Team:Imperial College London/Project Chemotaxis Design

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<h1>Design</h1>
<h1>Design</h1>
-
<p>We aim to ensure that the <a href="https://2011.igem.org/Team:Imperial_College_London/Project_Chemotaxis_Specifications">specifications</a> that were drawn up are considered in the design.
+
<p>We aim to ensure that the <a href="https://2011.igem.org/Team:Imperial_College_London/Project_Chemotaxis_Specifications"><b>specifications</b></a> that were drawn up are considered in the design.
<p><b>1. The bacteria should sense malate and actively move towards roots.</b></p>
<p><b>1. The bacteria should sense malate and actively move towards roots.</b></p>
<ul class="a">
<ul class="a">
-
<li><p>While malate-responsive sensors do not naturally occur in <i>E. coli</i>, they have been identified in several other bacteria species, including the soil microbes <i>Pseudomonas aeruginosa</i> PA01 strain & <i>Pseudomonas putida</i> KT2440 strain. PA2652 is a malate responsive chemoreceptor found in <i>P. aeruginosa</i> & mcpS is a receptor found in <i>P. putida</i> responding to a number of TCA cycle intermediates including malate<sup>[1][2]</sup>. The molecular mechanism of chemotaxis in <i>Pseudomonas aeruginosa</i> & <i>P. putida</i> are different to that of <i>E. coli</i>, however there is high degree of structural similarity between the proteins that make up the chemotaxis pathway. Since these proteins are structurally similar, it is reasonable to assume that the PA2652 or mcpS domain will interact with the native chemotaxis pathway in <i>E. coli</i> and the bacteria will be able to perform chemotactic response upon malate binding to an introduced receptor. </li>
+
<li><p>While malate-responsive sensors do not naturally occur in <i>E. coli</i>, they have been identified in several other bacterial species, including the soil microbes <i>Pseudomonas aeruginosa</i> PA01 strain & <i>Pseudomonas putida</i> KT2440 strain. PA2652 is a malate-responsive chemoreceptor found in <i>P. aeruginosa</i> and mcpS is a receptor found in <i>P. putida</i> that responds to a number of TCA cycle intermediates including malate<sup>[1][2]</sup>. The molecular mechanisms of chemotaxis in <i>Pseudomonas aeruginosa</i> and <i>P. putida</i> differ from that of <i>E. coli</i>. However, there is a high degree of structural similarity between the proteins that make up the chemotaxis pathways in these organisms. It is therefore reasonable to assume that the PA2652 or mcpS domain will interact with the native chemotaxis pathway in <i>E. coli</i> and that the bacteria will be able to perform a chemotactic response upon malate binding to an introduced receptor.</p></li>
</ul>
</ul>
-
<p><b>2. The construct must be as modular as possible.</b></p>  
+
<p><b>2. Efficient expression in our chassis.</b></p>  
<ul class="a">
<ul class="a">
-
<li><p> Genetic constructs for expression of malate chemoreceptor are not complicated, however they are modular. Expression of the constructs is under constitutive expression of promoter J23100. It is provided from the registry in the biobrick K398500, in the backbone vector pSB1C3. The coding sequence has been removed from this construct using PCR, providing us with backbone vector with the constitutive promoter, to be assembled using Gibson or CPEC assembly. Ribosome binding sites (RBS) have been generated using Salis RBS calculator with relative translation initiation rates (TIR): 44050 & 42010 for mcpS and PA2652 respectively. The coding sequences for mcpS and  for PA2652 have been codon optimised using our codon optimisation software, so that sequence is codon optimised for <i>E. coli</i>. We exploit terminator embedded in the backbone vector pSB1C3 for termination of transcription.  In addition, modularity of our system is ensured by putting 15 bp insulator sequence between the RBS and the promoter. This sequence was specifically designed to make the promoter interchangeable without affecting the RBS strength and therefore translation initation rate is not affected by changing a different promoter in front of the insulator sequence. This allows us to change the promoter strength simply by changing promoter without modifying the whole genetic construct, thus it improves modularity of the construct. The insulator sequence acts as a starting point for PCR and it can be used to amplify the receptor sequence without the promoter.</p></li>
+
<li><p> We have used our <a href="https://2011.igem.org/Team:Imperial_College_London/Software"><b>codon optimisation software</b></a> to optimise the PA2652 construct (<a href="http://partsregistry.org/Part:BBa_K515102"><b>BBa_K515102</b></a>) for expression in <i>E. coli</i>.</p></li>
</ul>
</ul>
-
<p><b>3. Uptake of bacteria into roots.</b> </p>
+
<p><b>3. The construct must be as modular as possible.</b></p>  
<ul class="a">
<ul class="a">
-
<li><p>Root uptake of both <i>E. coli</i> and the bakers’s yeast <i>S. cerevisiae</i> can be observed in the model organism <i>Arabidopsis thaliana</i> and in tomato plants. It is a process that occurs naturally (althought it yet remains to be observed in soil) and we do not need to incorporate additional genes into our design. </p></li>
+
<li><p> Genetic constructs for expression of the malate chemoreceptor are not complicated. However, they are modular. At present, expression of the constructs is regulated by the constitutive promoter <a href="http://partsregistry.org/Part:BBa_J23100"><b>J23100</b></a>. This is one of the strongest constitutive promoters in the <a href="http://partsregistry.org/Main_Page"><b>Parts Registry</b></a>. We chose this promoter as it is easier to tune down the expression of a construct than to tune it up. To tune the expression of the genetic construct we have introduced a 15 bp insulator sequence, which allows easy interchangeability of the promoter through the use of PCR and thus fine-tuning of expression levels. It ensures that the ribosome binding site's TIR (translation initiation rate) does not change when the promoter is replaced. The translation initiation rates for PA2652 and mcpS are 42010 and 44050, respectively.</p></li>
</ul>
</ul>
-
<p><b>4. Designing insulator sequences to enable promoter switching.</b> </p>
+
 
