with pET3a-R-bchHI that is already digested with SpeI and NsiI(9/17/07)<br><br />
*4.5uL pET3a-R-bchHI<br />
*12.5uL R-bchD fragment<br />
*2uL T4 ligase buffer<br />
*1uL T4 ligase<br />
6.)Transform into NovaBlue (9/17/07)<br><br />
7.)Inoculate to LB media (pick 10 colonies) (9/18/07)<br><br />
8.)Miniprep cells with pET3a-R-bchH (9/19/07)<br><br />
9.)Sequence</div>KonniamChanhttp://2007.igem.org/wiki/index.php/Berkeley_LBL/ResultsBerkeley LBL/Results2007-10-27T02:49:00Z<p>KonniamChan: /* Discussion */</p>
<hr />
<div>{| border="0" cellspacing="8px" cellpadding="15" width="80%"<br />
|-<br />
|[[Berkeley_LBL|Home]]<br />
|[[Berkeley_LBL/Project|Project Description]]<br />
|[[Berkeley_LBL/Methods|Methods]]<br />
|[[Berkeley_LBL/Notebook|Notebook]]<br />
|[[Berkeley_LBL/Results|Results and Discussion]]<br />
|[[Berkeley_LBL/Resources|Resources]]<br />
|}<br />
<br />
== Results ==<br />
<br />
[[Berkeley_LBL/Ladder|Invitrogen BenchMark Protein Ladder]]<br />
<br />
[[Berkeley_LBL/Key|Plasmid Key for Protein Gels]]<br />
<br />
[[Image:ProteinGel1.jpg]]<br />
<br />
[[Image:Gel1Key.jpg]]<br />
<br />
[[Image:ProteinGel2.jpg]]<br />
<br />
[[Image:Gel2Key.jpg]]<br />
<br />
[[Image:ProteinGel3.jpg]]<br />
<br />
[[Image:Gel3Key.jpg]]<br />
<br />
[[Image:ProteinGel4.jpg]]<br />
<br />
[[Image:Gel4Key.jpg]]<br />
<br />
<br />
== Discussion ==<br />
<br />
Prior to running the protein gels, we expected to see protein bands for the constructs that contained the -H and -I gene(~140kDa and ~38kDa, respectively) and were also induced with IPTG . We did not expect to see bands that coded for the -D gene (~70kDa) and for all constructs that were not induced with IPTG. Although the induced constructs that contained the -H gene had strong bands at ~140kDa, the uninduced constructs also showed strong bands in the same area. This gene was able to be expressed with or without induction of IPTG. In addition, protein bands that coded for the -I gene showed a strong band; however, when expressed with the -H gene, it did not clearly show a strong band at ~38 kDa in comparison to the benchmark ladder. As expected, protein bands for the -D gene also did not show, alone nor in conjunction with the -H and -I gene. Although our protein gels did not convey expression of neither the -I nor -D genes, our genes may still be expressed and may have enough activity to catalyze the Mg-chelatase enzyme to produce Mg-protoporphyrin IX.</div>KonniamChanhttp://2007.igem.org/wiki/index.php/Berkeley_LBL/MethodsBerkeley LBL/Methods2007-10-27T02:47:22Z<p>KonniamChan: /* Expression Protocols */</p>
<hr />
<div>{| border="0" cellspacing="8px" cellpadding="15" width="80%"<br />
|-<br />
|[[Berkeley_LBL|Home]]<br />
|[[Berkeley_LBL/Project|Project Description]]<br />
|[[Berkeley_LBL/Methods|Methods]]<br />
|[[Berkeley_LBL/Notebook|Notebook]]<br />
|[[Berkeley_LBL/Results|Results and Discussion]]<br />
|[[Berkeley_LBL/Resources|Resources]]<br />
|}<br />
<br />
<br />
==Experimental==<br />
<br />
=== Subcloning ===<br />
<br />
The genes of interest from ''Rhodobacter sphaeroides'', ''Synechocystis sp'', and ''Heliobacillus mobilis'' were amplified using PCR (the details of which can be found in Protocols). The PCR fragments were then digested and ligated with the T7 expression vector pET3a in various ways, resulting in the constructs shown below. They were transformed into ''E.Coli'' (either DH10B or NovaBlue) and glycerol stocks for these cells were saved.<br />
<br />
=== Constructs ===<br />
The T7 expression vector pET3a was used for the subcloning of the genes for magnesium-chelatase. The following constructs were built by inserting various fragments genes from the three organisms into pET3a.<br />
<br />
'''Magnesium-Chelatase'''<br><br />
''Rhodobacter sphaeroides''<br />
{| border="1"<br />
|-<br />
!Construct Name<br />
!Construct Details<br />
|-<br />
|pET3a-R-bchH<br />
|T7p-rbs-bchH-T7Term<br />
|-<br />
|pET3a-R-bchI<br />
|T7p-rbs-bchI-T7Term<br />
|-<br />
|pET3a-R-bchD<br />
|T7p-rbs-bchD-T7Term<br />
|-<br />
|pET3a-R-bchHID<br />
|T7p-rbs-bchH-rbs-bchI-rbs-bchD-T7Term<br />
|}<br />
<br />
<br />
''Synechocystis sp.''<br />
{| border="1"<br />
|-<br />
!Construct Name<br />
!Construct Details<br />
|-<br />
|pET3a-S-chlH<br />
|T7p-rbs-chlH-T7Term<br />
|-<br />
|pET3a-S-chlI<br />
|T7p-rbs-chlI-T7Term<br />
|-<br />
|pET3a-S-chlD<br />
|T7p-rbs-chlD-T7Term<br />
|-<br />
|pET3a-S-chlHID<br />
|T7p-rbs-chlH-rbs-chlI-rbs-chlD-T7Term<br />
|}<br />
<br />
'''Enzymes for reactions after Mg-insertion step'''<br><br />
<br />
''Heliobacillus mobilis''<br />
<br />
=== Expression ===<br />
<br />
Once the constructs are inside DH10B or NovaBlue, they were minipreped and transformed in BL21 (DE3). These cells were then induced to overexpress the proteins of interest. After protein expression, the cell cultures were centrifuged at 10,000rpm for 20 minutes. The resulting cell pellets were dissolved in TRIS buffer (pH 7.8) and sonicated. The sonicated cell extracts were then centrifuged again, with the supernatant containing the soluble proteins. The soluble protein extract was used for enzyme activity assays. Along with the soluble protein extract, the total sonicated cell extract and the insoluble cell extract were analyzed on a SDS-PAGE gel.<br />
<br />
=== Enzyme Activity Assays ===<br />
<br />
The E.Coli cells with the constructs pET3a-R-bchHID and pET3a-S-bchHID have all the peptides necessary for the enzyme Mg-chelatase. The activity of these enzymes can be measured by the concentration of the reaction product, Mg-protoporphyrin IX. The concentrations were measured by the UV-vis spectroscopy and fluorescence emission spectroscopy, utilizing the Beer-Lambert law. By assuming that the concentration of the product is proportional to the activity of the enzymes, we can determine which enzyme (either from Rhodobacter sphaeroides or Synechocystis sp) is more efficient.<br />
<br />
<br />
== '''Subcloning Protocols''' ==<br />
<br />
'''''Polymerase Chain Reaction (PCR) Protocols:'''''<br />
<br />
[[Berkeley_LBL/PCRphusion|PCR (Using Phusion Polymerase)]]<br />
<br />
[[Berkeley_LBL/PCRextaq|PCR (Using TaKaRa Ex Taq Polymerase)]]<br />
<br />
'''''Clean Up/ Purification Protocols:'''''<br />
<br />
[[Berkeley_LBL/PCRcleanup|PCR Clean Up/Purification]]<br />
<br />
[[Berkeley_LBL/GelExtraction|Gel Extraction]]<br />
<br />
'''''DNA Gel Eletrophoresis Protocol:''''' <br />
<br />
[[Berkeley_LBL/DNAGelElectrophoresis|DNA Gel Electrophoresis]]<br />
<br />
''''''Restriction Digestion Protocols:''''''<br />
<br />
[[Berkeley_LBL/Digestion|Digestion for PCR Product or Miniprepped DNA]]<br />
<br />
[[Berkeley_LBL/Digestion2|Analytic Digestion]]<br />
<br />
''''''Ligation Protocol:''''''<br />
<br />
[[Berkeley_LBL/Ligation|Ligation]]<br />
<br />
'''''Transformation Protocols:'''''<br />
<br />
[[Berkeley_LBL/CompetentCell|KCM Competent Cell Production]]<br />
<br />
[[Berkeley_LBL/CompetentCellTransformation|KCM Competent Cell Transformation]]<br />
<br />
[[Berkeley_LBL/Electroporation|Electroporation Transformation]]<br />
<br />
'''''Miniprep Protocol:'''''<br />
<br />
[[Berkeley_LBL/Miniprep|Miniprep]]<br />
<br />
<br />
== '''Expression Protocols''' ==<br />
<br />
'''''Protein Expression Protocols:'''''<br />
<br />
[[Berkeley_LBL/Overexpression|Overexpression]]<br />
<br />
'''''Protein Analysis Protocols:'''''<br />
<br />
[[Berkeley_LBL/Sonication|Sonication]]<br />
<br />
[[Berkeley_LBL/SDS-PAGE|SDS-PAGE]]</div>KonniamChanhttp://2007.igem.org/wiki/index.php/Berkeley_LBL/ResultsBerkeley LBL/Results2007-10-27T02:46:40Z<p>KonniamChan: /* Discussion */</p>
<hr />
<div>{| border="0" cellspacing="8px" cellpadding="15" width="80%"<br />
|-<br />
|[[Berkeley_LBL|Home]]<br />
|[[Berkeley_LBL/Project|Project Description]]<br />
|[[Berkeley_LBL/Methods|Methods]]<br />
|[[Berkeley_LBL/Notebook|Notebook]]<br />
|[[Berkeley_LBL/Results|Results and Discussion]]<br />
|[[Berkeley_LBL/Resources|Resources]]<br />
|}<br />
<br />
== Results ==<br />
<br />
[[Berkeley_LBL/Ladder|Invitrogen BenchMark Protein Ladder]]<br />
<br />
[[Berkeley_LBL/Key|Plasmid Key for Protein Gels]]<br />
<br />
[[Image:ProteinGel1.jpg]]<br />
<br />
[[Image:Gel1Key.jpg]]<br />
<br />
[[Image:ProteinGel2.jpg]]<br />
<br />
[[Image:Gel2Key.jpg]]<br />
<br />
[[Image:ProteinGel3.jpg]]<br />
<br />
[[Image:Gel3Key.jpg]]<br />
<br />
[[Image:ProteinGel4.jpg]]<br />
<br />
[[Image:Gel4Key.jpg]]<br />
<br />
<br />
== Discussion ==<br />
<br />
Prior to running the protein gels, we expected to see protein bands for the constructs that contained the -H and -I gene(~140kDa and ~38kDa, respectively) and were also induced with IPTG . We did not expect to see bands that coded for the -D gene (~70kDa) and for constructs that were not induced with IPTG. Although the induced constructs that contained the -H gene had strong bands at ~140kDa, the uninduced constructs also showed strong bands in the same area. This gene was able to be expressed with or without induction of IPTG. In addition, protein bands that coded for the -I gene showed a strong band; however, when expressed with the -H gene, it did not clearly show a strong band at ~38 kDa in comparison to the benchmark ladder. As expected, protein bands for the -D gene also did not show, alone nor in conjunction with the -H and -I gene. Although our protein gels did not convey expression of neither the -I nor -D genes, our genes may still be expressed and may have enough activity to catalyze the Mg-chelatase enzyme to produce Mg-protoporphyrin IX.</div>KonniamChanhttp://2007.igem.org/wiki/index.php/Berkeley_LBL/KonniamNotebookBerkeley LBL/KonniamNotebook2007-10-27T02:44:05Z<p>KonniamChan: /* Construction of pET3a-R-bchHI */</p>
