Difference between revisions of "Part:BBa K2278001"
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This DNA biobrick was designed in order to produce the C8-CAI-1of Vibrio harveyi  in E. coli strain.   
 
This DNA biobrick was designed in order to produce the C8-CAI-1of Vibrio harveyi  in E. coli strain.   
  
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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.  
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<p>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. The CqsA catalyzes the following reaction :</p>
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This enzyme catalyzes the production of C8-CAI-1, an analog of CAI-1 quorum sensing inducer from V. cholerae.  
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The CqsA catalyzes the following reaction :
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<figure><p style="text-align:center;"> <img src ="https://static.igem.org/mediawiki/parts/7/7c/CqsA_simplified.png" width = "600" /> <figcaption> Figure 1: <b>C8-CAI-1 simplified production mechanism. </b> 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) </figcaption> </figure>
 
<figure><p style="text-align:center;"> <img src ="https://static.igem.org/mediawiki/parts/7/7c/CqsA_simplified.png" width = "600" /> <figcaption> Figure 1: <b>C8-CAI-1 simplified production mechanism. </b> 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) </figcaption> </figure>
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<p>The BBa_K1934060 part conceived by the 2016  INSA-Lyon team and synthesized by Genecust was cloned into pUC57 and transformed into the  E. <i>coli</i> NM522 strain</p>
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Revision as of 21:07, 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

1- Biological background

This DNA biobrick was designed in order to produce the C8-CAI-1of Vibrio harveyi in E. coli strain.

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. 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)

2- Usage in iGEM projects

The BBa_K2278001 cames from the sensing module of the Croc’n cholera project (team INSA-UPS-France 2017) It 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.

The part includes Vibrio harveyi’s cqsA synthase 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 the cqsA 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.

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).