Team:Hong Kong-CUHK/PROJECT RESULTS

From 2012.igem.org

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       <p>&nbsp;</p>
       <p>&nbsp;</p>
       <p class="aloveofthunder" style="line-height:normal; margin-bottom:35px">RESULTS</p>
       <p class="aloveofthunder" style="line-height:normal; margin-bottom:35px">RESULTS</p>
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      <p><strong>Data for Our Favourite New Parts</strong></p>
 +
      <ol>
 +
        <li><a href="http://partsregistry.org/Part:BBa_K786001">Main Page</a> – <strong> Promoter efficiency for  BBa_K786001. BBa_K786002, BBa_K786003 </strong></li>
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      </ol>
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      <p>To test the expression of sensory rhodopsin triggered by constitutive promoter BBa_J23100 to sense light, </p>
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      <p align="center"><center><img width="200" height="66" src="https://static.igem.org/mediawiki/2012/1/1f/R1.png" /></center></p>
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      <p> </p>
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      <p>we need to test the effect conferred by different <em>E. coli</em> strains to expression of red fluorescence protein reporter downstream of BBa_J23100 to different bacterial strains. It allows us to select the suitable strain(s) for this constitutive promoter for expressing sensory rhodopsin.</p>
 +
      <p>Florescence plate reader was used to take readings of fluorescence emission of 635nm and absorbance at 600nm (OD600) between time intervals of 12 hours on each strain. The measurements were started when the cultures reached a OD600 of around 0.4 that represents log phase of active proliferation. Growth curve and fluorescence intensity against time were plotted to compare cell growth and protein expression on different strains.</p>
 +
      <p>Three independent experiments were conducted. No significant difference was observed on the growth curves, indicating a similar growth rate among the three bacterial strains with BBa_J23100 transformed. It implies the promoter does not cause cell toxicity or growth inhibition of these three bacterial strains.</p>
 +
<p><center><img border="0" width="417" height="334" src="https://static.igem.org/mediawiki/2012/e/e0/R2.png" alt="Description: ::Desktop:iGEM 2012:Characterization on Bba_J23100 OD600.jpg" /></center></p>
 +
      <p>For the protein expression, the results showed that the fluorescence intensity of reporter in DH5α was significantly lower compared with TOP10 and BL21(DE3).</p>
 +
      <p><center><img border="0" width="417" height="330" src="https://static.igem.org/mediawiki/2012/f/f5/R3.png" alt="Description: ::Desktop:iGEM 2012:Characterization on Bba_J23100-fluoresence.jpg" /></center></p>
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      <p>&nbsp;</p>
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      <p>To conclude, DH5α is not an optimal strain to utilize promoter BBa_J23100, while TOP10 and BL 21(DE3) can effectively express the reporter. Therefore, in downstream application of our light sensing biobricks (BBa_K786001, BBa_K786002, BBa_K786003) in which BBa_J23100 was used, DH5α are not used.</p>
 +
      <p>&nbsp;</p>
 +
      <ol>
 +
        <li><a href="http://partsregistry.org/Part:BBa_K786002">Main Page</a> – <strong>Positive Phototactic Construct for Blue Light Detection </strong><strong>(BBa_K786002)</strong></li>
 +
      </ol>
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      <p>To evaluate whether our biobrick BBa_K786002 causes cell movement under blue light exposure, we transferred transformed bacteria on soft agar and observe if it moves under blue light. It is known that bacteria can swim on soft agar.</p>
 +
      <p>Soft agar (0.4%) plate was prepared. Cell cultures transformed with BBa_K786002 (6, 12 and 25 µl) with OD600 ~2 were pipetted on each semi-agar plate. Duplicate aliquots were done on each plate and four plates were made. Two of the plates were placed in dark and two were placed under blue LED light of 200 mW (with spectra covering from 430-480 nm).<br />
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        <center><img border="0" width="300" height="352" src="https://static.igem.org/mediawiki/2012/b/bf/R4.png" alt="Description: C:\Users\Ricky\Documents\igem\rhodopsin\result\blue light (1).PNG" /></center><br />
 +
        We placed the plates overnight for 12 h at 25oC and compared the diameter differences between the plates with or without blue light exposure. We used paired <em>t</em>-test for analyzing the collected data. A significant difference was observed between the plates (*** indicates <em>p</em> &lt; 0.001). The average diameters of three clones exposed under blue light are bigger than the counterparts in dark. Blue light triggers a change in diameter of 180 ± 40 %, while there is no significant change in diameter for those without BBa_K786002. When we put the bacteria transformed with BBa_K786002 under light, the blue light stimulates SRII and switches on the hisitine kinase CheY by decreasing the phosphorylation level of CheY (CheY-P). Therefore, a prolonged running period in bacteria is resulted as CheY-P causes cell tumbling, a random movement. When the cell tumbles and faces towards light, SRII is stimulated again. The process repeats and the cell will eventually travel towards blue light.<br />
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<center> <img border="0" width="450" height="338" src="https://static.igem.org/mediawiki/2012/f/fe/R5.png" alt="Description: ::Desktop:iGEM 2012:Protein B with light on semi-agar.jpg" /></center></p>
 +
      <ol>
 +
        <li><a href="http://partsregistry.org/Part:BBa_K786003">Main Page</a> – <strong>Phototactic Construct for Orange Light Detection</strong><strong>&nbsp;</strong><strong>(</strong><strong>BBa_K786003</strong><strong>)</strong></li>
 +
      </ol>
 +
      <p>See our <a href="https://2012.igem.org/Team:Hong_Kong-CUHK/PROJECT_MODELING"><strong>modeling</strong></a> page for more details.</p>
 +
      <p><strong>Data for Pre-existing Parts</strong></p>
 +
      <ol>
 +
        <li>Experience – <strong>Constitutive promoter family</strong><strong> (BBa_J23100)</strong></li>
 +
      </ol>
 +
      <p>We tested the expression level of reporter protein under J23100 promoter in 3 typically used E. coli strains, BL21, TOP10 and DH5α. Our data showed that all except DH5α are suitable to express protein under J23100.</p>
 +
      <p>See our <a href="https://2012.igem.org/Team:Hong_Kong-CUHK/BIOBRICKS_CHAR"><strong>characterization</strong></a> page for more details.</p>
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Revision as of 16:41, 26 September 2012