<hr />
<div>==Construction of pET3A Derivatives Containing R-bchHID==<br />
===Construction of pET3a-R-bchH===<br />
1.)PCR the gene bchH from Rhodobacter sphaeroides<br><br />
Materials:<br />
*1uL R-genomic DNA<br />
*10uL 5x GC buffer<br />
*5uL primer<br />
*0.5uL Phusion<br />
*5uL DMSO<br />
*1uL dNTP<br />
*27.5uL H2O<br />
Conditions:<br />
*98C 30s<br />
*98C 10s*<br />
*63C 30s*<br />
*72C 1:50 min*<br />
*Repeat cycles with * 29x<br />
*72C 10 min<br />
*4C forever<br />
<br />
2.)Run PCR fragments on gel to make sure they are the correct size, then PCR purify<br><br />
3.)Digest R-bchH (8/21/07)<br><br />
*42uL PCR purified fragment<br />
*5uL NEB3<br />
*1.8uL NdeI<br />
*1.2uL BglII<br />
4.)Run the digested PCR fragment on gel, checking that the size is correct (8/21/07)<br><br />
5.)Ligate R-bchH with pET3a that is already digested with NdeI and BamHI (8/21/07)<br><br />
*4.5uL pET3a<br />
*12.5uL R-bchH fragment<br />
*2uL T4 ligase buffer<br />
*1uL T4 ligase<br />
6.)Transform into NovaBlue (8/21/07)<br><br />
7.)Inoculate to LB media (pick 10 colonies) (8/22/07)<br><br />
8.)Miniprep cells with pET3a-R-bchH (8/23/07)<br><br />
9.)Sequence<br />
<br />
===Construction of pET3a-R-bchHI===<br />
1.)PCR the gene bchI from Rhodobacter sphaeroides (7/19/07)<br><br />
Materials:<br />
*1uL R-genomic DNA<br />
*10uL 5x HF buffer<br />
*5uL primer<br />
*0.5uL Phusion<br />
*25uL DMSO<br />
*1uL dNTP<br />
*30uL H2O<br />
Conditions:<br />
*98C 30s<br />
*98C 10s*<br />
*62C 30s*<br />
*72C 32s min*<br />
*Repeat cycles with * 29x<br />
*72C 10 min<br />
*4C forever<br />
<br />
2.)Run PCR fragments on gel to make sure they are the correct size, then PCR purify<br><br />
3.)Digest R-bchI (7/20/07) with KpnI and BglII<br><br />
4.)Run the digested PCR fragment on gel, checking that the size is correct (7/21/07)<br><br />
5.)Ligate R-bchH with pET3a-R-bchH that is already digested with KpnI and NsiI(8/28/07)<br><br />
*4.5uL pET3a-R-bchH<br />
*12.5uL R-bchI fragment<br />
*2uL T4 ligase buffer<br />
*1uL T4 ligase<br />
6.)Transform into NovaBlue (9/3/07)<br><br />
7.)Inoculate to LB media (pick 10 colonies) (9/4/07)<br><br />
8.)Miniprep cells with pET3a-R-bchH (9/14/07)<br><br />
9.)Sequence</div>KonniamChanhttp://2007.igem.org/wiki/index.php/Berkeley_LBL/KonniamNotebookBerkeley LBL/KonniamNotebook2007-10-27T02:43:55Z<p>KonniamChan: /* Construction of pET3a-R-bchH */</p>
<hr />
<div>==Construction of pET3A Derivatives Containing R-bchHID==<br />
===Construction of pET3a-R-bchH===<br />
1.)PCR the gene bchH from Rhodobacter sphaeroides<br><br />
Materials:<br />
*1uL R-genomic DNA<br />
*10uL 5x GC buffer<br />
*5uL primer<br />
*0.5uL Phusion<br />
*5uL DMSO<br />
*1uL dNTP<br />
*27.5uL H2O<br />
Conditions:<br />
*98C 30s<br />
*98C 10s*<br />
*63C 30s*<br />
*72C 1:50 min*<br />
*Repeat cycles with * 29x<br />
*72C 10 min<br />
*4C forever<br />
<br />
2.)Run PCR fragments on gel to make sure they are the correct size, then PCR purify<br><br />
3.)Digest R-bchH (8/21/07)<br><br />
*42uL PCR purified fragment<br />
*5uL NEB3<br />
*1.8uL NdeI<br />
*1.2uL BglII<br />
4.)Run the digested PCR fragment on gel, checking that the size is correct (8/21/07)<br><br />
5.)Ligate R-bchH with pET3a that is already digested with NdeI and BamHI (8/21/07)<br><br />
*4.5uL pET3a<br />
*12.5uL R-bchH fragment<br />
*2uL T4 ligase buffer<br />
*1uL T4 ligase<br />
6.)Transform into NovaBlue (8/21/07)<br><br />
7.)Inoculate to LB media (pick 10 colonies) (8/22/07)<br><br />
8.)Miniprep cells with pET3a-R-bchH (8/23/07)<br><br />
9.)Sequence<br />
<br />
==Construction of pET3a-R-bchHI==<br />
1.)PCR the gene bchI from Rhodobacter sphaeroides (7/19/07)<br><br />
Materials:<br />
*1uL R-genomic DNA<br />
*10uL 5x HF buffer<br />
*5uL primer<br />
*0.5uL Phusion<br />
*25uL DMSO<br />
*1uL dNTP<br />
*30uL H2O<br />
Conditions:<br />
*98C 30s<br />
*98C 10s*<br />
*62C 30s*<br />
*72C 32s min*<br />
*Repeat cycles with * 29x<br />
*72C 10 min<br />
*4C forever<br />
<br />
2.)Run PCR fragments on gel to make sure they are the correct size, then PCR purify<br><br />
3.)Digest R-bchI (7/20/07) with KpnI and BglII<br><br />
4.)Run the digested PCR fragment on gel, checking that the size is correct (7/21/07)<br><br />
5.)Ligate R-bchH with pET3a-R-bchH that is already digested with KpnI and NsiI(8/28/07)<br><br />
*4.5uL pET3a-R-bchH<br />
*12.5uL R-bchI fragment<br />
*2uL T4 ligase buffer<br />
*1uL T4 ligase<br />
6.)Transform into NovaBlue (9/3/07)<br><br />
7.)Inoculate to LB media (pick 10 colonies) (9/4/07)<br><br />
8.)Miniprep cells with pET3a-R-bchH (9/14/07)<br><br />
9.)Sequence</div>KonniamChanhttp://2007.igem.org/wiki/index.php/Berkeley_LBL/KonniamNotebookBerkeley LBL/KonniamNotebook2007-10-27T02:43:00Z<p>KonniamChan: /* Construction of pET3a-R-bchH */</p>
<hr />
<div>==Construction of pET3A Derivatives Containing R-bchHID==<br />
==Construction of pET3a-R-bchH==<br />
1.)PCR the gene bchH from Rhodobacter sphaeroides<br><br />
Materials:<br />
*1uL R-genomic DNA<br />
*10uL 5x GC buffer<br />
*5uL primer<br />
*0.5uL Phusion<br />
*5uL DMSO<br />
*1uL dNTP<br />
*27.5uL H2O<br />
Conditions:<br />
*98C 30s<br />
*98C 10s*<br />
*63C 30s*<br />
*72C 1:50 min*<br />
*Repeat cycles with * 29x<br />
*72C 10 min<br />
*4C forever<br />
<br />
2.)Run PCR fragments on gel to make sure they are the correct size, then PCR purify<br><br />
3.)Digest R-bchH (8/21/07)<br><br />
*42uL PCR purified fragment<br />
*5uL NEB3<br />
*1.8uL NdeI<br />
*1.2uL BglII<br />
4.)Run the digested PCR fragment on gel, checking that the size is correct (8/21/07)<br><br />
5.)Ligate R-bchH with pET3a that is already digested with NdeI and BamHI (8/21/07)<br><br />
*4.5uL pET3a<br />
*12.5uL R-bchH fragment<br />
*2uL T4 ligase buffer<br />
*1uL T4 ligase<br />
6.)Transform into NovaBlue (8/21/07)<br><br />
7.)Inoculate to LB media (pick 10 colonies) (8/22/07)<br><br />
8.)Miniprep cells with pET3a-R-bchH (8/23/07)<br><br />
9.)Sequence<br />
<br />
==Construction of pET3a-R-bchHI==<br />
1.)PCR the gene bchI from Rhodobacter sphaeroides (7/19/07)<br><br />
Materials:<br />
*1uL R-genomic DNA<br />
*10uL 5x HF buffer<br />
*5uL primer<br />
*0.5uL Phusion<br />
*25uL DMSO<br />
*1uL dNTP<br />
*30uL H2O<br />
Conditions:<br />
*98C 30s<br />
*98C 10s*<br />
*62C 30s*<br />
*72C 32s min*<br />
*Repeat cycles with * 29x<br />
*72C 10 min<br />
*4C forever<br />
<br />
2.)Run PCR fragments on gel to make sure they are the correct size, then PCR purify<br><br />
3.)Digest R-bchI (7/20/07) with KpnI and BglII<br><br />
4.)Run the digested PCR fragment on gel, checking that the size is correct (7/21/07)<br><br />
5.)Ligate R-bchH with pET3a-R-bchH that is already digested with KpnI and NsiI(8/28/07)<br><br />
*4.5uL pET3a-R-bchH<br />
*12.5uL R-bchI fragment<br />
*2uL T4 ligase buffer<br />
*1uL T4 ligase<br />
6.)Transform into NovaBlue (9/3/07)<br><br />
7.)Inoculate to LB media (pick 10 colonies) (9/4/07)<br><br />
8.)Miniprep cells with pET3a-R-bchH (9/14/07)<br><br />
9.)Sequence</div>KonniamChanhttp://2007.igem.org/wiki/index.php/Berkeley_LBL/KonniamNotebookBerkeley LBL/KonniamNotebook2007-10-27T02:42:40Z<p>KonniamChan: /* Construction of pET3a-R-bchHI */</p>
<hr />
<div>==Construction of pET3A Derivatives Containing R-bchHID==<br />
==Construction of pET3a-R-bchH==<br />
1.)PCR the gene bchH from Rhodobacter sphaeroides<br><br />
Materials:<br />
*1uL R-genomic DNA<br />
*10uL 5x GC buffer<br />
*5uL primer<br />
*0.5uL Phusion<br />
*5uL DMSO<br />
*27.5uL H2O<br />
Conditions:<br />
*98C 30s<br />
*98C 10s*<br />
*63C 30s*<br />
*72C 1:50 min*<br />
*Repeat cycles with * 29x<br />
*72C 10 min<br />
*4C forever<br />
<br />
2.)Run PCR fragments on gel to make sure they are the correct size, then PCR purify<br><br />
3.)Digest R-bchH (8/21/07)<br><br />
*42uL PCR purified fragment<br />
*5uL NEB3<br />
*1.8uL NdeI<br />
*1.2uL BglII<br />
4.)Run the digested PCR fragment on gel, checking that the size is correct (8/21/07)<br><br />
5.)Ligate R-bchH with pET3a that is already digested with NdeI and BamHI (8/21/07)<br><br />
*4.5uL pET3a<br />
*12.5uL R-bchH fragment<br />
*2uL T4 ligase buffer<br />
*1uL T4 ligase<br />
6.)Transform into NovaBlue (8/21/07)<br><br />
7.)Inoculate to LB media (pick 10 colonies) (8/22/07)<br><br />
8.)Miniprep cells with pET3a-R-bchH (8/23/07)<br><br />
9.)Sequence<br />
<br />
==Construction of pET3a-R-bchHI==<br />
1.)PCR the gene bchI from Rhodobacter sphaeroides (7/19/07)<br><br />
Materials:<br />
*1uL R-genomic DNA<br />
*10uL 5x HF buffer<br />
*5uL primer<br />
*0.5uL Phusion<br />
*25uL DMSO<br />
*1uL dNTP<br />
*30uL H2O<br />
Conditions:<br />
*98C 30s<br />
*98C 10s*<br />
*62C 30s*<br />
*72C 32s min*<br />
*Repeat cycles with * 29x<br />
*72C 10 min<br />
*4C forever<br />
<br />
2.)Run PCR fragments on gel to make sure they are the correct size, then PCR purify<br><br />
3.)Digest R-bchI (7/20/07) with KpnI and BglII<br><br />
4.)Run the digested PCR fragment on gel, checking that the size is correct (7/21/07)<br><br />
5.)Ligate R-bchH with pET3a-R-bchH that is already digested with KpnI and NsiI(8/28/07)<br><br />
*4.5uL pET3a-R-bchH<br />
*12.5uL R-bchI fragment<br />
*2uL T4 ligase buffer<br />
*1uL T4 ligase<br />
6.)Transform into NovaBlue (9/3/07)<br><br />
7.)Inoculate to LB media (pick 10 colonies) (9/4/07)<br><br />
8.)Miniprep cells with pET3a-R-bchH (9/14/07)<br><br />
9.)Sequence</div>KonniamChanhttp://2007.igem.org/wiki/index.php/Berkeley_LBL/KonniamNotebookBerkeley LBL/KonniamNotebook2007-10-27T02:41:52Z<p>KonniamChan: /* Construction of pET3a-R-bchHI */</p>
<hr />
<div>==Construction of pET3A Derivatives Containing R-bchHID==<br />
