Construction and Testing

From 2007.igem.org

(Difference between revisions)
(Parts Submitted to the Registry)
 
(30 intermediate revisions not shown)
Line 1: Line 1:
-
==Construction==
+
==Construction Tree==
 +
The diagram below outlines our parallel construction plan, from individual registry parts to the final half-adder. Because many people were working on the project at once, it is designed to keep everyone organised.
 +
 
[[Image:UW_Construction_Chart.jpg|thumb|500px|left|Construction Chart]]
[[Image:UW_Construction_Chart.jpg|thumb|500px|left|Construction Chart]]
 +
 +
Diagram details:
 +
*Expected fragment sizes are shown at every node so gel results can be analyzed quickly.
 +
*Vector resistances for each part/construct are shown, with blue representing ampicillin resistance and red representing kanamycin resistance. Purple represents a resistance to both.
 +
*Parts and developing constructs all have abbreviated names for ease of labeling. To make the subtle distinction between a coding sequence and a coding sequence with a ribosome binding site, we use a ' on the computer and an underline when writing. A double ' or double underline indicates a coding sequence with a ribosome binding site and a transcriptional terminator.
 +
*Arrows show the direction of DNA cloning and specify which restriction enzymes to use for cutting. Hollow arrows are present when two parts are ligated from vectors of the same resistance, requiring additional methods such as the use of alkaline phosphatase, gel extraction, or a three-way ligation into a vector with different resistance.
 +
*The numbers 1-2-3-3.5-4 indicate which major protocols need to be carried out at each step in the assembly cycle:
 +
1) Transform selected DNA.
 +
 +
2) Streak colonies from transformation spread plates. Patch on to plates of the opposite resistance. Innoculate culture tubes.
 +
 +
3) Use the patches to discard colonies which are incorrect. Miniprep from the culture tubes of potentially correct colonies. Digest DNA with NotI and run on a gel. Discard colonies which have incorrect sizes. Innoculate tubes from correct colonies.
 +
 +
3.5) Perform a full digest using appropriate enzymes on correct parts. Ligate parts together.
 +
 +
4) Create a glycerol stock from the innoculation.
<br style="clear:both;"/>
<br style="clear:both;"/>
-
==Testing/Results==
+
==Testing Constructs==
-
===Testing Constructs===
+
-
Making use of flurorescent proteins(GFP and RFP) as reporter genes
+
We use the fluorescent proteins GFP and RFP as reporter genes to test the functionality of some of the components and constructs of our half-adder design. Where possible, we make use of the <i>lac</i> inducible promoter to control expression of the enhancing or repressing elements.
-
- use of indicuble promoters to...
+
-
====Comparing basal and induced expression levels for the quorum sensing promoters====
+
===Comparing basal and induced expression levels for the quorum sensing promoters===
<table width="100%" style="border-collapse: collapse; padding-top:15px; padding-bottom:15px;">   
<table width="100%" style="border-collapse: collapse; padding-top:15px; padding-bottom:15px;">   
Line 18: Line 34:
   <tr valign="middle" style="border-top: 1px solid black; border-bottom: 1px solid black;">
   <tr valign="middle" style="border-top: 1px solid black; border-bottom: 1px solid black;">
     <td>[[Image:UW_testing_constructs1AB.png|400x100px]]</td>
     <td>[[Image:UW_testing_constructs1AB.png|400x100px]]</td>
-
     <td><b>The quorum-sensing promoters: How active is 'unactivated' Plas and Plux?</B><BR>
+
     <td><b>The quorum-sensing promoters: How active is 'unactive'?</B> <br>
-
*preliminary test for the basal levels of activity of the positively-controlled quorum sensing promoters </td>
+
*Preliminary test for the basal levels of activity of the positively-controlled quorum sensing promoters, Plux  and Plas</td>
