Construction and Testing

==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]]

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 at each step in the assembly cycle.
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==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 <i>lac</i> inducible promoter to control expression of the enhancing or repressing elements.

===Comparing basal and induced expression levels for the quorum sensing promoters===
<table width="100%" style="border-collapse: collapse; padding-top:15px; padding-bottom:15px;">  

  <tr style="background-color:#CCCCCC; text-align:left;">
    <td width="400px"><b>CONSTRUCT</b></td>
    <td><b>AIMS</b></td>
  </tr>
  <tr valign="middle" style="border-top: 1px solid black; border-bottom: 1px solid black;">
    <td>[[Image:UW_testing_constructs1AB.png|400x100px]]</td>
    <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, Plux  and Plas</td>
  </tr>
  <tr valign="middle" style="border-top: 1px solid black;border-bottom: 1px solid black;">
    <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>
*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.</td>
  </tr>
</table>
<br>

===Testing functionality of the various promoters===
<table width="100%" style="border-collapse: collapse; padding-top:15px; padding-bottom:15px;">  
  <tr style="background-color:#CCCCCC; text-align:left;">
    <td width="400px"><b>CONSTRUCT</b></td>
    <td><b>AIMS</b></td>
  </tr>
  <tr valign="middle" style="border-top: 1px solid black; border-bottom: 1px solid black;">
    <td>[[Image:UW_testing_constructs2A1A2.png|400px]]</td>
    <td><b>The Omp promoter</b><br>  
*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 </td>
  </tr>
  <tr valign="middle" style="border-top: 1px solid black; border-bottom: 1px solid black;">
    <td>[[Image:UW_testing_constructs2B.png|400px]]</td>
    <td><b>The Tet promoter and operator </b><br> 
*Negatively-controlled promoter, repressed by TetR and derepressed by the inducer tetracycline
*Test for levels of expression during repressed versus derepressed state</td>
  </tr>   
  <tr valign="middle" style="border-top: 1px solid black; border-bottom: 1px solid black;">
    <td>[[Image:UW_testing_constructs2CD.png|400px]]</td>
    <td><b>The Plas and Plux quorum-sensing promoters </b><br> 
*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 valign="middle" style="border-top: 1px solid black; border-bottom: 1px solid black;">
    <td>[[Image:UW_testing_constructs2E.png|400px]]</td>
    <td><b>The PlasOcI promoter-operator fusion </b><br>
*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 <i>las</i> promoter activity </td>
  </tr>
</table>

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