==Construction of pET3a-R-bchH==<br />
1.)PCR the gene bchH from Rhodobacter sphaeroides<br><br />
Materials:<br />
*1uL R-genomic DNA<br />
*10uL 5x GC buffer<br />
*5uL primer<br />
*0.5uL Phusion<br />
*5uL DMSO<br />
*27.5uL H2O<br />
Conditions:<br />
*98C 30s<br />
*98C 10s*<br />
*63C 30s*<br />
*72C 1:50 min*<br />
*Repeat cycles with * 29x<br />
*72C 10 min<br />
*4C forever<br />
<br />
2.)Run PCR fragments on gel to make sure they are the correct size, then PCR purify<br><br />
3.)Digest R-bchH (8/21/07)<br><br />
*42uL PCR purified fragment<br />
*5uL NEB3<br />
*1.8uL NdeI<br />
*1.2uL BglII<br />
4.)Run the digested PCR fragment on gel, checking that the size is correct (8/21/07)<br><br />
5.)Ligate R-bchH with pET3a that is already digested with NdeI and BamHI (8/21/07)<br><br />
*4.5uL pET3a<br />
*12.5uL R-bchH fragment<br />
*2uL T4 ligase buffer<br />
*1uL T4 ligase<br />
6.)Transform into NovaBlue (8/21/07)<br><br />
7.)Inoculate to LB media (pick 10 colonies) (8/22/07)<br><br />
8.)Miniprep cells with pET3a-R-bchH (8/23/07)<br><br />
9.)Sequence<br />
<br />
==Construction of pET3a-R-bchHI==<br />
1.)PCR the gene bchI from Rhodobacter sphaeroides (7/19/07)<br><br />
Materials:<br />
*1uL R-genomic DNA<br />
*10uL 5x HF buffer<br />
*5uL primer<br />
*0.5uL Phusion<br />
*25uL DMSO<br />
*1uL dNTP<br />
*30uL H2O<br />
Conditions:<br />
*98C 30s<br />
*98C 10s*<br />
*63C 30s*<br />
*72C 1:50 min*<br />
*Repeat cycles with * 29x<br />
*72C 10 min<br />
*4C forever<br />
<br />
2.)Run PCR fragments on gel to make sure they are the correct size, then PCR purify<br><br />
3.)Digest R-bchI (7/20/07) with KpnI and BglII<br><br />
4.)Run the digested PCR fragment on gel, checking that the size is correct (7/21/07)<br><br />
5.)Ligate R-bchH with pET3a-R-bchH that is already digested with KpnI and NsiI(8/28/07)<br><br />
*4.5uL pET3a-R-bchH<br />
*12.5uL R-bchI fragment<br />
*2uL T4 ligase buffer<br />
*1uL T4 ligase<br />
6.)Transform into NovaBlue (9/3/07)<br><br />
7.)Inoculate to LB media (pick 10 colonies) (9/4/07)<br><br />
8.)Miniprep cells with pET3a-R-bchH (9/14/07)<br><br />
9.)Sequence</div>KonniamChanhttp://2007.igem.org/wiki/index.php/Berkeley_LBL/KonniamNotebookBerkeley LBL/KonniamNotebook2007-10-27T02:36:53Z<p>KonniamChan: </p>
<hr />
<div>==Construction of pET3A Derivatives Containing R-bchHID==<br />
==Construction of pET3a-R-bchH==<br />
1.)PCR the gene bchH from Rhodobacter sphaeroides<br><br />
Materials:<br />
*1uL R-genomic DNA<br />
*10uL 5x GC buffer<br />
*5uL primer<br />
*0.5uL Phusion<br />
*5uL DMSO<br />
*27.5uL H2O<br />
Conditions:<br />
*98C 30s<br />
*98C 10s*<br />
*63C 30s*<br />
*72C 1:50 min*<br />
*Repeat cycles with * 29x<br />
*72C 10 min<br />
*4C forever<br />
<br />
2.)Run PCR fragments on gel to make sure they are the correct size, then PCR purify<br><br />
3.)Digest R-bchH (8/21/07)<br><br />
*42uL PCR purified fragment<br />
*5uL NEB3<br />
*1.8uL NdeI<br />
*1.2uL BglII<br />
4.)Run the digested PCR fragment on gel, checking that the size is correct (8/21/07)<br><br />
5.)Ligate R-bchH with pET3a that is already digested with NdeI and BamHI (8/21/07)<br><br />
*4.5uL pET3a<br />
*12.5uL R-bchH fragment<br />
*2uL T4 ligase buffer<br />
*1uL T4 ligase<br />
6.)Transform into NovaBlue (8/21/07)<br><br />
7.)Inoculate to LB media (pick 10 colonies) (8/22/07)<br><br />
8.)Miniprep cells with pET3a-R-bchH (8/23/07)<br><br />
9.)Sequence<br />
<br />
==Construction of pET3a-R-bchHI==<br />
1.)PCR the gene bchI from Rhodobacter sphaeroides<br><br />
Materials:<br />
*1uL R-genomic DNA<br />
*10uL 5x GC buffer<br />
*5uL primer<br />
*0.5uL Phusion<br />
*5uL DMSO<br />
*27.5uL H2O<br />
Conditions:<br />
*98C 30s<br />
*98C 10s*<br />
*63C 30s*<br />
*72C 1:50 min*<br />
*Repeat cycles with * 29x<br />
*72C 10 min<br />
*4C forever<br />
<br />
2.)Run PCR fragments on gel to make sure they are the correct size, then PCR purify<br><br />
3.)Digest R-bchH (8/21/07)<br><br />
*42uL PCR purified fragment<br />
*5uL NEB3<br />
*1.8uL NdeI<br />
*1.2uL BglII<br />
4.)Run the digested PCR fragment on gel, checking that the size is correct (8/21/07)<br><br />
5.)Ligate R-bchH with pET3a that is already digested with NdeI and BamHI (8/21/07)<br><br />
*4.5uL pET3a<br />
*12.5uL R-bchH fragment<br />
*2uL T4 ligase buffer<br />
*1uL T4 ligase<br />
6.)Transform into NovaBlue (8/21/07)<br><br />
7.)Inoculate to LB media (pick 10 colonies) (8/22/07)<br><br />
8.)Miniprep cells with pET3a-R-bchH (8/23/07)<br><br />
9.)Sequence</div>KonniamChanhttp://2007.igem.org/wiki/index.php/Berkeley_LBL/KonniamNotebookBerkeley LBL/KonniamNotebook2007-10-27T02:33:44Z<p>KonniamChan: /* Construction of pET3a-R-bchH= */</p>
<hr />
<div>==Construction of pET3A Derivatives Containing R-bchHID==<br />
==Construction of pET3a-R-bchH==<br />
1.)PCR the gene bchH from Rhodobacter sphaeroides<br><br />
Materials:<br />
*1uL R-genomic DNA<br />
*10uL 5x GC buffer<br />
*5uL primer<br />
*0.5uL Phusion<br />
*5uL DMSO<br />
*27.5uL H2O<br />
Conditions:<br />
*98C 30s<br />
*98C 10s*<br />
*63C 30s*<br />
*72C 1:50 min*<br />
*Repeat cycles with * 29x<br />
*72C 10 min<br />
*4C forever<br />
<br />
2.)Run PCR fragments on gel to make sure they are the correct size, then PCR purify<br><br />
3.)Digest R-bchH (8/21/07)<br><br />
*42uL PCR purified fragment<br />
*5uL NEB3<br />
*1.8uL NdeI<br />
*1.2uL BglII<br />
4.)Run the digested PCR fragment on gel, checking that the size is correct (8/21/07)<br><br />
5.)Ligate R-bchH with pET3a that is already digested with NdeI and BamHI (8/21/07)<br><br />
*4.5uL pET3a<br />
*12.5uL R-bchH fragment<br />
*2uL T4 ligase buffer<br />
*1uL T4 ligase<br />
6.)Transform into NovaBlue (8/21/07)<br><br />
7.)Inoculate to LB media (pick 10 colonies) (8/22/07)<br><br />
8.)Miniprep cells with pET3a-R-bchH (8/23/07)<br><br />
9.)Sequence<br />
<br />
==Construction of pET3a-R-bchHI==</div>KonniamChanhttp://2007.igem.org/wiki/index.php/Berkeley_LBL/KonniamNotebookBerkeley LBL/KonniamNotebook2007-10-27T02:28:53Z<p>KonniamChan: /* Construction of pET3A Derivatives Containing R-bchHID */</p>
<hr />
<div>==Construction of pET3A Derivatives Containing R-bchHID==<br />
==Construction of pET3a-R-bchH===<br />
1.)PCR bchH, bchI, bchD genes from Rhodobacter sphaeroides<br><br />
2.)Run PCR fragments on gel to make sure they are the correct size, then PCR purify<br><br />
3.)Digest R-bchH (8/21/07)<br><br />
*42uL PCR purified fragment<br />
*5uL NEB3<br />
*1.8uL NdeI<br />
*1.2uL BglII<br />
4.)Run the digested PCR fragment on gel, checking that the size is correct (8/21/07)<br><br />
5.)Ligate R-bchH with pET3a that is already digested with NdeI and BamHI (8/21/07)<br><br />
*4.5uL pET3a<br />
*12.5uL R-bchH fragment<br />
*2uL T4 ligase buffer<br />
*1uL T4 ligase<br />
6.)Transform into NovaBlue (8/21/07)<br><br />
7.)Inoculate to LB media (pick 10 colonies) (8/22/07)<br><br />
8.)Miniprep cells with pET3a-R-bchH (8/23/07)<br><br />
9.)Sequence</div>KonniamChanhttp://2007.igem.org/wiki/index.php/Berkeley_LBL/KonniamNotebookBerkeley LBL/KonniamNotebook2007-10-27T02:20:13Z<p>KonniamChan: </p>
<hr />
<div>==Construction of pET3A Derivatives Containing R-bchHID==<br />
#PCR bchH, bchI, bchD genes from Rhodobacter sphaeroides<br />
#Run PCR fragments on gel to make sure they are the correct size, then PCR purify<br />
#</div>KonniamChanhttp://2007.igem.org/wiki/index.php/Berkeley_LBL/NotebookBerkeley LBL/Notebook2007-10-27T02:13:40Z<p>KonniamChan: </p>
<hr />
<div>{| border="0" cellspacing="8px" cellpadding="15" width="80%"<br />
|-<br />
|[[Berkeley_LBL|Home]]<br />
|[[Berkeley_LBL/Project|Project Description]]<br />
|[[Berkeley_LBL/Methods|Methods]]<br />
|[[Berkeley_LBL/Notebook|Notebook]]<br />
|[[Berkeley_LBL/Results|Results and Discussion]]<br />
|[[Berkeley_LBL/Resources|Resources]]<br />
|}<br />
<br />
<br />
<br />
[[Berkeley_LBL/KonniamNotebook|Konniam's Notebook]]<br />
<br />
[[Berkeley_LBL/JoyceNotebook|Joyce's Notebook]]<br />
<br />
[[Berkeley_LBL/MimiNotebook|Mimi's Notebook]]<br />
<br />
[[Berkeley_LBL/Laina_Notebook|Laina's Notebook]]<br />
<br />
[[Image:Image-Construct.jpg|left|thumb|Description]]</div>KonniamChanhttp://2007.igem.org/wiki/index.php/Berkeley_LBL/ResultsBerkeley LBL/Results2007-10-27T02:09:54Z<p>KonniamChan: </p>
<hr />
<div>{| border="0" cellspacing="8px" cellpadding="15" width="80%"<br />
|-<br />
|[[Berkeley_LBL|Home]]<br />
|[[Berkeley_LBL/Project|Project Description]]<br />
|[[Berkeley_LBL/Methods|Methods]]<br />
|[[Berkeley_LBL/Notebook|Notebook]]<br />
|[[Berkeley_LBL/Results|Results and Discussion]]<br />
|[[Berkeley_LBL/Resources|Resources]]<br />
|}<br />
<br />
== Results ==<br />
<br />
[[Berkeley_LBL/Ladder|Invitrogen BenchMark Protein Ladder]]<br />
<br />
[[Berkeley_LBL/Key|Plasmid Key for Protein Gels]]<br />
<br />
[[Image:ProteinGel1.jpg]]<br />
<br />
[[Image:Gel1Key.jpg]]<br />
<br />
[[Image:ProteinGel2.jpg]]<br />
<br />
[[Image:Gel2Key.jpg]]<br />
<br />
[[Image:ProteinGel3.jpg]]<br />
<br />
[[Image:Gel3Key.jpg]]<br />
<br />
[[Image:ProteinGel4.jpg]]<br />
<br />
[[Image:Gel4Key.jpg]]<br />
<br />
<br />
== Discussion ==</div>KonniamChanhttp://2007.igem.org/wiki/index.php/Berkeley_LBL/MethodsBerkeley LBL/Methods2007-10-27T02:05:14Z<p>KonniamChan: </p>
<hr />
<div>{| border="0" cellspacing="8px" cellpadding="15" width="80%"<br />
|-<br />
|[[Berkeley_LBL|Home]]<br />
|[[Berkeley_LBL/Project|Project Description]]<br />
|[[Berkeley_LBL/Methods|Methods]]<br />
|[[Berkeley_LBL/Notebook|Notebook]]<br />
|[[Berkeley_LBL/Results|Results and Discussion]]<br />
|[[Berkeley_LBL/Resources|Resources]]<br />
|}<br />
<br />
<br />
==Experimental==<br />
<br />