 

 

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RESULTS

Data for Our Favourite New Parts

  1. Main Page Promoter efficiency for BBa_K786001. BBa_K786002, BBa_K786003

To test the expression of sensory rhodopsin triggered by constitutive promoter BBa_J23100 to sense light,

we need to test the effect conferred by different E. coli strains to expression of red fluorescence protein reporter downstream of BBa_J23100 to different bacterial strains. It allows us to select the suitable strain(s) for this constitutive promoter for expressing sensory rhodopsin.

Florescence plate reader was used to take readings of fluorescence emission of 635nm and absorbance at 600nm (OD600) between time intervals of 12 hours on each strain. The measurements were started when the cultures reached a OD600 of around 0.4 that represents log phase of active proliferation. Growth curve and fluorescence intensity against time were plotted to compare cell growth and protein expression on different strains.

Three independent experiments were conducted. No significant difference was observed on the growth curves, indicating a similar growth rate among the three bacterial strains with BBa_J23100 transformed. It implies the promoter does not cause cell toxicity or growth inhibition of these three bacterial strains.

Description: ::Desktop:iGEM 2012:Characterization on Bba_J23100 OD600.jpg

For the protein expression, the results showed that the fluorescence intensity of reporter in DH5α was significantly lower compared with TOP10 and BL21(DE3).

Description: ::Desktop:iGEM 2012:Characterization on Bba_J23100-fluoresence.jpg

 

To conclude, DH5α is not an optimal strain to utilize promoter BBa_J23100, while TOP10 and BL 21(DE3) can effectively express the reporter. Therefore, in downstream application of our light sensing biobricks (BBa_K786001, BBa_K786002, BBa_K786003) in which BBa_J23100 was used, DH5α are not used.

 

  1. Main PagePositive Phototactic Construct for Blue Light Detection (BBa_K786002)

To evaluate whether our biobrick BBa_K786002 causes cell movement under blue light exposure, we transferred transformed bacteria on soft agar and observe if it moves under blue light. It is known that bacteria can swim on soft agar.

Soft agar (0.4%) plate was prepared. Cell cultures transformed with BBa_K786002 (6, 12 and 25 µl) with OD600 ~2 were pipetted on each semi-agar plate. Duplicate aliquots were done on each plate and four plates were made. Two of the plates were placed in dark and two were placed under blue LED light of 200 mW (with spectra covering from 430-480 nm).

Description: C:\Users\Ricky\Documents\igem\rhodopsin\result\blue light (1).PNG

We placed the plates overnight for 12 h at 25oC and compared the diameter differences between the plates with or without blue light exposure. We used paired t-test for analyzing the collected data. A significant difference was observed between the plates (*** indicates p < 0.001). The average diameters of three clones exposed under blue light are bigger than the counterparts in dark. Blue light triggers a change in diameter of 180 ± 40 %, while there is no significant change in diameter for those without BBa_K786002. When we put the bacteria transformed with BBa_K786002 under light, the blue light stimulates SRII and switches on the hisitine kinase CheY by decreasing the phosphorylation level of CheY (CheY-P). Therefore, a prolonged running period in bacteria is resulted as CheY-P causes cell tumbling, a random movement. When the cell tumbles and faces towards light, SRII is stimulated again. The process repeats and the cell will eventually travel towards blue light.
Description: ::Desktop:iGEM 2012:Protein B with light on semi-agar.jpg

  1. Main PagePhototactic Construct for Orange Light Detection (BBa_K786003)

See our modeling page for more details.

Data for Pre-existing Parts

  1. Experience – Constitutive promoter family (BBa_J23100)

We tested the expression level of reporter protein under J23100 promoter in 3 typically used E. coli strains, BL21, TOP10 and DH5α. Our data showed that all except DH5α are suitable to express protein under J23100.

See our characterization page for more details.


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