Difference between revisions of "Part:BBa K2278001"
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<p>The BBa_K2278001 part was designed to produce C8-CAI-1 to simulate the presence of V. cholerae in a water sample and so, to allow the validation of our V. cholerae quorum sensing based detection system.  
 
<p>The BBa_K2278001 part was designed to produce C8-CAI-1 to simulate the presence of V. cholerae in a water sample and so, to allow the validation of our V. cholerae quorum sensing based detection system.  
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It belongs to the sensing module in the Croc’n cholera project of iGEM INSA-UPS-France 2017 : http://2017.igem.org/Team:INSA-UPS_France  
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It cames from the sensing module of the Croc’n cholera project iGEM INSA-UPS-France 2017 : <a href="http://2017.igem.org/Team:INSA-UPS_France">here</a>
  
 
The part includes: Vibrio harveyi’s cqsA enzyme under the control of an IPTG inducible promoter.  
 
The part includes: Vibrio harveyi’s cqsA enzyme under the control of an IPTG inducible promoter.  

Revision as of 20:48, 9 October 2017

Vibrio harveyi C8-CAI-1 (quorum sensing inducer) generator

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 136
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Introduction

The production of the Vibrio harveyi quorum sensing inducer (C8-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 V. cholerae. This DNA biobrick was designed in order to produce the CAI-1 in E. coli strain. The production of the CAI-1 is under the control of the cqsA gene coding for the CqsA enzyme (Ng et al, 2011). The CqsA catalyzes the following reaction :

Figure 1: C8-CAI-1 simplified production mechanism. CqsA synthase catalyses the reaction between (S)-adenosylmethionine (SAM) and octanoyl-coenzyme A. Ea-C8-CAI-1 is then converted to C8-CAI-1 (Wei et al. 2011). In contrast to V. cholerae CqsA, V. harveyi CqsA is highly selective for the octanoyl CoA substrate which explains that V. harveyi only produces C8-CAI-1. (figure is adapted from Wei et al. 2011)

The BBa_K2278001 part was designed to produce C8-CAI-1 to simulate the presence of V. cholerae in a water sample and so, to allow the validation of our V. cholerae quorum sensing based detection system. It cames from the sensing module of the Croc’n cholera project iGEM INSA-UPS-France 2017 : here The part includes: Vibrio harveyi’s cqsA enzyme under the control of an IPTG inducible promoter. The C8-CAI-1 producing system is inducible in order to avoid toxicity problems and high metabolic activity during cells growth.

As this gene comes from V. harveyi which is a BSL1 organism, it can be used as substitute in experiment about the V. cholerae quorum sensing in BSL1 condition.

The BBa_K1934060 part conceived by the 2016 INSA-Lyon team and synthesized by Genecust was cloned into pUC57 and transformed into the E. coli NM522 strain

Characterization

3. Bioactivity of the C8-CAI-1 molecule

Figure 4: Solid bioluminescence assay. Control is made of Vibrio harveyi wild type directly plated, JMH626 (reporter strain) with supernatant of V.harveyi WT as a positive control. JMH626 without supernatant is a negative control. Empty means production of C8-CAI-1 with empty plasmid, and clone means E.coli that transformed with VhCqsA.

The molecule that we have produced has an activity in vivo because it activates the bioluminescence pathway of Vibrio harveyi JMH626. This vibrio strain used to detect C8-CAI-1 molecule because its deleted of other quorum sensing pathway and of the CqsA enzyme- as strong as the wild type strain or as our positive control (SN WT/JMH626). Still, a basal bioluminescence exist (Sn -/JMH626) but is really lower compared to SN with C8-CAI-1 (SN WT/JMH626).