=== Subcloning ===<br />
<br />
The genes of interest from ''Rhodobacter sphaeroides'', ''Synechocystis sp'', and ''Heliobacillus mobilis'' were amplified using PCR (the details of which can be found in Protocols). The PCR fragments were then digested and ligated with the T7 expression vector pET3a in various ways, resulting in the constructs shown below. They were transformed into ''E.Coli'' (either DH10B or NovaBlue) and glycerol stocks for these cells were saved.<br />
<br />
=== Constructs ===<br />
The T7 expression vector pET3a was used for the subcloning of the genes for magnesium-chelatase. The following constructs were built by inserting various fragments genes from the three organisms into pET3a.<br />
<br />
'''Magnesium-Chelatase'''<br><br />
''Rhodobacter sphaeroides''<br />
{| border="1"<br />
|-<br />
!Construct Name<br />
!Construct Details<br />
|-<br />
|pET3a-R-bchH<br />
|T7p-rbs-bchH-T7Term<br />
|-<br />
|pET3a-R-bchI<br />
|T7p-rbs-bchI-T7Term<br />
|-<br />
|pET3a-R-bchD<br />
|T7p-rbs-bchD-T7Term<br />
|-<br />
|pET3a-R-bchHID<br />
|T7p-rbs-bchH-rbs-bchI-rbs-bchD-T7Term<br />
|}<br />
<br />
<br />
''Synechocystis sp.''<br />
{| border="1"<br />
|-<br />
!Construct Name<br />
!Construct Details<br />
|-<br />
|pET3a-S-chlH<br />
|T7p-rbs-chlH-T7Term<br />
|-<br />
|pET3a-S-chlI<br />
|T7p-rbs-chlI-T7Term<br />
|-<br />
|pET3a-S-chlD<br />
|T7p-rbs-chlD-T7Term<br />
|-<br />
|pET3a-S-chlHID<br />
|T7p-rbs-chlH-rbs-chlI-rbs-chlD-T7Term<br />
|}<br />
<br />
'''Enzymes for reactions after Mg-insertion step'''<br><br />
<br />
''Heliobacillus mobilis''<br />
<br />
=== Expression ===<br />
<br />
Once the constructs are inside DH10B or NovaBlue, they were minipreped and transformed in BL21 (DE3). These cells were then induced to overexpress the proteins of interest. After protein expression, the cell cultures were centrifuged at 10,000rpm for 20 minutes. The resulting cell pellets were dissolved in TRIS buffer (pH 7.8) and sonicated. The sonicated cell extracts were then centrifuged again, with the supernatant containing the soluble proteins. The soluble protein extract was used for enzyme activity assays. Along with the soluble protein extract, the total sonicated cell extract and the insoluble cell extract were analyzed on a SDS-PAGE gel.<br />
<br />
=== Enzyme Activity Assays ===<br />
<br />
The E.Coli cells with the constructs pET3a-R-bchHID and pET3a-S-bchHID have all the peptides necessary for the enzyme Mg-chelatase. The activity of these enzymes can be measured by the concentration of the reaction product, Mg-protoporphyrin IX. The concentrations were measured by the UV-vis spectroscopy and fluorescence emission spectroscopy, utilizing the Beer-Lambert law. By assuming that the concentration of the product is proportional to the activity of the enzymes, we can determine which enzyme (either from Rhodobacter sphaeroides or Synechocystis sp) is more efficient.<br />
<br />
<br />
== '''Subcloning Protocols''' ==<br />
<br />
'''''Polymerase Chain Reaction (PCR) Protocols:'''''<br />
<br />
[[Berkeley_LBL/PCRphusion|PCR (Using Phusion Polymerase)]]<br />
<br />
[[Berkeley_LBL/PCRextaq|PCR (Using TaKaRa Ex Taq Polymerase)]]<br />
<br />
'''''Clean Up/ Purification Protocols:'''''<br />
<br />
[[Berkeley_LBL/PCRcleanup|PCR Clean Up/Purification]]<br />
<br />
[[Berkeley_LBL/GelExtraction|Gel Extraction]]<br />
<br />
'''''DNA Gel Eletrophoresis Protocol:''''' <br />
<br />
[[Berkeley_LBL/DNAGelElectrophoresis|DNA Gel Electrophoresis]]<br />
<br />
''''''Restriction Digestion Protocols:''''''<br />
<br />
[[Berkeley_LBL/Digestion|Digestion for PCR Product or Miniprepped DNA]]<br />
<br />
[[Berkeley_LBL/Digestion2|Analytic Digestion]]<br />
<br />
''''''Ligation Protocol:''''''<br />
<br />
[[Berkeley_LBL/Ligation|Ligation]]<br />
<br />
'''''Transformation Protocols:'''''<br />
<br />
[[Berkeley_LBL/CompetentCell|KCM Competent Cell Production]]<br />
<br />
[[Berkeley_LBL/CompetentCellTransformation|KCM Competent Cell Transformation]]<br />
<br />
[[Berkeley_LBL/Electroporation|Electroporation Transformation]]<br />
<br />
'''''Miniprep Protocol:'''''<br />
<br />
[[Berkeley_LBL/Miniprep|Miniprep]]<br />
<br />
<br />
== Expression Protocols ==<br />
<br />
'''''Protein Expression Protocols:'''''<br />
<br />
[[Berkeley_LBL/Overexpression|Overexpression]]<br />
<br />
'''''Protein Analysis Protocols:'''''<br />
<br />
[[Berkeley_LBL/Sonication|Sonication]]<br />
<br />
[[Berkeley_LBL/SDS-PAGE|SDS-PAGE]]</div>KonniamChanhttp://2007.igem.org/wiki/index.php/Berkeley_LBL/MethodsBerkeley LBL/Methods2007-10-27T01:40:56Z<p>KonniamChan: /* Constructs */</p>
<hr />
<div>{| border="0" cellspacing="8px" cellpadding="15" width="80%"<br />
|-<br />
|[[Berkeley_LBL|Home]]<br />
|[[Berkeley_LBL/Project|Project Description]]<br />
|[[Berkeley_LBL/Methods|Methods]]<br />
|[[Berkeley_LBL/Notebook|Notebook]]<br />
|[[Berkeley_LBL/Results|Results and Discussion]]<br />
|[[Berkeley_LBL/Resources|Resources]]<br />
|}<br />
<br />
<br />
==Experimental==<br />
<br />
=== Subcloning ===<br />
<br />
The genes of interest from Rhodobacter sphaeroides, Synechocystis sp, and Heliobacillus mobilis were amplified using PCR (the details of which can be found in Protocols). The PCR fragments were then digested and ligated with the T7 expression vector pET3a in various ways, resulting in the constructs shown below. They were transformed into E.Coli (either DH10B or NovaBlue) and glycerol stocks for these cells were saved. <br />
<br />
=== Constructs ===<br />
The T7 expression vector pET3a was used for the subcloning of the genes for magnesium-chelatase. The following constructs were built by inserting various fragments genes from the three organisms into pET3a.<br />
<br />
'''Magnesium-Chelatase'''<br><br />
''Rhodobacter sphaeroides''<br />
{| border="1"<br />
|-<br />
!Construct Name<br />
!Construct Details<br />
|-<br />
|pET3a-R-bchH<br />
|T7p-rbs-bchH-T7Term<br />
|-<br />
|pET3a-R-bchI<br />
|T7p-rbs-bchI-T7Term<br />
|-<br />
|pET3a-R-bchD<br />
|T7p-rbs-bchD-T7Term<br />
|-<br />
|pET3a-R-bchHID<br />
|T7p-rbs-bchH-rbs-bchI-rbs-bchD-T7Term<br />
|}<br />
<br />
<br />
''Synechocystis sp.''<br />
{| border="1"<br />
|-<br />
!Construct Name<br />
!Construct Details<br />
|-<br />
|pET3a-S-chlH<br />
|T7p-rbs-chlH-T7Term<br />
|-<br />
|pET3a-S-chlI<br />
|T7p-rbs-chlI-T7Term<br />
|-<br />
|pET3a-S-chlD<br />
|T7p-rbs-chlD-T7Term<br />
|-<br />
|pET3a-S-chlHID<br />
|T7p-rbs-chlH-rbs-chlI-rbs-chlD-T7Term<br />
|}<br />
<br />
'''Enzymes for reactions after Mg-insertion step'''<br><br />
<br />
''Heliobacillus mobilis''<br />
<br />
=== Expression ===<br />
<br />
Once the constructs are inside DH10B or NovaBlue, they were minipreped and transformed in BL21 (DE3). These cells were then induced to overexpress the proteins of interest. After protein expression, the cell cultures were centrifuged at 10,000rpm for 20 minutes. The resulting cell pellets were dissolved in TRIS buffer (pH 7.8) and sonicated. The sonicated cell extracts were then centrifuged again, with the supernatant containing the soluble proteins. The soluble protein extract was used for enzyme activity assays. Along with the soluble protein extract, the total sonicated cell extract and the insoluble cell extract were analyzed on a SDS-PAGE gel.<br />
<br />
=== Enzyme Activity Assays ===<br />
<br />
The E.Coli cells with the constructs pET3a-R-bchHID and pET3a-S-bchHID have all the peptides necessary for the enzyme Mg-chelatase. The activity of these enzymes can be measured by the concentration of the reaction product, Mg-protoporphyrin IX. The concentrations were measured by the UV-vis spectroscopy and fluorescence emission spectroscopy, utilizing the Beer-Lambert law. By assuming that the concentration of the product is proportional to the activity of the enzymes, we can determine which enzyme (either from Rhodobacter sphaeroides or Synechocystis sp) is more efficient.<br />
<br />
== Protocols ==<br />
<br />
[[Berkeley_LBL/PCRphusion|PCR (Using Phusion Polymerase)]]<br />
<br />
[[Berkeley_LBL/PCRextaq|PCR (Using TaKaRa Ex Taq Polymerase)]]<br />
<br />
[[Berkeley_LBL/PCRcleanup|PCR Clean Up/Purification]]<br />
<br />
[[Berkeley_LBL/DNAGelElectrophoresis|DNA Gel Electrophoresis]]<br />
<br />
[[Berkeley_LBL/Miniprep|Miniprep]]<br />
<br />
[[Berkeley_LBL/Digestion|Digestion for PCR Product or Miniprepped DNA]]<br />
<br />
[[Berkeley_LBL/Digestion2|Analytic Digestion]]<br />
<br />
[[Berkeley_LBL/GelExtraction|Gel Extraction]]<br />
<br />
[[Berkeley_LBL/Ligation|Ligation]]<br />
<br />
[[Berkeley_LBL/CompetentCell|KCM Competent Cell Production]]<br />
<br />
[[Berkeley_LBL/CompetentCellTransformation|KCM Competent Cell Transformation]]<br />
<br />
[[Berkeley_LBL/Electroporation|Electroporation Transformation]]<br />
<br />
[[Berkeley_LBL/Overexpression|Overexpression]]<br />
<br />
[[Berkeley_LBL/Sonication|Sonication]]<br />
<br />
[[Berkeley_LBL/SDS-PAGE|SDS-PAGE]]</div>KonniamChanhttp://2007.igem.org/wiki/index.php/Berkeley_LBL/MethodsBerkeley LBL/Methods2007-10-27T01:40:42Z<p>KonniamChan: /* Constructs */</p>
<hr />
<div>{| border="0" cellspacing="8px" cellpadding="15" width="80%"<br />
|-<br />
|[[Berkeley_LBL|Home]]<br />
|[[Berkeley_LBL/Project|Project Description]]<br />
|[[Berkeley_LBL/Methods|Methods]]<br />
|[[Berkeley_LBL/Notebook|Notebook]]<br />
|[[Berkeley_LBL/Results|Results and Discussion]]<br />
|[[Berkeley_LBL/Resources|Resources]]<br />
|}<br />
<br />
<br />
==Experimental==<br />
<br />
=== Subcloning ===<br />
<br />
