Difference between revisions of "Part:BBa K2278002"
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=='''Experiments'''==
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<h3 id="RT"> Molecular biology </h3>
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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. 
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The construct was cloned by conventional ligation into pSB1C3 plasmid and then transformed into E. coli Dh5 alpha strain. Three transformants were obtained.
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<b>Analysis of the restriction map </b>
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<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 1300bp insert)</figcaption></figure>

Revision as of 22:29, 17 October 2017

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 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.

As the cqsA gene comes from Vibrio cholerae 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.

To avoid safety issues, we recommand to use BBa_K2278001 which produce a quorum sensing analog molecule : C8-CAI-1

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_Vc cqsA restriction map. 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 1300bp insert)