Difference between revisions of "Team:SDU-CHINA/Demonstrate"

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<h1 id="main-title">Demonstration</h1>
 
<h1 id="main-title">Demonstration</h1>
  
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<p>This year we desired to create a light-induced metabolic flux redirection platform in engineered bacteria. To achieve this idea, we improved the light sensor CcaS-CcaR system. In addition, we measured the type-I-E CRSPRi system and combined it with advanced photoreceptor to control the metabolic flux dynamically.</p>
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<p>This year we desired to create a light-induced metabolic flux redirection platform in engineered bacteria. To achieve this idea, we improved the light sensor CcaS/CcaR system. In addition, we measured the type-I-E CRSPRi system and combined it with advanced photoreceptor to control the metabolic flux dynamically.</p>
  
 
<p>Now we are proud to present our results:</p>
 
<p>Now we are proud to present our results:</p>
  
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<p>(1) We successfully created three new types of CcaS-CcaR system: #3, #4, #10.</p>
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<p>(1) We successfully created three new types of CcaS/CcaR system: #3, #4, #10.<br>
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(2) We constructed a metabolic flux redirection platform dynamically controlled by light.<br>
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<p>(2) We constructed a metabolic flux redirection platform dynamically controlled by light.</p>
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(3) We demonstrated our system through the production of Polyhydroxybutyrate(PHB).<br>
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(4) We manufactured illuminated hardware through reconstructing the shakers suitable for further fermentation.</p>
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<p>(3) We demonstrated our system through the production of Polyhydroxybutyrate(PHB).</p>
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<p>(4) We manufactured illuminated hardware through reconstructing the shakers suitable for further fermentation. </p>
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<p>We removed two PAS domains in CcaS and acquired #3、#4、#10 variants of CcaS. We tested these three variants with the wild type and measured their expressions of sfGFP in the dark state or under light (green or red) state respectively. The measurements showed that #3 and #10 have similar response to green/red light and are better than the original one.</p>
 
<p>We removed two PAS domains in CcaS and acquired #3、#4、#10 variants of CcaS. We tested these three variants with the wild type and measured their expressions of sfGFP in the dark state or under light (green or red) state respectively. The measurements showed that #3 and #10 have similar response to green/red light and are better than the original one.</p>
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<img src="https://static.igem.org/mediawiki/2018/e/e2/T--SDU-CHINA--fluorescence1.png" width="600">
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<img src="https://static.igem.org/mediawiki/2018/e/e2/T--SDU-CHINA--fluorescence1.png" width="600" title="Figure 1. The fluorescence intensity of #3、#4、#10 under illumination.">
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<div style="text-align: center; font-size: 15px">Figure 1. The fluorescence intensity of wild type #3, #4, #10 under illumination.</div>
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<br>
  
 
<p>To demonstrate our dynamic light-activated redirection system, we tried to change the induction conditions at different stages of growth. The samples were illuminated by red light at the beginning, then induced with green light at early-log phase (2h), mid-log phase(5h) and late-log phase(9h). As you can see in the figure that among all conditions, bacteria growth was repressed the most obviously when induced at mid-log phase (5h), whereas induction at late-log phase (9h) had little impact on bacteria growth. Through this dynamic platform, we can arbitrarily regulate the expression of targeted gene at different stages to control the metabolism in bacteria to optimize the production of biological products.</p>
 
<p>To demonstrate our dynamic light-activated redirection system, we tried to change the induction conditions at different stages of growth. The samples were illuminated by red light at the beginning, then induced with green light at early-log phase (2h), mid-log phase(5h) and late-log phase(9h). As you can see in the figure that among all conditions, bacteria growth was repressed the most obviously when induced at mid-log phase (5h), whereas induction at late-log phase (9h) had little impact on bacteria growth. Through this dynamic platform, we can arbitrarily regulate the expression of targeted gene at different stages to control the metabolism in bacteria to optimize the production of biological products.</p>
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<img src="https://static.igem.org/mediawiki/2018/5/5c/T--SDU-CHINA--fluorescence2.png" width="600">
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<img src="https://static.igem.org/mediawiki/2018/5/5c/T--SDU-CHINA--fluorescence2.png" width="600" title="Figure 2. OD curves of E. coli induced at different stages of growth." alt="Figure 2. OD curves of E. coli induced at different stages of growth.">
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<div style="text-align: center; font-size: 15px">Figure 2. OD curves of <i>E. coli</i> induced at different stages of growth.</div>
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<br>
  
