Team:HKUST-Hong Kong/Characterization

From 2012.igem.org

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           <p>In our project, we have characterized two promoters and the cell death device by different methods. This tells us that our parts are functional and we can quantitatively adapt their activity by changing the experimental conditions.</p>
           <p>In our project, we have characterized two promoters and the cell death device by different methods. This tells us that our parts are functional and we can quantitatively adapt their activity by changing the experimental conditions.</p>
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           <div class="Section_Heading" align="center"><h3><p>Low Efficiency Constitutive Promoter pTms</p></h3></div>
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           <div class="Section_Heading" align="center"><h3><p>Detailed Plan</p></h3></div>
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              <h1>Low Efficiency Constitutive Promoter pTms</h1>
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          </div>
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          <p><strong>Background  Information <u>(link to construct page)</u></strong><br />
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(The basic reason of using this low  efficiency constitutive promoter is to enable our bacteria to express a low  level of antitoxin so that a certain amount of toxin can be balanced; thus the  BMP-2 whose expression is tightly linked with the toxin can be expressed at a  certain level as well.)</p>
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<p><strong>Objective</strong><br />
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  Our objective of characterizing this  promoter is to determine the relevant activity of it to the standard constitutive  promoter activity reference point given by part registry so that we and the  subsequent IGEM teams may have an idea how efficient this promoter is.</p>
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<p><strong>Method</strong><br />
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  Instead of measuring the absolute promoter activity,  our characterization was generally based on measuring the relevant in vivo activity  of this constitutive promoter. By adopting this method, we may be able to eliminate  the error caused by different experimental conditions and give a relatively  more convincing result.<br />
 +
  By linking the promoter with GFP (BBa_E0240),  the promoter activity was represented by the GFP synthesis rate which can be  easily measured. E.Coli carrying the right construct was then cultured to log  phase. During a time slot around the mid-log phase, the GFP intensity and OD595  value were measured to obtain the Relative Promoter Units (RPU).</p>
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<p><strong>Procedure</strong></p>
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<ol>
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  <li>Constructing BBa_K733009-pSB3K3 (pTms-BBa_E0240-pSB3K3); Transforming  BBa_I20260-pSB3K3 (Standard Constitutive Promoter Activity Reference Point) from  2012 Distribution;</li>
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  <li>Preparing supplemented M9 medium; <u>(link to recipe for  supplemented M9 medium)</u></li>
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  <li>Culturing E.Coli carrying BBa_K733009-pSB3K3 and E.Coli carrying BBa_I20260-pSB3K3 in supplemented M9 medium and  measuring the growth curve respectively;</li>
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  <li>Measuring the GFP intensity and ODA595 value every 15 minutes after  E.Coli carrying BBa_K733009-pSB3K3 and E.Coli carrying BBa_I20260 are cultured  to mid-log phase;</li>
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  <li>Calculating the Relative Promoter Unites (RPU) using the obtained  data;</li>
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  <li>Modifying the result.</li>
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</ol>
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<p><strong>Result</strong><br />
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  ^^^^^^^^^</p>
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Revision as of 09:59, 23 September 2012

Team:HKUST-Hong Kong - 2012.igem.org

CHARACTERIZATION

Introduction

In our project, we have characterized two promoters and the cell death device by different methods. This tells us that our parts are functional and we can quantitatively adapt their activity by changing the experimental conditions.

Detailed Plan

Low Efficiency Constitutive Promoter pTms

Background Information (link to construct page)
(The basic reason of using this low efficiency constitutive promoter is to enable our bacteria to express a low level of antitoxin so that a certain amount of toxin can be balanced; thus the BMP-2 whose expression is tightly linked with the toxin can be expressed at a certain level as well.)

Objective
Our objective of characterizing this promoter is to determine the relevant activity of it to the standard constitutive promoter activity reference point given by part registry so that we and the subsequent IGEM teams may have an idea how efficient this promoter is.

Method
Instead of measuring the absolute promoter activity, our characterization was generally based on measuring the relevant in vivo activity of this constitutive promoter. By adopting this method, we may be able to eliminate the error caused by different experimental conditions and give a relatively more convincing result.
By linking the promoter with GFP (BBa_E0240), the promoter activity was represented by the GFP synthesis rate which can be easily measured. E.Coli carrying the right construct was then cultured to log phase. During a time slot around the mid-log phase, the GFP intensity and OD595 value were measured to obtain the Relative Promoter Units (RPU).

Procedure

  1. Constructing BBa_K733009-pSB3K3 (pTms-BBa_E0240-pSB3K3); Transforming BBa_I20260-pSB3K3 (Standard Constitutive Promoter Activity Reference Point) from 2012 Distribution;
  2. Preparing supplemented M9 medium; (link to recipe for supplemented M9 medium)
  3. Culturing E.Coli carrying BBa_K733009-pSB3K3 and E.Coli carrying BBa_I20260-pSB3K3 in supplemented M9 medium and measuring the growth curve respectively;
  4. Measuring the GFP intensity and ODA595 value every 15 minutes after E.Coli carrying BBa_K733009-pSB3K3 and E.Coli carrying BBa_I20260 are cultured to mid-log phase;
  5. Calculating the Relative Promoter Unites (RPU) using the obtained data;
  6. Modifying the result.

Result
^^^^^^^^^

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