Difference between revisions of "Part:BBa K2278002"

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=='''Introduction'''==
 
=='''Introduction'''==
 
<html>
 
<html>
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This DNA biobrick was designed in order to produce CAI-1 of <i>Vibrio cholerae</i> in <i>E. coli</i> strain.  
+
This DNA biobrick was designed in order to produce CAI-1 of <i>Vibrio cholerae</i> in <i>E. coli</i> strain.  
  
 
<h3 id="RT"> 1- Biological background </h3>
 
<h3 id="RT"> 1- Biological background </h3>
  
−
<p>The production of the <i>Vibrio cholerae</i> quorum sensing inducer (CAI-1) is under the control of the cqsA gene coding for the CqsA synthase. This enzyme catalyzes the production of C8-CAI-1, an analog of CAI-1 quorum sensing inducer from <i>Vibrio cholerae</i>. The CqsA catalyzes the following reaction :</p>
+
<p>The production of the <i>Vibrio cholerae</i> quorum sensing inducer (CAI-1) is under the control of the <i>cqsA</i> gene coding for the CqsA synthase. This enzyme catalyzes the production of C8-CAI-1, an analog of CAI-1 quorum sensing inducer from <i>Vibrio cholerae</i>. The CqsA catalyzes the following reaction :</p>
  
−
<figure><p style="text-align:center;"> <img src ="https://static.igem.org/mediawiki/parts/0/03/VcCqsA.png" width = "600" /> <figcaption> Figure 1: <b>CAI-1 simplified production mechanism. </b> CqsA synthase catalyses the reaction between (S)-adenosylmethionine (SAM) and decanoyl-coenzyme A.  Ea-CAI-1 is then converted to CAI-1 (Wei et al. 2011). (figure is adapted from Wei et al. 2011) </figcaption> </figure>
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 +
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[[Image:T--INSA-UPS_France--VcCqsA.png|800px|thumb|center|'''Figure 1:''' <b>CAI-1 simplified production mechanism. </b> CqsA synthase catalyses the reaction between (S)-adenosylmethionine (SAM) and decanoyl-coenzyme A.  Ea-CAI-1 is then converted to CAI-1 (Wei <i>et al</i>. 2011). (figure is adapted from Wei <i>et al</i>. 2011) ]]
 +
<html>
  
 
<h3 id="RT"> 2- Usage in iGEM projects </h3>
 
<h3 id="RT"> 2- Usage in iGEM projects </h3>
  
 
<p> The BBa_K2278002 cames from the sensing module of the Croc’n cholera project <a href="http://2017.igem.org/Team:INSA-UPS_France">(team INSA-UPS-France 2017)</a>
 
<p> The BBa_K2278002 cames from the sensing module of the Croc’n cholera project <a href="http://2017.igem.org/Team:INSA-UPS_France">(team INSA-UPS-France 2017)</a>
−
It was designed to produce CAI-1 by an E.coli to simulate the presence of <i>Vibrio cholerae</i> in a water sample and so, to allow the validation of our <i>Vibrio cholerae</i> quorum sensing based detection system. </p>
+
It was designed to produce CAI-1 by an <i>E. coli</i> strain to simulate the presence of <i>Vibrio cholerae</i> in a water sample and so, to allow the validation of our <i>Vibrio cholerae</i> quorum sensing based detection system. </p>
  
−
<p>The part includes <i>Vibrio cholerae</i> cqsA synthase under the control of an IPTG inducible promoter.  
+
<p>The part includes <i>Vibrio cholerae cqsA</i> synthase under the control of an IPTG inducible promoter.  
 
The CAI-1 producing system is inducible in order to avoid toxicity problems and high metabolic activity during cells growth. </p>
 
The CAI-1 producing system is inducible in order to avoid toxicity problems and high metabolic activity during cells growth. </p>
−
 
−
<p><b>As the cqsA gene comes from <i>Vibrio cholerae</i> which is a BSL2 organism, it has to be manipulated under safe condition even if this gene is not directly involved in pathogenicity or antibiotic resistance.
 