   </tr>
   </tr>
   <tr valign="middle" style="border-top: 1px solid black;border-bottom: 1px solid black;">
   <tr valign="middle" style="border-top: 1px solid black;border-bottom: 1px solid black;">
     <td>[[Image:UW_testing_constructs1CD.png|400px]]</td>
     <td>[[Image:UW_testing_constructs1CD.png|400px]]</td>
-
     <td><b>The quorum-sensing constructs: How off is 'off' and how on is 'on'?</b></br>
+
     <td><b>The quorum-sensing constructs: How off is 'off' and how on is 'on'?</b><br>
-
*test for levels of expression in the unenhanced versus enhanced state
+
*Test for levels of expression in the unenhanced versus enhanced state.
-
*xR must be expressed in same strain as Px; xI can be expressed in a different strain since its AHL is diffusable
+
*lasR/luxR must be expressed in same strain as Plas/Plux (respectively) since these genes encode transcription activators regulated  by the products of lasI/luxI; lasI/luxI can be expressed in a different strain since their respective AHL molecules are diffusible through cell membranes.
-
*both strains grown in a single culture tube </td>
+
*The two strains are grown in a single culture tube for this test.</td>
   </tr>
   </tr>
</table>
</table>
<br>
<br>
-
====Testing functionality of the various promoters====
+
===Testing functionality of the various promoters===
<table width="100%" style="border-collapse: collapse; padding-top:15px; padding-bottom:15px;">   
<table width="100%" style="border-collapse: collapse; padding-top:15px; padding-bottom:15px;">   
   <tr style="background-color:#CCCCCC; text-align:left;">
   <tr style="background-color:#CCCCCC; text-align:left;">
Line 40: Line 56:
     <td>[[Image:UW_testing_constructs2A1A2.png|400px]]</td>
     <td>[[Image:UW_testing_constructs2A1A2.png|400px]]</td>
     <td><b>The Omp promoter</b><br>   
     <td><b>The Omp promoter</b><br>   
-
*postively-controlled promoter activated by endogenous OmpR protein (sensitive to salt concentrations via EnvZ protein)
+
*Positively-controlled promoter activated by endogenous OmpR protein (sensitive to salt concentrations via EnvZ protein)
-
*test for activity using high salt levels in EnvZ+ strain
+
*Test for activity using high salt levels in EnvZ+ strain
-
*test for activity using the photo-active red system in EnvZ- strain </td>
+
*Test for activity using the photo-active red system in EnvZ- strain </td>
   </tr>
   </tr>
   <tr valign="middle" style="border-top: 1px solid black; border-bottom: 1px solid black;">
   <tr valign="middle" style="border-top: 1px solid black; border-bottom: 1px solid black;">
     <td>[[Image:UW_testing_constructs2B.png|400px]]</td>
     <td>[[Image:UW_testing_constructs2B.png|400px]]</td>
     <td><b>The Tet promoter and operator </b><br>  
     <td><b>The Tet promoter and operator </b><br>  
-
*negatively-controlled promoter, repressed by TetR and derepressed by the inducer tetracycline
+
*Negatively-controlled promoter, repressed by TetR and derepressed by the inducer tetracycline
-
*test for levels of expression during repressed versus derepressed state</td>
+
*Test for levels of expression during repressed versus derepressed state</td>
   </tr>   
   </tr>   
   <tr valign="middle" style="border-top: 1px solid black; border-bottom: 1px solid black;">
   <tr valign="middle" style="border-top: 1px solid black; border-bottom: 1px solid black;">
     <td>[[Image:UW_testing_constructs2CD.png|400px]]</td>
     <td>[[Image:UW_testing_constructs2CD.png|400px]]</td>
-
     <td><b>The quorum-sensing promoters: Plas and Plux </b><br>  
+
     <td><b>The Plas and Plux quorum-sensing promoters </b><br>  
-
*as above</td>
+
*Test for levels of expression in the unenhanced versus enhanced state
 +
*lasR/luxR must be expressed in same strain as Plas/Plux since these genes encode transcription activators regulated  by the products of lasI/luxI; lasI/luxI can be expressed in a different strain since their respective AHL molecules are diffusible through cell membranes