The genes of interest from Rhodobacter sphaeroides, Synechocystis sp, and Heliobacillus mobilis were amplified using PCR (the details of which can be found in Protocols). The PCR fragments were then digested and ligated with the T7 expression vector pET3a in various ways, resulting in the constructs shown below. They were transformed into E.Coli (either DH10B or NovaBlue) and glycerol stocks for these cells were saved. <br />
<br />
=== Constructs ===<br />
The T7 expression vector pET3a was used for the subcloning of the genes for magnesium-chelatase. The following constructs were built by inserting various fragments genes from the three organisms into pET3a.<br />
<br />
'''Magnesium-Chelatase'''<br><br />
''Rhodobacter sphaeroides''<br />
{| border="1"<br />
|-<br />
!Construct Name<br />
!Construct Details<br />
|-<br />
|pET3a-R-bchH<br />
|T7p-rbs-bchH-T7Term<br />
|-<br />
|pET3a-R-bchI<br />
|T7p-rbs-bchI-T7Term<br />
|-<br />
|pET3a-R-bchD<br />
|T7p-rbs-bchD-T7Term<br />
|-<br />
|pET3a-R-bchHID<br />
|T7p-rbs-bchH-rbs-bchI-rbs-bchD-T7Term<br />
|}<br />
<br />
<br />
''Synechocystis sp.''<br />
{| border="1"<br />
|-<br />
!Construct Name<br />
!Construct Details<br />
|-<br />
|pET3a-S-chlH<br />
|T7p-rbs-chlH-T7Term<br />
|-<br />
|pET3a-S-chlI<br />
|T7p-rbs-chlI-T7Term<br />
|-<br />
|pET3a-S-chlD<br />
|T7p-rbs-chlD-T7Term<br />
|-<br />
|pET3a-S-chlHID<br />
|T7p-rbs-chlH-rbs-chlI-rbs-chlD-T7Term<br />
|}<br />
<br />
'''Enzymes for reactions after Mg-insertion step'''<br><br />
''Heliobacillus mobilis''<br />
<br />
=== Expression ===<br />
<br />
Once the constructs are inside DH10B or NovaBlue, they were minipreped and transformed in BL21 (DE3). These cells were then induced to overexpress the proteins of interest. After protein expression, the cell cultures were centrifuged at 10,000rpm for 20 minutes. The resulting cell pellets were dissolved in TRIS buffer (pH 7.8) and sonicated. The sonicated cell extracts were then centrifuged again, with the supernatant containing the soluble proteins. The soluble protein extract was used for enzyme activity assays. Along with the soluble protein extract, the total sonicated cell extract and the insoluble cell extract were analyzed on a SDS-PAGE gel.<br />
<br />
=== Enzyme Activity Assays ===<br />
<br />
The E.Coli cells with the constructs pET3a-R-bchHID and pET3a-S-bchHID have all the peptides necessary for the enzyme Mg-chelatase. The activity of these enzymes can be measured by the concentration of the reaction product, Mg-protoporphyrin IX. The concentrations were measured by the UV-vis spectroscopy and fluorescence emission spectroscopy, utilizing the Beer-Lambert law. By assuming that the concentration of the product is proportional to the activity of the enzymes, we can determine which enzyme (either from Rhodobacter sphaeroides or Synechocystis sp) is more efficient.<br />
<br />
== Protocols ==<br />
<br />
[[Berkeley_LBL/PCRphusion|PCR (Using Phusion Polymerase)]]<br />
<br />
[[Berkeley_LBL/PCRextaq|PCR (Using TaKaRa Ex Taq Polymerase)]]<br />
<br />
[[Berkeley_LBL/PCRcleanup|PCR Clean Up/Purification]]<br />
<br />
[[Berkeley_LBL/DNAGelElectrophoresis|DNA Gel Electrophoresis]]<br />
<br />
[[Berkeley_LBL/Miniprep|Miniprep]]<br />
<br />
[[Berkeley_LBL/Digestion|Digestion for PCR Product or Miniprepped DNA]]<br />
<br />
[[Berkeley_LBL/Digestion2|Analytic Digestion]]<br />
<br />
[[Berkeley_LBL/GelExtraction|Gel Extraction]]<br />
<br />
[[Berkeley_LBL/Ligation|Ligation]]<br />
<br />
[[Berkeley_LBL/CompetentCell|KCM Competent Cell Production]]<br />
<br />
[[Berkeley_LBL/CompetentCellTransformation|KCM Competent Cell Transformation]]<br />
<br />
[[Berkeley_LBL/Electroporation|Electroporation Transformation]]<br />
<br />
[[Berkeley_LBL/Overexpression|Overexpression]]<br />
<br />
[[Berkeley_LBL/Sonication|Sonication]]<br />
<br />
[[Berkeley_LBL/SDS-PAGE|SDS-PAGE]]</div>KonniamChanhttp://2007.igem.org/wiki/index.php/Berkeley_LBL/ProjectBerkeley LBL/Project2007-10-27T01:40:08Z<p>KonniamChan: </p>
<hr />
<div>{| border="0" cellspacing="8px" cellpadding="15" width="80%"<br />
|-<br />
|[[Berkeley_LBL|Home]]<br />
|[[Berkeley_LBL/Project|Project Description]]<br />
|[[Berkeley_LBL/Methods|Methods]]<br />
|[[Berkeley_LBL/Notebook|Notebook]]<br />
|[[Berkeley_LBL/Results|Results and Discussion]]<br />
|[[Berkeley_LBL/Resources|Resources]]<br />
|}<br />
<br />
{| border = "1"<br />
|-<br />
|[[Image:purpose.jpg]]<br />
|<br />
'''Purpose'''<br />
<br />
The purpose of the project is to engineer the most efficient pathway for the biosynthesis of bacteriochlorophyll using ''E. Coli'' as the host organism. <br />
<br />
|-<br />
|[[image:Solar_Energy.jpg]]<br />
|<br />
<br />
'''Background'''<br />
<br />
The specific goal for this project is to utilize ''E. Coli'' to produce chlorophyll efficiently. This is accomplished by controlling the first set of genes in the first branch point of the native ''E. Coli'' pathway and the chlorophyll biosynthesis pathway. However, the production of chlorophyll can lead to the eventual goal of engineering ''E. Coli'' to be a photosynthetic organism.<br />
<br />
Photosynthetic organisms that can be easily manipulated bring tremendous value to the current alternative energy research. Solar energy is available on Earth at a rate of 3850 x 10^21 Joules per year, while the worldwide energy consumption is only 0.471 x 10^21 Joules per year([http://en.wikipedia.org/wiki/Solar_energy Wikipedia Solar Energy]).<br />
<br />
Photosynthetic organisms can capture this untapped source of solar energy and transform them into chemical energy as biofuels.<br />
<br />
<br />
|-<br />
|[[Image:photosynthesis.jpg]]<br />
| <br />
'''Photosynthesis'''<br />
<br />
Photosynthesis is the process by which light energy is converted to chemical energy in form of glucose. The chemical equation of the process is: 6CO2 + 12H2O --> C6H12O6 + 6O2 + 6H2O, with the input of light energy required for the forward reaction. As shown in the equation, carbon dioxide is fixed in photosynthesis to produce a more complicated organic compound, glucose.<br />
<br />
|-<br />
|[[Image:organisms.jpg]]<br />
|<br />
<br />
'''Photosynthetic Organisms'''<br />
<br />
Plants and many species of bacteria are photosynthetic. Bacterial photosynthesis would be the focus in this description. The main classes of photosynthetic bacteria include '''cyanobacteria''', '''purple bacteria''', and '''heliobacteria'''. <br />
<br />
While plants have chloroplasts as a center for photosynthetic activity, bacteria do not. For bacteria, photosynthesis takes place within the cell. To perform the required photoreactions, a variety of pigments and membrane proteins are necessary. Out of the pigments, chlorophyll is the essential one.<br />
<br />
<br />
|-<br />
|[[Image:approach.jpg]]<br />
|<br />
<br />
'''Approach'''<br />
<br />
The goal of the project is to produce bacteriochlorophyll by establishing a strong metabolic pathway in ''E.Coli''. A generic metabolic pathway for chlorophyll synthesis in plants is given [https://2007.igem.org/Image:Chlorophyll_Biosynthesis.gif here (Generic Chlorophyll Biosynthesis)]. The biosynthesis of bacteriochlorophyll is similar; the part that our project focused on is shown [https://2007.igem.org/Image:Chlorophyll_Biosynthesis2.JPG here(Bacteriochlorophyll Biosynthesis)].<br />
<br />
In order to channel the flux of carbon through the bacteriochlorophyll biosynthetic pathway, the flux through the first branch point between the native ''E.Coli'' pathway and the chlorophyll pathway must be optimized. The enzyme magnesium-chelatase is responsible for converting protoporphyrin IX to Mg-protoporphyrin IX. Because this reaction must occur to a large degree, we want to use an enzyme that has the most enhanced activity. Since all photosynthetic bacteria utilize very similar bacteriochlorophyll synthesis pathways, they all have their own versions of Mg-chelatase to perform the Mg-insertion reaction. A large part of the project is to subclone the genes for Mg-chelatse, for three photosynthetic bacteria—''Rhodobacter sphaeroides'' (purple bacteria), ''Synechocystis sp.'' (cyanobacteria), and ''Heliobacillus mobilis'' (heliobacteria). After subcloning, the enzymes are overexpressed and their activities are measured. <br />
<br />
Besides the establishment of a strong initial input of flux through the bacteriochlorophyll photosynthesis pathway, we have also explored the latter parts of the pathway. These latter steps are crucial for catalyzing reactions that would lead to the final product—bacteriochlorophyll.<br />
<br />
<br />
|-<br />
|[[Image:application.jpg]]<br />
|<br />
<br />
'''Applications'''<br />
<br />
Applications for the use of photosynthetic ''E.Coli'' are plentiful. The most important of which is the industrial-scale production of biofuels using bacteria. One can set up a bioreactor, filled with photosynthetic ''E.Coli'', running under sunlight to produce biofuels. The costs of producing biofuels using such bioreactors should be much more inexpensive than current methods due to the fact that solar energy is directly captured and utilized.<br />
<br />
The second application is the production of chlorophyll for research purposes. Once the most efficient metabolic pathway to produce chlorophylls is established, the synthesis of chlorophyll and its derivatives can be done easily.<br />
<br />
The third application can be the use of novel enzymes to insert specific metal ions into compounds such as protoporphyrin IX. These new organometallic complexes may have novel reactivity and be useful in many contexts. <br />
<br />
<br />
|}</div>KonniamChanhttp://2007.igem.org/wiki/index.php/Berkeley_LBL/ProjectBerkeley LBL/Project2007-10-27T01:38:42Z<p>KonniamChan: </p>
<hr />
<div>{| border="0" cellspacing="8px" cellpadding="15" width="80%"<br />
|-<br />