−
<p>To avoid safety issues, we recommand to use BBa_K2278001 which produce a quorum sensing analog molecule : C8-CAI-1</b></p>
 
  
 
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<h3 id="RT"> Molecular biology </h3>
 
<h3 id="RT"> Molecular biology </h3>
 
<p>
 
<p>
−
The cqsA coding gene was placed in silico under the control of the plac promoter (BBa_R0040), a strong RBS (BBa_B0034) and a terminator (BBa_B1006). IDT performed the DNA synthesis and delivered the part as gBlock. 
+
The <i>cqsA</i> coding gene was placed in silico under the control of the plac promoter (BBa_R0040), a strong RBS (BBa_B0034) and a terminator (BBa_B1006). IDT performed the DNA synthesis and delivered the part as gBlock. 
−
The construct was cloned by conventional ligation into pSB1C3 plasmid and then transformed into E. coli Dh5 alpha strain. Three transformants were obtained.  
+
The construct was cloned by conventional ligation into pSB1C3 plasmid and then transformed into <i>E. coli</i> Dh5-alpha strain. Three transformants were obtained.  
 
</p>
 
</p>
 
<b>Analysis of the restriction map </b>
 
<b>Analysis of the restriction map </b>
−
<figure><p style="text-align:center;"><img src="https://static.igem.org/mediawiki/parts/1/19/Gel_Vc.png" width = "600"/><figcaption> Figure 2: <b>Analyses of pSB1C3_Vc cqsA restriction map.  </b> Digested plasmids are electrophoresed through an 0.7% agarose gel. The desired plasmids lengths are in parentheses. pSB1C3 (2029bp the other band correspond to cqsA gene (1300bp).</figcaption></figure>
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 +
 
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</html>
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[[Image:T--INSA-UPS_France--_gelVcCqsA1.png|800px|thumb|center|'''Figure 2:''' <b>Analyses of pSB1C3-VcCqsA restriction map.  </b> Digested plasmids (EcoRI and Pst1) are electrophoresed through a 1% agarose gel. The desired fragment lengths are in brackets. After digestion, the 2029 bp fragment corresponds to the pSB1C3 vector while the 1318 bp fragment corresponds to the <i>cqsA</i> gene.]]
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<html>
  
 
<p><b>Perceptives:</b></p>
 
<p><b>Perceptives:</b></p>
−
The biobrick can be validated by a bioluminescence assay with V. harveyi JMH626 with deleted cqsA.
+
The biobrick can be validated by a bioluminescence assay but this will require manipulating <i>Vibrio cholerae</i> in a BSL2 facility. Alternatively, the completion of the <i>V. harveyi</i> sensor as designed by the INSA-UPS France iGEM Team 2017 could permit to do the assay in BSL1 facility (<a href="http://2017.igem.org/Team:INSA-UPS_France">team INSA-UPS-France 2017</a>)
  
 
</html>
 
</html>
  
 
===Design Notes===
 
===Design Notes===
−
Terminator : BBa_B1006* is mutated substitution at the 35th pair (initial A->T) due to IDT complexity requirements for gBlocks synthesis. The effect of the mutation was not investigated.
+
Terminator : BBa_B1006* pressent for this construction a mutated substitution at the 35th pair (initial A->T) due to IDT complexity requirements for gBlocks synthesis. The effect of the mutation was not investigated.
  
 
<p> This biobrick is very close to Part:BBa_K356001. We proposed an alternative design by inversing the RBS and promoter place in the initial design. The promoter is now before the RBS and we used an inducible promoter in order to avoid toxicity problems and high metabolism activity during cell growth.
 
<p> This biobrick is very close to Part:BBa_K356001. We proposed an alternative design by inversing the RBS and promoter place in the initial design. The promoter is now before the RBS and we used an inducible promoter in order to avoid toxicity problems and high metabolism activity during cell growth.
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===Source===
 
===Source===
  
−
This enzyme cames from Vibrio cholerae DNA genomic sequence.  
+
This enzyme cames from <i>Vibrio cholerae</i> DNA genomic sequence.  
  
 
===References===
 
===References===
  
−
<p>Ng W-L, Perez LJ, Wei Y, Kraml C, Semmelhack MF & Bassler BL (2011) Signal production and detection specificity in Vibrio CqsA/CqsS quorum-sensing systems: Vibrio quorum-sensing systems. Molecular Microbiology 79 1407–1417
+
Ng W-L, Perez LJ, Wei Y, Kraml C, Semmelhack MF & Bassler BL (2011) Signal production and detection specificity in Vibrio CqsA/CqsS quorum-sensing systems: Vibrio quorum-sensing systems. Molecular Microbiology 79 1407–1417
 
https://www.ncbi.nlm.nih.gov/pubmed/21219472 </p>
 
https://www.ncbi.nlm.nih.gov/pubmed/21219472 </p>
 
<p> Wei, Y., Perez, L., Ng, W., Semmelhack, M. and Bassler, B. (2011). Mechanism of Vibrio cholerae Autoinducer-1 Biosynthesis. ACS Chemical Biology, 6(4), pp.356-365.
 