 +
*The two strains are grown in a single culture tube for this test</td>
   </tr>   
   </tr>   
   <tr valign="middle" style="border-top: 1px solid black; border-bottom: 1px solid black;">
   <tr valign="middle" style="border-top: 1px solid black; border-bottom: 1px solid black;">
     <td>[[Image:UW_testing_constructs2E.png|400px]]</td>
     <td>[[Image:UW_testing_constructs2E.png|400px]]</td>
     <td><b>The PlasOcI promoter-operator fusion </b><br>
     <td><b>The PlasOcI promoter-operator fusion </b><br>
-
*negatively- and positively-controlled promoter in the final half adder design, repressed by cI and enhanced by LasR  
+
*Both negatively- and positively-controlled promoter in the final half adder design, repressed by cI and enhanced by LasR  
-
*testfor activity in the repressed versus enhanced state
+
*Test for activity in the repressed versus enhanced state
-
*test in addition to above construct since operator fusion may affect promoter activity </td>
+
*Test in addition to above construct since cI operator fusion may affect <i>las</i> promoter activity </td>
   </tr>
   </tr>
</table>
</table>
 +
 +
 +
 +
==Parts Submitted to the Registry==
 +
 +
The following is a table listing all of the parts submitted to the registry.
 +
 +
<table style="border-width:1px;border-style:solid;border-spacing:0px;border-collapse:separate;width:100%">
 +
  <tr style="font-weight:bold">
 +
    <td style="border-bottom:1px solid black;padding:5px"> Name </td>
 +
    <td style="border-bottom:1px solid black;padding:5px"> Part Number </td>
 +
    <td style="border-bottom:1px solid black;padding:5px"> Constructs </td>
 +
    <td style="border-bottom:1px solid black;padding:5px;width:33%"> Comments </td>
 +
  </tr>
 +
  <tr>
 +
    <td style="border-bottom:1px solid black;padding:5px"> rfp'</td>
 +
    <td style="border-bottom:1px solid black;padding:5px"> [http://partsregistry.org/Part:BBa_S03873 BBa_S03873] </td>
 +
    <td style="border-bottom:1px solid black;padding:5px"> [[Image:UW_1.PNG]] </td>
 +
    <td style="border-bottom:1px solid black;padding:5px"> confirmed (functional after promoter) </td>
 +
  </tr>
 +
  <tr>
 +
    <td style="border-bottom:1px solid black;padding:5px"> ho1' </td>
 +
    <td style="border-bottom:1px solid black;padding:5px"> [http://partsregistry.org/Part:BBa_S03877 BBa_S03877]</td>
 +
    <td style="border-bottom:1px solid black;padding:5px"> [[Image:UW_3.png]] </td>
 +
    <td style="border-bottom:1px solid black;padding:5px">  </td>
 +
  </tr>
 +
  <tr>
 +
    <td style="border-bottom:1px solid black;padding:5px"> lasI' ' </td>
 +
    <td style="border-bottom:1px solid black;padding:5px"> [http://partsregistry.org/Part:BBa_S03878 BBa_S03878] </td>
 +
    <td style="border-bottom:1px solid black;padding:5px"> [[Image:UW_4.PNG]] </td>
 +
    <td style="border-bottom:1px solid black;padding:5px"> LasI protein generator </td>
 +
  </tr>
 +
  <tr>
 +
    <td style="border-bottom:1px solid black;padding:5px"> lasR' ' </td>
 +
    <td style="border-bottom:1px solid black;padding:5px"> [http://partsregistry.org/Part:BBa_S03882 BBa_S03882] </td>
 +
    <td style="border-bottom:1px solid black;padding:5px"> [[Image:UW_14.PNG]] </td>
 +
    <td style="border-bottom:1px solid black;padding:5px"> LasR protein generator </td>
 +
  </tr>
 +
  <tr>
 +
    <td style="border-bottom:1px solid black;padding:5px"> pcyA' </td>
 +
    <td style="border-bottom:1px solid black;padding:5px"> [http://partsregistry.org/Part:BBa_S03874 BBa_S03874] </td>
 +
    <td style="border-bottom:1px solid black;padding:5px"> [[Image:UW_12.PNG]] </td>
 +
    <td style="border-bottom:1px solid black;padding:5px">  </td>
 +
  </tr>
 +
  <tr>
 +
    <td style="border-bottom:1px solid black;padding:5px"> pcyA' ' </td>
 +