|[[Berkeley_LBL|Home]]<br />
|[[Berkeley_LBL/Project|Project Description]]<br />
|[[Berkeley_LBL/Methods|Methods]]<br />
|[[Berkeley_LBL/Notebook|Notebook]]<br />
|[[Berkeley_LBL/Results|Results and Discussion]]<br />
|[[Berkeley_LBL/Resources|Resources]]<br />
|}<br />
<br />
{| border = "1"<br />
|-<br />
|[[Image:purpose.jpg]]<br />
|<br />
'''Purpose'''<br />
<br />
The purpose of the project is to engineer the most efficient pathway for the biosynthesis of bacteriochlorophyll using ''E. Coli'' as the host organism. <br />
<br />
|-<br />
|[[image:Solar_Energy.jpg]]<br />
|<br />
<br />
'''Background'''<br />
<br />
The specific goal for this project is to utilize ''E. Coli'' to produce chlorophyll efficiently. This is accomplished by controlling the first set of genes in the first branch point of the native ''E. Coli'' pathway and the chlorophyll biosynthesis pathway. However, the production of chlorophyll can lead to the eventual goal of engineering ''E. Coli'' to be a photosynthetic organism.<br />
<br />
Photosynthetic organisms that can be easily manipulated bring tremendous value to the current alternative energy research. Solar energy is available on Earth at a rate of 3850 x 10^21 Joules per year, while the worldwide energy consumption is only 0.471 x 10^21 Joules per year([http://en.wikipedia.org/wiki/Solar_energy Wikipedia Solar Energy]).<br />
<br />
Photosynthetic organisms can capture this untapped source of solar energy and transform them into chemical energy as biofuels.<br />
<br />
<br />
|-<br />
|[[Image:photosynthesis.jpg]]<br />
| <br />
'''Photosynthesis'''<br />
<br />
Photosynthesis is the process by which light energy is converted to chemical energy in form of glucose. The chemical equation of the process is: 6CO2 + 12H2O --> C6H12O6 + 6O2 + 6H2O, with the input of light energy required for the forward reaction. As shown in the equation, carbon dioxide is fixed in photosynthesis to produce a more complicated organic compound, glucose.<br />
<br />
|-<br />
|[[Image:organisms.jpg]]<br />
|<br />
<br />
'''Photosynthetic Organisms'''<br />
<br />
Plants and many species of bacteria are photosynthetic. Bacterial photosynthesis would be the focus in this description. The main classes of photosynthetic bacteria include '''cyanobacteria''', '''purple bacteria''', and '''heliobacteria'''. <br />
<br />
While plants have chloroplasts as a center for photosynthetic activity, bacteria do not. For bacteria, photosynthesis takes place within the cell. To perform the required photoreactions, a variety of pigments and membrane proteins are necessary. Out of the pigments, chlorophyll is the essential one.<br />
<br />
<br />
|-<br />
|[[Image:approach.jpg]]<br />
|<br />
<br />
'''Approach'''<br />
<br />
The goal of the project is to produce chlorophyll by establishing a strong metabolic pathway in ''E.Coli''. A generic metabolic pathway for chlorophyll synthesis in plants is given [https://2007.igem.org/Image:Chlorophyll_Biosynthesis.gif here (Generic Chlorophyll Biosynthesis)]. The biosynthesis of bacteriochlorophyll is similar; the part that our project focused on is shown [https://2007.igem.org/Image:Chlorophyll_Biosynthesis2.JPG here(Bacteriochlorophyll Biosynthesis)].<br />
<br />
In order to channel the flux of carbon through the chlorophyll biosynthetic pathway, the flux through the first branch point between the native ''E.Coli'' pathway and the chlorophyll pathway must be optimized. The enzyme magnesium-chelatase is responsible for converting protoporphyrin IX to Mg-protoporphyrin IX. Because this reaction must occur to a large degree, we want to use an enzyme that has the most enhanced activity. Since all photosynthetic bacteria utilize very similar bacteriochlorophyll synthesis pathways, they all have their own versions of Mg-chelatase to perform the Mg-insertion reaction. A large part of the project is to subclone the genes for Mg-chelatse, for three photosynthetic bacteria—''Rhodobacter sphaeroides'' (purple bacteria), ''Synechocystis sp.'' (cyanobacteria), and ''Heliobacillus mobilis'' (heliobacteria). After subcloning, the enzymes are overexpressed and their activities are measured. <br />
<br />
Besides the establishment of a strong initial input of flux through the bacteriochlorophyll photosynthesis pathway, we have also explored the latter parts of the pathway. These latter steps are crucial for catalyzing reactions that would lead to the final product—bacteriochlorophyll.<br />
<br />
<br />
|-<br />
|[[Image:application.jpg]]<br />
|<br />
<br />
'''Applications'''<br />
<br />
Applications for the use of photosynthetic ''E.Coli'' are plentiful. The most important of which is the industrial-scale production of biofuels using bacteria. One can set up a bioreactor, filled with photosynthetic ''E.Coli'', running under sunlight to produce biofuels. The costs of producing biofuels using such bioreactors should be much more inexpensive than current methods due to the fact that solar energy is directly captured and utilized.<br />
<br />
The second application is the production of chlorophyll for research purposes. Once the most efficient metabolic pathway to produce chlorophylls is established, the synthesis of chlorophyll and its derivatives can be done easily.<br />
<br />
The third application can be the use of novel enzymes to insert specific metal ions into compounds such as protoporphyrin IX. These new organometallic complexes may have novel reactivity and be useful in many contexts. <br />
<br />
<br />
|}</div>KonniamChanhttp://2007.igem.org/wiki/index.php/Berkeley_LBL/MethodsBerkeley LBL/Methods2007-10-27T01:34:43Z<p>KonniamChan: /* Constructs */</p>
<hr />
<div>{| border="0" cellspacing="8px" cellpadding="15" width="80%"<br />
|-<br />
|[[Berkeley_LBL|Home]]<br />
|[[Berkeley_LBL/Project|Project Description]]<br />
|[[Berkeley_LBL/Methods|Methods]]<br />
|[[Berkeley_LBL/Notebook|Notebook]]<br />
|[[Berkeley_LBL/Results|Results and Discussion]]<br />
|[[Berkeley_LBL/Resources|Resources]]<br />
|}<br />
<br />
<br />
==Experimental==<br />
<br />
=== Subcloning ===<br />
<br />
The genes of interest from Rhodobacter sphaeroides, Synechocystis sp, and Heliobacillus mobilis were amplified using PCR (the details of which can be found in Protocols). The PCR fragments were then digested and ligated with the T7 expression vector pET3a in various ways, resulting in the constructs shown below. They were transformed into E.Coli (either DH10B or NovaBlue) and glycerol stocks for these cells were saved. <br />
<br />
=== Constructs ===<br />
The T7 expression vector pET3a was used for the subcloning of the genes for magnesium-chelatase. The following constructs were built by inserting various fragments genes from the three organisms into pET3a.<br />
<br />
'''Magnesium-Chelatase'''<br><br />
''Rhodobacter sphaeroides''<br />
{| border="1"<br />
|-<br />
!Construct Name<br />
!Construct Details<br />
|-<br />
|pET3a-R-bchH<br />
|T7p-rbs-bchH-T7Term<br />
|-<br />
|pET3a-R-bchI<br />
|T7p-rbs-bchI-T7Term<br />
|-<br />
|pET3a-R-bchD<br />
|T7p-rbs-bchD-T7Term<br />
|-<br />
|pET3a-R-bchHID<br />
|T7p-rbs-bchH-rbs-bchI-rbs-bchD-T7Term<br />
|}<br />
<br />
<br />
''Synechocystis sp.''<br />
{| border="1"<br />
|-<br />
!Construct Name<br />
!Construct Details<br />
|-<br />
|pET3a-S-chlH<br />
|T7p-rbs-chlH-T7Term<br />
|-<br />
|pET3a-S-chlI<br />
|T7p-rbs-chlI-T7Term<br />
|-<br />
|pET3a-S-chlD<br />
|T7p-rbs-chlD-T7Term<br />
|-<br />
|pET3a-S-chlHID<br />
|T7p-rbs-chlH-rbs-chlI-rbs-chlD-T7Term<br />
|}<br />
<br />
'''Enzymes for reactions after Mg-insertion step'''<br />
''Heliobacillus mobilis''<br />
<br />
=== Expression ===<br />
<br />
Once the constructs are inside DH10B or NovaBlue, they were minipreped and transformed in BL21 (DE3). These cells were then induced to overexpress the proteins of interest. After protein expression, the cell cultures were centrifuged at 10,000rpm for 20 minutes. The resulting cell pellets were dissolved in TRIS buffer (pH 7.8) and sonicated. The sonicated cell extracts were then centrifuged again, with the supernatant containing the soluble proteins. The soluble protein extract was used for enzyme activity assays. Along with the soluble protein extract, the total sonicated cell extract and the insoluble cell extract were analyzed on a SDS-PAGE gel.<br />
<br />
=== Enzyme Activity Assays ===<br />
<br />
The E.Coli cells with the constructs pET3a-R-bchHID and pET3a-S-bchHID have all the peptides necessary for the enzyme Mg-chelatase. The activity of these enzymes can be measured by the concentration of the reaction product, Mg-protoporphyrin IX. The concentrations were measured by the UV-vis spectroscopy and fluorescence emission spectroscopy, utilizing the Beer-Lambert law. By assuming that the concentration of the product is proportional to the activity of the enzymes, we can determine which enzyme (either from Rhodobacter sphaeroides or Synechocystis sp) is more efficient.<br />
<br />
== Protocols ==<br />
<br />
[[Berkeley_LBL/PCRphusion|PCR (Using Phusion Polymerase)]]<br />
<br />
[[Berkeley_LBL/PCRextaq|PCR (Using TaKaRa Ex Taq Polymerase)]]<br />
<br />
[[Berkeley_LBL/PCRcleanup|PCR Clean Up/Purification]]<br />
<br />
[[Berkeley_LBL/DNAGelElectrophoresis|DNA Gel Electrophoresis]]<br />
<br />
[[Berkeley_LBL/Miniprep|Miniprep]]<br />
<br />
[[Berkeley_LBL/Digestion|Digestion for PCR Product or Miniprepped DNA]]<br />
<br />
[[Berkeley_LBL/Digestion2|Analytic Digestion]]<br />
<br />
[[Berkeley_LBL/GelExtraction|Gel Extraction]]<br />
<br />
[[Berkeley_LBL/Ligation|Ligation]]<br />
<br />
[[Berkeley_LBL/CompetentCell|KCM Competent Cell Production]]<br />
<br />
