Difference between revisions of "Part:BBa K817033:Experience"
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how you used this part and how it worked out.
 
how you used this part and how it worked out.
  
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===Applications of BBa_K817033===
 
  
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===User Reviews===
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==Improvement==
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Designed by NTHU_Taiwan 2019. (See the part at BBa_K3040007)
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===Team NTHU Taiwan 2019: Effects and inducing of fatty acid to E.coli and Relative Fluorescence to Anderson Promoter===
 
  
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====Overview====
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===Background===
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This  has an improvement on the natural acyl-CoA responsive promoter pfadBA submitted by iGEM12_NTU-Taida (BBa_K817033). However, according to their result, this promoter has a massive leakage, which has very high value of downstream reporter gene baseline expression. Besides, the fold change after 0.04% of oleic acid induction has only achieved 1.67-fold, considered no significant signal rise. Thus, we coupled an endogenous thioesterase coding gene with this promoter. By doing so, we are able to perform a tunable and dynamic gene expression.
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===Mechanism===
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Tes A is a heterologous thioesterase, which is able to hydrolyze fatty acyl-CoAs to free fatty acids. Here, we combine Tes A with pFadBA (wild type) together as a new promoter, Tes A pFadBA, to see if the sensitivity range of fatty acid concentration lowers due to the increase of endogenous fatty acid.
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<br>
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In the microbial, carbon source such as glucose or fatty acid will be metabolized to acetyl-CoA. When fatty acid is needed to be catabolized into other macromolecules, the acetyl-CoA will be converted into Acyl-ACP and finally formed free fatty acid. The following is the detailed mechanism of the biosynthetic and degradation pathway of fatty acid. [1]
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From previous study, NTU-Taida found that pFadBA can be induce by adding fatty acid. We’d like to understand more about this promoter by answering the following questions:<br>
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**Does fatty acid affect the growth of cell?
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**Will higher fatty acid concentration induce more fluorescence?
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**How strong is the promoter pFadBA?
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              <img style="margin:20px auto 5px auto;" src="https://2019.igem.org/wiki/images/8/86/T--NTHU_Taiwan--Improve1.gif" width="60%">
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              <div class="fig_title"> The mechansim system</div>
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      <div class="col-lg-3" style=" margin:auto;text-align:center;"></div>
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====Effect of Fatty acid to E. coli====
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Nevertheless, the accumulation of Acyl-ACP will negatively inhibit the conversion of malonyl-CoA to Acyl-ACP and thus repress biosynthesis of free fatty acid. Thus, endogenous TesA gene which encodes a thioesterase will hydrolyze these Acyl-ACP and subsequently produce a significant level of free fatty acid.
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We discuss the relationship between fatty acid and the growth of E. coli to check suitable conditions for future experiments. First, we check whether fatty acid affect our experiments. Second, we choose the best concentration for future experiments.<br>
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In general, relative cell concentration does not change very much. It indicates that under 0.4mM and 3.125mM fatty acid, E. coli might grow better than control. However, when the concentration is 25mM, it inhibits growth of E. coli. We can notice that low concentration of fatty acid is somehow benefit for cell growth. Therefore, we choose concentration lower than 10 mM as our highest concentration to induce the promoter in the following experiments.<br>
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===Concept of design===
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<br><img src="https://2019.igem.org/wiki/images/a/a4/T--NTHU_Taiwan--characterization1.png"><br>
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As you know, high concentration of fatty acid will promote β-oxidation but not synthesis of fatty acid. However, overexpression of TesA can help the E. coli to deplete the Acyl-ACP and thus rescue the production of free fatty acid. The more fatty acid present, the more acyl-CoA can be converted and thus the higher transcription rate can pfadBA can achieved. The following was the mechanism proposed.  
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      <div class="row">
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              <img style="margin:20px auto 5px auto;" src="https://2019.igem.org/wiki/images/0/01/T--NTHU_Taiwan--liya8.png" width="60%">  
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              <div class="fig_title"> The mechansim system</div>
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      </div>
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      <div class="col-lg-3" style=" margin:auto;text-align:center;"></div>
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      </div>
 
</html>
 
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<br>
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Some previous literature has reported that the strain carries TesA overexpression is capable of showing 10 to 25-fold of fluorescence than the native promoter [2]. This result matched our proposed model.
  