    <td style="border-bottom:1px solid black;padding:5px"> [http://partsregistry.org/Part:BBa_S03875 BBa_S03875] </td>
 +
    <td style="border-bottom:1px solid black;padding:5px"> [[Image:UW_2.PNG]] </td>
 +
    <td style="border-bottom:1px solid black;padding:5px"> PcyA protein generator </td>
 +
  </tr>
 +
  <tr>
 +
    <td style="border-bottom:1px solid black;padding:5px"> tetR' </td>
 +
    <td style="border-bottom:1px solid black;padding:5px"> [http://partsregistry.org/Part:BBa_S03879 BBa_S03879] </td>
 +
    <td style="border-bottom:1px solid black;padding:5px"> [[Image:UW_13.PNG]] </td>
 +
    <td style="border-bottom:1px solid black;padding:5px">  </td>
 +
  </tr>
 +
  <tr>
 +
    <td style="border-bottom:1px solid black;padding:5px"> tetR' ' </td>
 +
    <td style="border-bottom:1px solid black;padding:5px"> [http://partsregistry.org/Part:BBa_S03880 BBa_S03880] </td>
 +
    <td style="border-bottom:1px solid black;padding:5px"> [[Image:UW_5.PNG]] </td>
 +
    <td style="border-bottom:1px solid black;padding:5px"> TetR protein generator </td>
 +
  </tr>
 +
  <tr>
 +
    <td style="border-bottom:1px solid black;padding:5px"> Plas rfp' </td>
 +
    <td style="border-bottom:1px solid black;padding:5px"> [http://partsregistry.org/Part:BBa_S03885 BBa_S03885] </td>
 +
    <td style="border-bottom:1px solid black;padding:5px"> [[Image:UW_8.PNG]] </td>
 +
    <td style="border-bottom:1px solid black;padding:5px"> </td>
 +
  </tr>
 +
  <tr>
 +
    <td style="border-bottom:1px solid black;padding:5px"> Plac lasr' ' </td>
 +
    <td style="border-bottom:1px solid black;padding:5px"> [http://partsregistry.org/Part:BBa_S03883 BBa_S03883] </td>
 +
    <td style="border-bottom:1px solid black;padding:5px"> [[Image:UW_7.PNG]] </td>
 +
    <td style="border-bottom:1px solid black;padding:5px"> </td>
 +
  </tr>
 +
  <tr>
 +
    <td style="border-bottom:1px solid black;padding:5px"> Ptet rfp' </td>
 +
    <td style="border-bottom:1px solid black;padding:5px"> [http://partsregistry.org/Part:BBa_S03887 BBa_S03887] </td>
 +
    <td style="border-bottom:1px solid black;padding:5px"> [[Image:UW_9.PNG]] </td>
 +
    <td style="border-bottom:1px solid black;padding:5px"> functionality confirmed </td>
 +
  </tr>
 +
  <tr>
 +
    <td style="border-bottom:1px solid black;padding:5px"> Pomp rfp' </td>
 +
    <td style="border-bottom:1px solid black;padding:5px"> [http://partsregistry.org/Part:BBa_S03888 BBa_S03888] </td>
 +
    <td style="border-bottom:1px solid black;padding:5px"> [[Image:UW_10.PNG]] </td>
 +
    <td style="border-bottom:1px solid black;padding:5px"> functionality confirmed </td>
 +
  </tr>
 +
  <tr>
 +
    <td style="border-bottom:1px solid black;padding:5px"> Plux rfp' </td>
 +
    <td style="border-bottom:1px solid black;padding:5px"> [http://partsregistry.org/Part:BBa_S03889 BBa_S03889] </td>
 +
    <td style="border-bottom:1px solid black;padding:5px"> [[Image:UW_11.PNG]] </td>
 +
    <td style="border-bottom:1px solid black;padding:5px"> functionality confirmed </td>
 +
  </tr>
 +
  <tr>
 +
    <td style="border-bottom:1px solid black;padding:5px"> PlasOcI rfp' </td>
 +
    <td style="border-bottom:1px solid black;padding:5px"> [http://partsregistry.org/Part:BBa_S03881 BBa_S03881] </td>
 +
    <td style="border-bottom:1px solid black;padding:5px"> [[Image:UW_6.PNG]] </td>
 +
    <td style="border-bottom:1px solid black;padding:5px"> functionality confirmed </td>
 +
  </tr>
 +
</table>
 +
 +
Notes:
 +
* ' denotes that the construct contains the ribosome binding site (RBS) and the coding region for that gene.
 +
* ' ' denotes that the construct contains the ribosome binding site, coding region for that gene, and a transcriptional terminator.
 +
 +
<br clear="all">
 +
 +
<center>
 +
  [[Waterloo | Home ]] | [[Project | Project]] | [[Modelling | Mathematical Modelling]] | [[Construction_and_Testing | Construction and Testing]] | [[Future_Work | Future Work]]
 +
</center>