[[Berkeley_LBL/CompetentCellTransformation|KCM Competent Cell Transformation]]<br />
<br />
[[Berkeley_LBL/Electroporation|Electroporation Transformation]]<br />
<br />
[[Berkeley_LBL/Overexpression|Overexpression]]<br />
<br />
[[Berkeley_LBL/Sonication|Sonication]]<br />
<br />
[[Berkeley_LBL/SDS-PAGE|SDS-PAGE]]</div>KonniamChanhttp://2007.igem.org/wiki/index.php/Berkeley_LBL/MethodsBerkeley LBL/Methods2007-10-27T01:23:59Z<p>KonniamChan: </p>
<hr />
<div>{| border="0" cellspacing="8px" cellpadding="15" width="80%"<br />
|-<br />
|[[Berkeley_LBL|Home]]<br />
|[[Berkeley_LBL/Project|Project Description]]<br />
|[[Berkeley_LBL/Methods|Methods]]<br />
|[[Berkeley_LBL/Notebook|Notebook]]<br />
|[[Berkeley_LBL/Results|Results and Discussion]]<br />
|[[Berkeley_LBL/Resources|Resources]]<br />
|}<br />
<br />
<br />
==Experimental==<br />
<br />
=== Subcloning ===<br />
<br />
The genes of interest from Rhodobacter sphaeroides, Synechocystis sp, and Heliobacillus mobilis were amplified using PCR (the details of which can be found in Protocols). The PCR fragments were then digested and ligated with the T7 expression vector pET3a in various ways, resulting in the constructs shown below. They were transformed into E.Coli (either DH10B or NovaBlue) and glycerol stocks for these cells were saved. <br />
<br />
=== Constructs ===<br />
The T7 expression vector pET3a was used for the subcloning of the genes for magnesium-chelatase. The following constructs were built by inserting various fragments genes from the three organisms into pET3a. (Note: The pET3a vector contains a ribosome binding site downstream of the T7 promoter region; this rbs would not be indicated below)<br />
<br />
'''Magnesium-Chelatase'''<br><br />
Rhodobacter sphaeroides<br><br />
pET3a-bchH<br><br />
pET3a-bchI<br><br />
pET3a-bchD<br><br />
pET3a-bchHID<br><br />
Synechocystis sp.<br><br />
pET3a-chlH<br><br />
pET3a-chlI<br><br />
pET3a-chlD<br><br />
pET3a-chlHID<br />
<br />
Heliobacillus mobilis<br><br />
<br />
<br />
=== Expression ===<br />
<br />
Once the constructs are inside DH10B or NovaBlue, they were minipreped and transformed in BL21 (DE3). These cells were then induced to overexpress the proteins of interest. After protein expression, the cell cultures were centrifuged at 10,000rpm for 20 minutes. The resulting cell pellets were dissolved in TRIS buffer (pH 7.8) and sonicated. The sonicated cell extracts were then centrifuged again, with the supernatant containing the soluble proteins. The soluble protein extract was used for enzyme activity assays. Along with the soluble protein extract, the total sonicated cell extract and the insoluble cell extract were analyzed on a SDS-PAGE gel.<br />
<br />
=== Enzyme Activity Assays ===<br />
<br />
The E.Coli cells with the constructs pET3a-R-bchHID and pET3a-S-bchHID have all the peptides necessary for the enzyme Mg-chelatase. The activity of these enzymes can be measured by the concentration of the reaction product, Mg-protoporphyrin IX. The concentrations were measured by the UV-vis spectroscopy and fluorescence emission spectroscopy, utilizing the Beer-Lambert law. By assuming that the concentration of the product is proportional to the activity of the enzymes, we can determine which enzyme (either from Rhodobacter sphaeroides or Synechocystis sp) is more efficient.<br />
<br />
== Protocols ==<br />
<br />
[[Berkeley_LBL/PCRphusion|PCR (Using Phusion Polymerase)]]<br />
<br />
[[Berkeley_LBL/PCRextaq|PCR (Using TaKaRa Ex Taq Polymerase)]]<br />
<br />
[[Berkeley_LBL/PCRcleanup|PCR Clean Up/Purification]]<br />
<br />
[[Berkeley_LBL/DNAGelElectrophoresis|DNA Gel Electrophoresis]]<br />
<br />
[[Berkeley_LBL/Miniprep|Miniprep]]<br />
<br />
[[Berkeley_LBL/Digestion|Digestion for PCR Product or Miniprepped DNA]]<br />
<br />
[[Berkeley_LBL/Digestion2|Analytic Digestion]]<br />
<br />
[[Berkeley_LBL/GelExtraction|Gel Extraction]]<br />
<br />
[[Berkeley_LBL/Ligation|Ligation]]<br />
<br />
[[Berkeley_LBL/CompetentCell|KCM Competent Cell Production]]<br />
<br />
[[Berkeley_LBL/CompetentCellTransformation|KCM Competent Cell Transformation]]<br />
<br />
[[Berkeley_LBL/Electroporation|Electroporation Transformation]]<br />
<br />
[[Berkeley_LBL/Overexpression|Overexpression]]<br />
<br />
[[Berkeley_LBL/Sonication|Sonication]]<br />
<br />
[[Berkeley_LBL/SDS-PAGE|SDS-PAGE]]</div>KonniamChanhttp://2007.igem.org/wiki/index.php/Berkeley_LBL/MethodsBerkeley LBL/Methods2007-10-27T01:22:55Z<p>KonniamChan: /* Constructs */</p>
<hr />
<div>{| border="0" cellspacing="8px" cellpadding="15" width="80%"<br />
|-<br />
|[[Berkeley_LBL|Home]]<br />
|[[Berkeley_LBL/Project|Project Description]]<br />
|[[Berkeley_LBL/Methods|Methods]]<br />
|[[Berkeley_LBL/Notebook|Notebook]]<br />
|[[Berkeley_LBL/Results|Results and Discussion]]<br />
|[[Berkeley_LBL/Resources|Resources]]<br />
|}<br />
<br />
==Experimental==<br />
<br />
==Experimental==<br />
<br />
== Subcloning ==<br />
<br />
The genes of interest from Rhodobacter sphaeroides, Synechocystis sp, and Heliobacillus mobilis were amplified using PCR (the details of which can be found in Protocols). The PCR fragments were then digested and ligated with the T7 expression vector pET3a in various ways, resulting in the constructs shown below. They were transformed into E.Coli (either DH10B or NovaBlue) and glycerol stocks for these cells were saved. <br />
<br />
===Constructs===<br />
The T7 expression vector pET3a was used for the subcloning of the genes for magnesium-chelatase. The following constructs were built by inserting various fragments genes from the three organisms into pET3a. (Note: The pET3a vector contains a ribosome binding site downstream of the T7 promoter region; this rbs would not be indicated below)<br />
<br />
'''Magnesium-Chelatase'''<br><br />
Rhodobacter sphaeroides<br><br />
pET3a-bchH<br><br />
pET3a-bchI<br><br />
pET3a-bchD<br><br />
pET3a-bchHID<br><br />
Synechocystis sp.<br><br />
pET3a-chlH<br><br />
pET3a-chlI<br><br />
pET3a-chlD<br><br />
pET3a-chlHID<br />
<br />
Heliobacillus mobilis<br><br />
<br />
<br />
== Expression ==<br />
<br />
Once the constructs are inside DH10B or NovaBlue, they were minipreped and transformed in BL21 (DE3). These cells were then induced to overexpress the proteins of interest. After protein expression, the cell cultures were centrifuged at 10,000rpm for 20 minutes. The resulting cell pellets were dissolved in TRIS buffer (pH 7.8) and sonicated. The sonicated cell extracts were then centrifuged again, with the supernatant containing the soluble proteins. The soluble protein extract was used for enzyme activity assays. Along with the soluble protein extract, the total sonicated cell extract and the insoluble cell extract were analyzed on a SDS-PAGE gel.<br />
<br />
== Enzyme Activity Assays ==<br />
<br />
The E.Coli cells with the constructs pET3a-R-bchHID and pET3a-S-bchHID have all the peptides necessary for the enzyme Mg-chelatase. The activity of these enzymes can be measured by the concentration of the reaction product, Mg-protoporphyrin IX. The concentrations were measured by the UV-vis spectroscopy and fluorescence emission spectroscopy, utilizing the Beer-Lambert law. By assuming that the concentration of the product is proportional to the activity of the enzymes, we can determine which enzyme (either from Rhodobacter sphaeroides or Synechocystis sp) is more efficient.<br />
<br />
==Protocols==<br />
<br />
[[Berkeley_LBL/PCRphusion|PCR (Using Phusion Polymerase)]]<br />
<br />
[[Berkeley_LBL/PCRextaq|PCR (Using TaKaRa Ex Taq Polymerase)]]<br />
<br />
[[Berkeley_LBL/PCRcleanup|PCR Clean Up/Purification]]<br />
<br />
[[Berkeley_LBL/DNAGelElectrophoresis|DNA Gel Electrophoresis]]<br />
<br />
[[Berkeley_LBL/Miniprep|Miniprep]]<br />
<br />
[[Berkeley_LBL/Digestion|Digestion for PCR Product or Miniprepped DNA]]<br />
<br />
[[Berkeley_LBL/Digestion2|Analytic Digestion]]<br />
<br />
[[Berkeley_LBL/GelExtraction|Gel Extraction]]<br />
<br />
[[Berkeley_LBL/Ligation|Ligation]]<br />
<br />
[[Berkeley_LBL/CompetentCell|KCM Competent Cell Production]]<br />
<br />
[[Berkeley_LBL/CompetentCellTransformation|KCM Competent Cell Transformation]]<br />
<br />
[[Berkeley_LBL/Electroporation|Electroporation Transformation]]<br />
<br />
[[Berkeley_LBL/Overexpression|Overexpression]]<br />
<br />
[[Berkeley_LBL/Sonication|Sonication]]<br />
<br />
[[Berkeley_LBL/SDS-PAGE|SDS-PAGE]]</div>KonniamChanhttp://2007.igem.org/wiki/index.php/Berkeley_LBL/ProjectBerkeley LBL/Project2007-10-27T00:51:51Z<p>KonniamChan: </p>
<hr />
<div>{| border="0" cellspacing="8px" cellpadding="15" width="80%"<br />
|-<br />
|[[Berkeley_LBL|Home]]<br />
|[[Berkeley_LBL/Project|Project Description]]<br />
|[[Berkeley_LBL/Methods|Methods]]<br />
|[[Berkeley_LBL/Notebook|Notebook]]<br />
|[[Berkeley_LBL/Results|Results and Discussion]]<br />
|[[Berkeley_LBL/Resources|Resources]]<br />
|}<br />
<br />
{| border = "1"<br />
|-<br />
|[[Image:purpose.jpg]]<br />
|<br />
'''Purpose'''<br />
<br />
The purpose of the project is to engineer the most efficient pathway for the biosynthesis of bacteriochlorophyll using ''E. Coli'' as the host organism. <br />
<br />
|-<br />
|[[image:Solar_Energy.jpg]]<br />
|<br />
<br />
'''Background'''<br />
<br />
The specific goal for this project is to utilize ''E. Coli'' to produce chlorophyll efficiently. This is accomplished by controlling the first set of genes in the first branch point of the native ''E. Coli'' pathway and the chlorophyll biosynthesis pathway. However, the production of chlorophyll can lead to the eventual goal of engineering ''E. Coli'' to be a photosynthetic organism.<br />
<br />