 +
<p><b>4. Uptake of bacteria into roots.</b> </p>
<ul class="a">
<ul class="a">
-
<li><p>To enable tweaking of IAA production by using promoters of different strengths, we are designing an insulator sequence in front of the ribosome binding sites of both IaaM and IaaH to facilitate promoter switching without affecting the RBS strength. </p></li>
+
<li><p>Root uptake of both <i>E. coli</i> and bakers’s yeast <i>Saccharomyces cerevisiae</i> can be observed in the model organism <i>Arabidopsis thaliana</i> and also in tomato plants. This is a process that occurs naturally (althought it yet remains to be observed in soil) and we do not need to incorporate additional genes into our design for uptake to take place. </p></li>
-
</ul>
+
-
<p><b>5. Joint codon optimisation for <i>E. coli</i> and <i>B. subtilis</i>
+
-
<ul class="a">
+
-
<li><p>We made a codon optimising software to optimise the IaaM and IaaH sequences for both chassis to provide flexibility in the future, although we are using <i>E. coli</i> as our chassis.</p></li>
+
-
</ul>
+
-
<p><b>1. The bacteria should sense malate and actively move towards roots.</b></p>
+
-
<p><ul class="a">
+
-
<li><p>While malate-responsive sensors do not naturally occur in <i>E. coli</i>, they have been identified in several other bacteria species, including the soil microbes <i>Pseudomonas aeruginosa</i> PA01 strain & <i>Pseudomonas putida</i> KT2440 strain. PA2652 is a malate responsive chemoreceptor found in <i>P. aeruginosa</i> & mcpS is a receptor found in <i>P. putida</i> responding to a number of TCA cycle intermediates including malate<sup>[1][2]</sup>. The molecular mechanism of chemotaxis in <i>Pseudomonas aeruginosa</i> & <i>P. putida</i> are different to that of <i>E. coli</i>, however there is high degree of structural similarity between the proteins that make up the chemotaxis pathway. Since these proteins are structurally similar, it is reasonable to assume that the PA2652 or mcpS domain will interact with the native chemotaxis pathway in <i>E. coli</i> and the bacteria will be able to perform chemotactic response upon malate binding to an introduced receptor. </p></p>
+
-
<p><b>2. The construct must be as modular as possible.</b></p>
+
-
<p><ul class="a">
+
-
<p>Genetic constructs for expression of malate chemoreceptor are not complicated, however they are modular. Expression of the constructs is under constitutive expression of promoter J23100. It is provided from the registry in the biobrick K398500, in the backbone vector pSB1C3. The coding sequence has been removed from this construct using PCR, providing us with backbone vector with the constitutive promoter, to be assembled using Gibson or CPEC assembly. Ribosome binding sites (RBS) have been generated using Salis RBS calculator with relative translation initiation rates (TIR): 44050 & 42010 for mcpS and PA2652 respectively. The coding sequences for mcpS and  for PA2652 have been codon optimised using our codon optimisation software, so that sequence is codon optimised for <i>E. coli</i>. We exploit terminator embedded in the backbone vector pSB1C3 for termination of transcription.  In addition, modularity of our system is ensured by putting 15 bp insulator sequence between the RBS and the promoter. This sequence was specifically designed to make the promoter interchangeable without affecting the RBS strength and therefore translation initation rate is not affected by changing a different promoter in front of the insulator sequence. This allows us to change the promoter strength simply by changing promoter without modifying the whole genetic construct, thus it improves modularity of the construct. The insulator sequence acts as a starting point for PCR and it can be used to amplify the receptor sequence without the promoter.</p>
+
-
</ul>
+
-
<p><b>3. Uptake of bacteria into roots.</b>
+
-
<p><ul class="a">
+
-
<li><p>Root uptake of both <i>E. coli</i> and the bakers’s yeast <i>S. cerevisiae</i> can be observed in the model organism <i>Arabidopsis thaliana</i> and in tomato plants. It is a process that occurs naturally (althought it yet remains to be observed in soil) and we do not need to incorporate additional genes into our design.</p>
+
</ul>
</ul>
 +
<h2>References</h2>
 +
<p>[1] Lacal J, Alfonso C, Liu X et al. (2010) Identification of a chemoreceptor for tricarboxylic acid cycle intermediates: differential chemotactic response towards receptor ligands. <i>Journal of Biological Chemistry</i> <b>285 (30)</b> 23126–23136.</p>
 +
<p>
 +
[2] Alvarez-Ortega C and Harwood CS (2007) Identification of malate chemoreceptor in <i>Pseudomonas aeruginosa</i> by screening for chemotaxis defects in an energy taxis-deficient mutant. <i>Applied and Environmental Microbiology</i> <b>73</b> 7793-7795.</p>
 +
 +
<h2>
 +
<a href="https://2011.igem.org/Team:Imperial_College_London/Project_Chemotaxis_Specifications" style="text-decoration:none;color:#728F1D;float:left;">
 +
<img src="https://static.igem.org/mediawiki/2011/8/8e/ICL_PreviousBtn.png" width="40px" style="float;left;"/>
 +
M1: Specifications
 +
</a>
 +
<a href="https://2011.igem.org/Team:Imperial_College_London/Project_Chemotaxis_Modelling" style="text-decoration:none;color:#728F1D;float:right;">
 +
M1: Modelling
 +
<img src="https://static.igem.org/mediawiki/2011/9/90/ICL_NextBtn.png" width="40px" style="float;right;"/>
 +
</a>
 +
</h2>
 +
<br/>
 +
<br/>
-
<h1>References</h1>
 