Latest revision as of 03:43, 27 October 2007

Contents

Construction Tree

The diagram below outlines our parallel construction plan, from individual registry parts to the final half-adder. Because many people were working on the project at once, it is designed to keep everyone organised.

Construction Chart

Diagram details:

  • Expected fragment sizes are shown at every node so gel results can be analyzed quickly.
  • Vector resistances for each part/construct are shown, with blue representing ampicillin resistance and red representing kanamycin resistance. Purple represents a resistance to both.
  • Parts and developing constructs all have abbreviated names for ease of labeling. To make the subtle distinction between a coding sequence and a coding sequence with a ribosome binding site, we use a ' on the computer and an underline when writing. A double ' or double underline indicates a coding sequence with a ribosome binding site and a transcriptional terminator.
  • Arrows show the direction of DNA cloning and specify which restriction enzymes to use for cutting. Hollow arrows are present when two parts are ligated from vectors of the same resistance, requiring additional methods such as the use of alkaline phosphatase, gel extraction, or a three-way ligation into a vector with different resistance.
  • The numbers 1-2-3-3.5-4 indicate which major protocols need to be carried out at each step in the assembly cycle:

1) Transform selected DNA.

2) Streak colonies from transformation spread plates. Patch on to plates of the opposite resistance. Innoculate culture tubes.

3) Use the patches to discard colonies which are incorrect. Miniprep from the culture tubes of potentially correct colonies. Digest DNA with NotI and run on a gel. Discard colonies which have incorrect sizes. Innoculate tubes from correct colonies.

3.5) Perform a full digest using appropriate enzymes on correct parts. Ligate parts together.

4) Create a glycerol stock from the innoculation.

Testing Constructs

We use the fluorescent proteins GFP and RFP as reporter genes to test the functionality of some of the components and constructs of our half-adder design. Where possible, we make use of the lac inducible promoter to control expression of the enhancing or repressing elements.