<p> Wei, Y., Perez, L., Ng, W., Semmelhack, M. and Bassler, B. (2011). Mechanism of Vibrio cholerae Autoinducer-1 Biosynthesis. ACS Chemical Biology, 6(4), pp.356-365.

Latest revision as of 14:21, 23 February 2018

Vibrio cholerae CAI-1 (quorum sensing inducer) generator

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 619
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Introduction

This DNA biobrick was designed in order to produce CAI-1 of Vibrio cholerae in E. coli strain.

1- Biological background

The production of the Vibrio cholerae quorum sensing inducer (CAI-1) is under the control of the cqsA gene coding for the CqsA synthase. This enzyme catalyzes the production of C8-CAI-1, an analog of CAI-1 quorum sensing inducer from Vibrio cholerae. The CqsA catalyzes the following reaction :

Figure 1: CAI-1 simplified production mechanism. CqsA synthase catalyses the reaction between (S)-adenosylmethionine (SAM) and decanoyl-coenzyme A. Ea-CAI-1 is then converted to CAI-1 (Wei et al. 2011). (figure is adapted from Wei et al. 2011)

2- Usage in iGEM projects

The BBa_K2278002 cames from the sensing module of the Croc’n cholera project (team INSA-UPS-France 2017) It was designed to produce CAI-1 by an E. coli strain to simulate the presence of Vibrio cholerae in a water sample and so, to allow the validation of our Vibrio cholerae quorum sensing based detection system.

The part includes Vibrio cholerae cqsA synthase under the control of an IPTG inducible promoter. The CAI-1 producing system is inducible in order to avoid toxicity problems and high metabolic activity during cells growth.

Experiments

Molecular biology

The cqsA coding gene was placed in silico under the control of the plac promoter (BBa_R0040), a strong RBS (BBa_B0034) and a terminator (BBa_B1006). IDT performed the DNA synthesis and delivered the part as gBlock.  The construct was cloned by conventional ligation into pSB1C3 plasmid and then transformed into E. coli Dh5-alpha strain. Three transformants were obtained.

Analysis of the restriction map

Figure 2: Analyses of pSB1C3-VcCqsA restriction map. Digested plasmids (EcoRI and Pst1) are electrophoresed through a 1% agarose gel. The desired fragment lengths are in brackets. After digestion, the 2029 bp fragment corresponds to the pSB1C3 vector while the 1318 bp fragment corresponds to the cqsA gene.

Perceptives:

The biobrick can be validated by a bioluminescence assay but this will require manipulating Vibrio cholerae in a BSL2 facility. Alternatively, the completion of the V. harveyi sensor as designed by the INSA-UPS France iGEM Team 2017 could permit to do the assay in BSL1 facility (team INSA-UPS-France 2017)

Design Notes

Terminator : BBa_B1006* pressent for this construction a mutated substitution at the 35th pair (initial A->T) due to IDT complexity requirements for gBlocks synthesis. The effect of the mutation was not investigated.

This biobrick is very close to Part:BBa_K356001. We proposed an alternative design by inversing the RBS and promoter place in the initial design. The promoter is now before the RBS and we used an inducible promoter in order to avoid toxicity problems and high metabolism activity during cell growth.

Source

This enzyme cames from Vibrio cholerae DNA genomic sequence.

References

Ng W-L, Perez LJ, Wei Y, Kraml C, Semmelhack MF & Bassler BL (2011) Signal production and detection specificity in Vibrio CqsA/CqsS quorum-sensing systems: Vibrio quorum-sensing systems. Molecular Microbiology 79 1407–1417

https://www.ncbi.nlm.nih.gov/pubmed/21219472

Wei, Y., Perez, L., Ng, W., Semmelhack, M. and Bassler, B. (2011). Mechanism of Vibrio cholerae Autoinducer-1 Biosynthesis. ACS Chemical Biology, 6(4), pp.356-365.