-
<p>[1] Lacal J, Alfonso C, Liu X et al. (2010) Identification of a chemoreceptor for tricarboxylic acid cycle intermediates: differential chemotactic response towards receptor ligands. <i>Journal of Biological Chemistry</i> <b>285(30)</b> 23126–23136.</p>
 
-
<p>
 
-
[2] Alvarez-Ortega C and Harwood CS (2007) Identification of malate chemoreceptor in <i>Pseudomonas aeruginosa</i> by screening for chemotaxis defects in an energy taxis-deficient mutant. <i>Applied and Environmental Microbiology</i> <b>73</b> 7793-7795.<br>
 
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Latest revision as of 03:42, 29 October 2011



Module 1: Phyto-Route

Chemotaxis is the movement of bacteria based on attraction or repulsion of chemicals. Roots secrete a variety of compounds that E. coli are not attracted to naturally. Accordingly, we engineered a chemoreceptor into our chassis that can sense malate, a common root exudate, so that it can swim towards the root. Additionally, E. coli are actively taken up by plant roots, which will allow targeted IAA delivery into roots by our system.





Design

We aim to ensure that the specifications that were drawn up are considered in the design.

1. The bacteria should sense malate and actively move towards roots.

  • While malate-responsive sensors do not naturally occur in E. coli, they have been identified in several other bacterial species, including the soil microbes Pseudomonas aeruginosa PA01 strain & Pseudomonas putida KT2440 strain. PA2652 is a malate-responsive chemoreceptor found in P. aeruginosa and mcpS is a receptor found in P. putida that responds to a number of TCA cycle intermediates including malate[1][2]. The molecular mechanisms of chemotaxis in Pseudomonas aeruginosa and P. putida differ from that of E. coli. However, there is a high degree of structural similarity between the proteins that make up the chemotaxis pathways in these organisms. It is therefore reasonable to assume that the PA2652 or mcpS domain will interact with the native chemotaxis pathway in E. coli and that the bacteria will be able to perform a chemotactic response upon malate binding to an introduced receptor.

2. Efficient expression in our chassis.

3. The construct must be as modular as possible.

  • Genetic constructs for expression of the malate chemoreceptor are not complicated. However, they are modular. At present, expression of the constructs is regulated by the constitutive promoter J23100. This is one of the strongest constitutive promoters in the Parts Registry. We chose this promoter as it is easier to tune down the expression of a construct than to tune it up. To tune the expression of the genetic construct we have introduced a 15 bp insulator sequence, which allows easy interchangeability of the promoter through the use of PCR and thus fine-tuning of expression levels. It ensures that the ribosome binding site's TIR (translation initiation rate) does not change when the promoter is replaced. The translation initiation rates for PA2652 and mcpS are 42010 and 44050, respectively.

4. Uptake of bacteria into roots.

  • Root uptake of both E. coli and bakers’s yeast Saccharomyces cerevisiae can be observed in the model organism Arabidopsis thaliana and also in tomato plants. This is a process that occurs naturally (althought it yet remains to be observed in soil) and we do not need to incorporate additional genes into our design for uptake to take place.

References

[1] Lacal J, Alfonso C, Liu X et al. (2010) Identification of a chemoreceptor for tricarboxylic acid cycle intermediates: differential chemotactic response towards receptor ligands. Journal of Biological Chemistry 285 (30) 23126–23136.

[2] Alvarez-Ortega C and Harwood CS (2007) Identification of malate chemoreceptor in Pseudomonas aeruginosa by screening for chemotaxis defects in an energy taxis-deficient mutant. Applied and Environmental Microbiology 73 7793-7795.

M1: Specifications M1: Modelling



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