Photosynthetic organisms that can be easily manipulated bring tremendous value to the current alternative energy research. Solar energy is available on Earth at a rate of 3850 x 10^21 Joules per year, while the worldwide energy consumption is only 0.471 x 10^21 Joules per year([http://en.wikipedia.org/wiki/Solar_energy Wikipedia Solar Energy]).<br />
<br />
Photosynthetic organisms can capture this untapped source of solar energy and transform them into chemical energy as biofuels.<br />
<br />
<br />
|-<br />
|[[Image:photosynthesis.jpg]]<br />
| <br />
'''Photosynthesis'''<br />
<br />
Photosynthesis is the process by which light energy is converted to chemical energy in form of glucose. The chemical equation of the process is: 6CO2 + 12H2O --> C6H12O6 + 6O2 + 6H2O, with the input of light energy required for the forward reaction. As shown in the equation, carbon dioxide is fixed in photosynthesis to produce a more complicated organic compound, glucose.<br />
<br />
|-<br />
|[[Image:organisms.jpg]]<br />
|<br />
<br />
'''Photosynthetic Organisms'''<br />
<br />
Plants and many species of bacteria are photosynthetic. Bacterial photosynthesis would be the focus in this description. The main classes of photosynthetic bacteria include cyanobacteria, purple bacteria, and heliobacteria. <br />
<br />
While plants have chloroplasts as a center for photosynthetic activity, bacteria do not. For bacteria, photosynthesis takes place within the cell. To perform the required photoreactions, a variety of pigments and membrane proteins are necessary. Out of the pigments, chlorophyll is the essential one.<br />
<br />
<br />
|-<br />
|[[Image:approach.jpg]]<br />
|<br />
<br />
'''Approach'''<br />
<br />
The goal of the project is to produce chlorophyll by establishing a strong metabolic pathway in ''E.Coli''. A generic metabolic pathway for chlorophyll synthesis in plants is given [https://2007.igem.org/Image:Chlorophyll_Biosynthesis.gif here (Generic Chlorophyll Biosynthesis)]. The biosynthesis of bacteriochlorophyll is similar; the part that our project focused on is shown [https://2007.igem.org/Image:Chlorophyll_Biosynthesis2.JPG here(Bacteriochlorophyll Biosynthesis)].<br />
<br />
In order to channel the flux of carbon through the chlorophyll biosynthetic pathway, the flux through the first branch point between the native ''E.Coli'' pathway and the chlorophyll pathway must be optimized. The enzyme magnesium-chelatase is responsible for converting protoporphyrin IX to Mg-protoporphyrin IX. Because this reaction must occur to a large degree, we want to use an enzyme that has the most enhanced activity. Since all photosynthetic bacteria utilize the very similar bacteriochlorophyll synthesis pathways, they all have their own versions of Mg-chelatase to perform the Mg-insertion reaction. A large part of the project is to subclone the genes for Mg-chelatse, for three photosynthetic bacteria—''Rhodobacter sphaeroides'' (purple bacteria), ''Synechocystis sp.'' (cyanobacteria), and ''Heliobacillus mobilis'' (heliobacteria). After subcloning, the enzymes are overexpressed and their activities are measured. <br />
<br />
Besides the establishment of a strong initial input of flux through the bacteriochlorophyll photosynthesis pathway, we have also explored the latter parts of the pathway. These latter steps are crucial for catalyzing reactions that would lead to the final product—bacteriochlorophyll.<br />
<br />
<br />
|-<br />
|[[Image:application.jpg]]<br />
|<br />
<br />
'''Applications'''<br />
<br />
Applications for the use of photosynthetic ''E.Coli'' are plentiful. The most important of which is the industrial-scale production of biofuels using bacteria. One can set up a bioreactor, filled with photosynthetic ''E.Coli'', running under sunlight to produce biofuels. The costs of producing biofuels using such bioreactors should be much more inexpensive than current methods due to the fact that solar energy is directly captured and utilized.<br />
<br />
The second application is the production of chlorophyll for research purposes. Once the most efficient metabolic pathway to produce chlorophylls is established, the synthesis of chlorophyll and its derivatives can be done easily.<br />
<br />
The third application can be the use of novel enzymes to insert specific metal ions into compounds such as protoporphyrin IX. These new organometallic complexes may have novel reactivity and be useful in many contexts. <br />
<br />
<br />
|}</div>KonniamChanhttp://2007.igem.org/wiki/index.php/File:Gel4Key.jpgFile:Gel4Key.jpg2007-10-26T22:47:59Z<p>KonniamChan: </p>
<hr />
<div></div>KonniamChanhttp://2007.igem.org/wiki/index.php/Berkeley_LBL/ResultsBerkeley LBL/Results2007-10-26T22:47:47Z<p>KonniamChan: </p>
<hr />
<div>{| border="0" cellspacing="8px" cellpadding="15" width="80%"<br />
|-<br />
|[[Berkeley_LBL|Home]]<br />
|[[Berkeley_LBL/Project|Project Description]]<br />
|[[Berkeley_LBL/Methods|Methods]]<br />
|[[Berkeley_LBL/Notebook|Notebook]]<br />
|[[Berkeley_LBL/Results|Results and Discussion]]<br />
|[[Berkeley_LBL/Resources|Resources]]<br />
|}<br />
<br />
== Results and Discussion ==<br />
<br />
[[Berkeley_LBL/Ladder|Invitrogen BenchMark Protein Ladder]]<br />
<br />
[[Berkeley_LBL/Key|Plasmid Key for Protein Gels]]<br />
<br />
[[Image:ProteinGel1.jpg]]<br />
<br />
[[Image:Gel1Key.jpg]]<br />
<br />
[[Image:ProteinGel2.jpg]]<br />
<br />
[[Image:Gel2Key.jpg]]<br />
<br />
[[Image:ProteinGel3.jpg]]<br />
<br />
[[Image:Gel3Key.jpg]]<br />
<br />
[[Image:ProteinGel4.jpg]]<br />
<br />
[[Image:Gel4Key.jpg]]</div>KonniamChanhttp://2007.igem.org/wiki/index.php/File:Gel3Key.jpgFile:Gel3Key.jpg2007-10-26T22:45:51Z<p>KonniamChan: </p>
<hr />
<div></div>KonniamChanhttp://2007.igem.org/wiki/index.php/Berkeley_LBL/ResultsBerkeley LBL/Results2007-10-26T22:45:38Z<p>KonniamChan: </p>
<hr />
<div>{| border="0" cellspacing="8px" cellpadding="15" width="80%"<br />
|-<br />
|[[Berkeley_LBL|Home]]<br />
|[[Berkeley_LBL/Project|Project Description]]<br />
|[[Berkeley_LBL/Methods|Methods]]<br />
|[[Berkeley_LBL/Notebook|Notebook]]<br />
|[[Berkeley_LBL/Results|Results and Discussion]]<br />
|[[Berkeley_LBL/Resources|Resources]]<br />
|}<br />
<br />
== Results and Discussion ==<br />
<br />
[[Berkeley_LBL/Ladder|Invitrogen BenchMark Protein Ladder]]<br />
<br />
[[Berkeley_LBL/Key|Plasmid Key for Protein Gels]]<br />
<br />
[[Image:ProteinGel1.jpg]]<br />
<br />
[[Image:Gel1Key.jpg]]<br />
<br />
[[Image:ProteinGel2.jpg]]<br />
<br />
[[Image:Gel2Key.jpg]]<br />
<br />
[[Image:ProteinGel3.jpg]]<br />
<br />
[[Image:Gel3Key.jpg]]<br />
<br />
[[Image:ProteinGel4.jpg]]</div>KonniamChanhttp://2007.igem.org/wiki/index.php/File:Gel2Key.jpgFile:Gel2Key.jpg2007-10-26T22:41:58Z<p>KonniamChan: </p>
<hr />
<div></div>KonniamChanhttp://2007.igem.org/wiki/index.php/Berkeley_LBL/ResultsBerkeley LBL/Results2007-10-26T22:41:43Z<p>KonniamChan: </p>
<hr />
<div>{| border="0" cellspacing="8px" cellpadding="15" width="80%"<br />
|-<br />
|[[Berkeley_LBL|Home]]<br />
|[[Berkeley_LBL/Project|Project Description]]<br />
|[[Berkeley_LBL/Methods|Methods]]<br />
|[[Berkeley_LBL/Notebook|Notebook]]<br />
|[[Berkeley_LBL/Results|Results and Discussion]]<br />
|[[Berkeley_LBL/Resources|Resources]]<br />
|}<br />
<br />
== Results and Discussion ==<br />
<br />
[[Berkeley_LBL/Ladder|Invitrogen BenchMark Protein Ladder]]<br />
<br />
[[Berkeley_LBL/Key|Plasmid Key for Protein Gels]]<br />
<br />
[[Image:ProteinGel1.jpg]]<br />
<br />
[[Image:Gel1Key.jpg]]<br />
<br />
[[Image:ProteinGel2.jpg]]<br />
<br />
[[Image:Gel2Key.jpg]]<br />
<br />
[[Image:ProteinGel3.jpg]]<br />
<br />
[[Image:ProteinGel4.jpg]]</div>KonniamChanhttp://2007.igem.org/wiki/index.php/Berkeley_LBL/ResultsBerkeley LBL/Results2007-10-26T22:34:57Z<p>KonniamChan: </p>
<hr />
<div>{| border="0" cellspacing="8px" cellpadding="15" width="80%"<br />
|-<br />
|[[Berkeley_LBL|Home]]<br />
|[[Berkeley_LBL/Project|Project Description]]<br />
|[[Berkeley_LBL/Methods|Methods]]<br />
|[[Berkeley_LBL/Notebook|Notebook]]<br />
|[[Berkeley_LBL/Results|Results and Discussion]]<br />
|[[Berkeley_LBL/Resources|Resources]]<br />
|}<br />
<br />
== Results and Discussion ==<br />
<br />
[[Berkeley_LBL/Ladder|Invitrogen BenchMark Protein Ladder]]<br />
<br />
[[Berkeley_LBL/Key|Plasmid Key for Protein Gels]]<br />
<br />
[[Image:ProteinGel1.jpg]]<br />
<br />
[[Image:Gel1Key.jpg]]<br />
<br />
[[Image:ProteinGel2.jpg]]<br />
<br />
[[Image:ProteinGel3.jpg]]<br />
<br />
[[Image:ProteinGel4.jpg]]</div>KonniamChanhttp://2007.igem.org/wiki/index.php/File:Gel1Key.jpgFile:Gel1Key.jpg2007-10-26T22:24:20Z<p>KonniamChan: </p>
<hr />
<div></div>KonniamChanhttp://2007.igem.org/wiki/index.php/Berkeley_LBL/ResultsBerkeley LBL/Results2007-10-26T22:21:53Z<p>KonniamChan: </p>
<hr />
<div>{| border="0" cellspacing="8px" cellpadding="15" width="80%"<br />
|-<br />
|[[Berkeley_LBL|Home]]<br />
|[[Berkeley_LBL/Project|Project Description]]<br />
|[[Berkeley_LBL/Methods|Methods]]<br />
|[[Berkeley_LBL/Notebook|Notebook]]<br />
|[[Berkeley_LBL/Results|Results and Discussion]]<br />
|[[Berkeley_LBL/Resources|Resources]]<br />
|}<br />
<br />
== Results and Discussion ==<br />
<br />
[[Berkeley_LBL/Ladder|Invitrogen BenchMark Protein Ladder]]<br />
<br />
[[Berkeley_LBL/Key|Plasmid Key for Protein Gels]]<br />
<br />
[[Image:ProteinGel1.jpg]]<br />
<br />
[[Image:ProteinGel2.jpg]]<br />
<br />
[[Image:ProteinGel3.jpg]]<br />
<br />
[[Image:ProteinGel4.jpg]]</div>KonniamChanhttp://2007.igem.org/wiki/index.php/Berkeley_LBL/KeyBerkeley LBL/Key2007-10-26T22:21:05Z<p>KonniamChan: </p>
<hr />
<div>[[Image:Key.jpg]]</div>KonniamChanhttp://2007.igem.org/wiki/index.php/Berkeley_LBL/LadderBerkeley LBL/Ladder2007-10-26T22:19:21Z<p>KonniamChan: </p>
<hr />
<div>[[Image:BenchMark.jpg]]</div>KonniamChan