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===Result===
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As we predicted that the fluorescence fold change after induction of fatty acid will be greater as tesA has produce more fatty acid. The result shows that the fold change of fluorescence after 16 hours 5mM oleic acid induction can come up to 9-fold. This has greatly improved the native strength of the promoter since it can only increase to about 2-fold. This modification helped us to control the strength of promoter more precisely compared to the native pfadBA since the induced-transcription range of the promoter has been broaden.
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              <img style="margin:20px auto 5px auto;" src="https://2019.igem.org/wiki/images/2/2f/T--NTHU_Taiwan--liya9.png" width="60%">
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              <div class="fig_title"> Figure 7. Protein expression of fatty acid promoter TesA-FadBA after 16 hours of induction under different fatty acid concentration (n=3).</div>
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===Future work===
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According to the previous research, the fold change should be able to reached 10 to 25-fold. We deduce that the problem is we used a weaker promoter placed before TesA, thus the fatty acid produced endogenously is fewer than previously reported. Hence, we proposed to insert the TesA sequence directly at the downstream of pfadBA promoter and made it regulated by this promoter. Once we have added the fatty acid, then pfadBA promoter will be activated, RFP and tesA will be produced. Tes A will further catalyze the formation of fatty acid through the dissociation of Acyl-ACP. The more fatty acid, the more pfadBA will be activated and more tesA protein will be produced. This will form a positive feedback loop and thus the fold change will become higher.
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===Reference===
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[1] Janßen, H. J., & Steinbüchel, A. (2014). Fatty acid synthesis in Escherichia coli and its applications towards the production of fatty acid based biofuels. Biotechnology for biofuels, 7(1), 7.
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<br>
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[2] Zhang, F., Carothers, J. M., & Keasling, J. D. (2012). Design of a dynamic sensor-regulator system for production of chemicals and fuels derived from fatty acids. Nature biotechnology, 30(4), 354.
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===User Reviews===
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<!-- DON'T DELETE --><partinfo>BBa_K817033 StartReviews</partinfo>
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<!-- Template for a user review-->

Latest revision as of 05:03, 21 October 2019


This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.


Improvement

Designed by NTHU_Taiwan 2019. (See the part at BBa_K3040007)


Background

This has an improvement on the natural acyl-CoA responsive promoter pfadBA submitted by iGEM12_NTU-Taida (BBa_K817033). However, according to their result, this promoter has a massive leakage, which has very high value of downstream reporter gene baseline expression. Besides, the fold change after 0.04% of oleic acid induction has only achieved 1.67-fold, considered no significant signal rise. Thus, we coupled an endogenous thioesterase coding gene with this promoter. By doing so, we are able to perform a tunable and dynamic gene expression.

Mechanism

Tes A is a heterologous thioesterase, which is able to hydrolyze fatty acyl-CoAs to free fatty acids. Here, we combine Tes A with pFadBA (wild type) together as a new promoter, Tes A pFadBA, to see if the sensitivity range of fatty acid concentration lowers due to the increase of endogenous fatty acid.
In the microbial, carbon source such as glucose or fatty acid will be metabolized to acetyl-CoA. When fatty acid is needed to be catabolized into other macromolecules, the acetyl-CoA will be converted into Acyl-ACP and finally formed free fatty acid. The following is the detailed mechanism of the biosynthetic and degradation pathway of fatty acid. [1]

The mechansim system

Nevertheless, the accumulation of Acyl-ACP will negatively inhibit the conversion of malonyl-CoA to Acyl-ACP and thus repress biosynthesis of free fatty acid. Thus, endogenous TesA gene which encodes a thioesterase will hydrolyze these Acyl-ACP and subsequently produce a significant level of free fatty acid.

Concept of design

As you know, high concentration of fatty acid will promote β-oxidation but not synthesis of fatty acid. However, overexpression of TesA can help the E. coli to deplete the Acyl-ACP and thus rescue the production of free fatty acid. The more fatty acid present, the more acyl-CoA can be converted and thus the higher transcription rate can pfadBA can achieved. The following was the mechanism proposed.

The mechansim system

Some previous literature has reported that the strain carries TesA overexpression is capable of showing 10 to 25-fold of fluorescence than the native promoter [2]. This result matched our proposed model.

Result

As we predicted that the fluorescence fold change after induction of fatty acid will be greater as tesA has produce more fatty acid. The result shows that the fold change of fluorescence after 16 hours 5mM oleic acid induction can come up to 9-fold. This has greatly improved the native strength of the promoter since it can only increase to about 2-fold. This modification helped us to control the strength of promoter more precisely compared to the native pfadBA since the induced-transcription range of the promoter has been broaden.

Figure 7. Protein expression of fatty acid promoter TesA-FadBA after 16 hours of induction under different fatty acid concentration (n=3).

Future work

According to the previous research, the fold change should be able to reached 10 to 25-fold. We deduce that the problem is we used a weaker promoter placed before TesA, thus the fatty acid produced endogenously is fewer than previously reported. Hence, we proposed to insert the TesA sequence directly at the downstream of pfadBA promoter and made it regulated by this promoter. Once we have added the fatty acid, then pfadBA promoter will be activated, RFP and tesA will be produced. Tes A will further catalyze the formation of fatty acid through the dissociation of Acyl-ACP. The more fatty acid, the more pfadBA will be activated and more tesA protein will be produced. This will form a positive feedback loop and thus the fold change will become higher.

Reference

[1] Janßen, H. J., & Steinbüchel, A. (2014). Fatty acid synthesis in Escherichia coli and its applications towards the production of fatty acid based biofuels. Biotechnology for biofuels, 7(1), 7.
[2] Zhang, F., Carothers, J. M., & Keasling, J. D. (2012). Design of a dynamic sensor-regulator system for production of chemicals and fuels derived from fatty acids. Nature biotechnology, 30(4), 354.



User Reviews

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