 
<p>In addition, we analyzed the metabolic flux before and after the shift through the Flux Balance Analysis model, and the computational result also certifies that original metabolic flux was affected by targeting gltA gene as well as TCA Cycle.</p>
 
<p>In addition, we analyzed the metabolic flux before and after the shift through the Flux Balance Analysis model, and the computational result also certifies that original metabolic flux was affected by targeting gltA gene as well as TCA Cycle.</p>
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<img src="https://static.igem.org/mediawiki/2018/e/ed/T--SDU-CHINA--hdwr1.png" width="600">
 
<img src="https://static.igem.org/mediawiki/2018/e/ed/T--SDU-CHINA--hdwr1.png" width="600">
 
<img src="https://static.igem.org/mediawiki/2018/5/53/T--SDU-CHINA--hdwr2.png" width="600">
 
<img src="https://static.igem.org/mediawiki/2018/5/53/T--SDU-CHINA--hdwr2.png" width="600">
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<div style="text-align: center; font-size: 15px">Figure 3. Fermentation under illumination.</div>
  
 
<p>In a conclusion, we successfully constructed a dynamic photoactivated metabolic flux redirection platform. Our idea worked out for PHB production. (I hope, I hope, too…) </p>
 
<p>In a conclusion, we successfully constructed a dynamic photoactivated metabolic flux redirection platform. Our idea worked out for PHB production. (I hope, I hope, too…) </p>

Revision as of 05:51, 17 October 2018

Demonstration

This year we desired to create a light-induced metabolic flux redirection platform in engineered bacteria. To achieve this idea, we improved the light sensor CcaS/CcaR system. In addition, we measured the type-I-E CRSPRi system and combined it with advanced photoreceptor to control the metabolic flux dynamically.

Now we are proud to present our results:

(1) We successfully created three new types of CcaS/CcaR system: #3, #4, #10.
(2) We constructed a metabolic flux redirection platform dynamically controlled by light.
(3) We demonstrated our system through the production of Polyhydroxybutyrate(PHB).
(4) We manufactured illuminated hardware through reconstructing the shakers suitable for further fermentation.

We removed two PAS domains in CcaS and acquired #3、#4、#10 variants of CcaS. We tested these three variants with the wild type and measured their expressions of sfGFP in the dark state or under light (green or red) state respectively. The measurements showed that #3 and #10 have similar response to green/red light and are better than the original one.

Figure 1. The fluorescence intensity of wild type #3, #4, #10 under illumination.

To demonstrate our dynamic light-activated redirection system, we tried to change the induction conditions at different stages of growth. The samples were illuminated by red light at the beginning, then induced with green light at early-log phase (2h), mid-log phase(5h) and late-log phase(9h). As you can see in the figure that among all conditions, bacteria growth was repressed the most obviously when induced at mid-log phase (5h), whereas induction at late-log phase (9h) had little impact on bacteria growth. Through this dynamic platform, we can arbitrarily regulate the expression of targeted gene at different stages to control the metabolism in bacteria to optimize the production of biological products.

Figure 2. OD curves of E. coli induced at different stages of growth.
Figure 2. OD curves of E. coli induced at different stages of growth.

In addition, we analyzed the metabolic flux before and after the shift through the Flux Balance Analysis model, and the computational result also certifies that original metabolic flux was affected by targeting gltA gene as well as TCA Cycle.

To introduce this system into metabolic engineering and demonstrate that it would make sense in production, we took the fermentation of producing PHB as an example. The results show…

(waiting for the result. I still don’t have it…)

For the convenience of the experiments and further fermentation, we also reconstructed the shakers for erlenmeyers in which the volume ranging from 50ml to 1000ml. This equipment can change the light color, light intensity, even the irradiation frequency. Such a multi-functional ameliorated hardware provides a solid foundation for further study in light.

Figure 3. Fermentation under illumination.

In a conclusion, we successfully constructed a dynamic photoactivated metabolic flux redirection platform. Our idea worked out for PHB production. (I hope, I hope, too…)