Comparing basal and induced expression levels for the quorum sensing promoters

CONSTRUCT AIMS
UW testing constructs1AB.png The quorum-sensing promoters: How active is 'unactive'?
  • Preliminary test for the basal levels of activity of the positively-controlled quorum sensing promoters, Plux and Plas
UW testing constructs1CD.png The quorum-sensing constructs: How off is 'off' and how on is 'on'?
  • Test for levels of expression in the unenhanced versus enhanced state.
  • lasR/luxR must be expressed in same strain as Plas/Plux (respectively) since these genes encode transcription activators regulated by the products of lasI/luxI; lasI/luxI can be expressed in a different strain since their respective AHL molecules are diffusible through cell membranes.
  • The two strains are grown in a single culture tube for this test.


Testing functionality of the various promoters

CONSTRUCT AIMS
UW testing constructs2A1A2.png The Omp promoter
  • Positively-controlled promoter activated by endogenous OmpR protein (sensitive to salt concentrations via EnvZ protein)
  • Test for activity using high salt levels in EnvZ+ strain
  • Test for activity using the photo-active red system in EnvZ- strain
UW testing constructs2B.png The Tet promoter and operator
  • Negatively-controlled promoter, repressed by TetR and derepressed by the inducer tetracycline
  • Test for levels of expression during repressed versus derepressed state
UW testing constructs2CD.png The Plas and Plux quorum-sensing promoters
  • Test for levels of expression in the unenhanced versus enhanced state
  • lasR/luxR must be expressed in same strain as Plas/Plux since these genes encode transcription activators regulated by the products of lasI/luxI; lasI/luxI can be expressed in a different strain since their respective AHL molecules are diffusible through cell membranes
  • The two strains are grown in a single culture tube for this test
UW testing constructs2E.png The PlasOcI promoter-operator fusion
  • Both negatively- and positively-controlled promoter in the final half adder design, repressed by cI and enhanced by LasR
  • Test for activity in the repressed versus enhanced state
  • Test in addition to above construct since cI operator fusion may affect las promoter activity


Parts Submitted to the Registry

The following is a table listing all of the parts submitted to the registry.

Name Part Number Constructs Comments
rfp' [http://partsregistry.org/Part:BBa_S03873 BBa_S03873] UW 1.PNG confirmed (functional after promoter)
ho1' [http://partsregistry.org/Part:BBa_S03877 BBa_S03877] UW 3.png
lasI' ' [http://partsregistry.org/Part:BBa_S03878 BBa_S03878] UW 4.PNG LasI protein generator
lasR' ' [http://partsregistry.org/Part:BBa_S03882 BBa_S03882] UW 14.PNG LasR protein generator
pcyA' [http://partsregistry.org/Part:BBa_S03874 BBa_S03874] UW 12.PNG
pcyA' ' [http://partsregistry.org/Part:BBa_S03875 BBa_S03875] UW 2.PNG PcyA protein generator
tetR' [http://partsregistry.org/Part:BBa_S03879 BBa_S03879] UW 13.PNG
tetR' ' [http://partsregistry.org/Part:BBa_S03880 BBa_S03880] UW 5.PNG TetR protein generator
Plas rfp' [http://partsregistry.org/Part:BBa_S03885 BBa_S03885] UW 8.PNG
Plac lasr' ' [http://partsregistry.org/Part:BBa_S03883 BBa_S03883] UW 7.PNG
Ptet rfp' [http://partsregistry.org/Part:BBa_S03887 BBa_S03887] UW 9.PNG functionality confirmed
Pomp rfp' [http://partsregistry.org/Part:BBa_S03888 BBa_S03888] UW 10.PNG functionality confirmed
Plux rfp' [http://partsregistry.org/Part:BBa_S03889 BBa_S03889] UW 11.PNG functionality confirmed
PlasOcI rfp' [http://partsregistry.org/Part:BBa_S03881 BBa_S03881] UW 6.PNG functionality confirmed

Notes:

  • ' denotes that the construct contains the ribosome binding site (RBS) and the coding region for that gene.
  • ' ' denotes that the construct contains the ribosome binding site, coding region for that gene, and a transcriptional terminator.


  Home  |  Project |  Mathematical Modelling |  Construction and Testing |  Future Work