Difference between revisions of "Part:BBa K2278001"

Revision as of 03:17, 16 October 2017

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

Sequence and Features

Assembly Compatibility:

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

Introduction

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

1- Biological background

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 Vibrio 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 *Vibrio cholerae* CqsA, *Vibrio harveyi* CqsA is highly selective for the octanoyl CoA substrate which explains that *Vibrio 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 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 harveyi 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 Vibrio harveyi which is a BSL1 organism, it can be used as substitute in experiment about the Vibrio cholerae quorum sensing in BSL1 condition.

Experiments

1- 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_Vh 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 a 700bp insert)

Sequencing

Sequencing revealed that the VhCqsA construction slightly differs from the initial design, with a loss of the 9 last amino acid of the protein (position 382 to 391). However this mutation does not seem to affect the catalytic activity of the enzyme.

2- Expression of CqsA to produce in vivo C8-CAI-1

Proteins expression

E. coli cells freshly transformed with BBa_K2278001 in pSB1C3 and pSB1C3 empty vector (negative control)  were pre cultured in LB-Cm (5 mL) supplemented with glucose (10g/L) at 37°C O/N.

V. harveyi WT and JMH626 (∆cqsA) precultures were made at 30°C O/N in LM.

Fresh LB-Cm-Glc(10 g/L) flasks were inoculated to reach OD = 0.1 to set up exponential phase growth.  At OD = 0.3 , IPTG induction was made at final concentration of 0.5 mM.At the end of the exponential phase, the supernatants is collected and filtrated through a 0.2 µm filter. The supernatant is stored at -20°C it will be used for NMR and MS analysis and for bioluminescence assay

NMR and MS analysis

The supernatant is extracted by liquid liquid extraction with dichloromethane. The sample is diluted in Chloroform for the analysis.

Bioluminescence assay

V.harveyi preculutre culture were inoculated to reach OD=0,1 and incubated at 30°C with shaking to ensure a good stability of cqsA molecule (Wei et al. ) . At OD= 0.7, the cells are pelleted by centrifugation at 4500 rpm for 6 min.

The pellets were resuspended with 5 mL of fresh LB medium and 5 mL of E.coli transformed cells.

Characterization

1- Validation of the production of C8-CAI-1

We use MS and NMR analysis approaches to show that we are able to produce C8-CAI-1 using E.coli

MS spectra :

[Image MS avec le pic qui correspond à la masse de C8-CAI-1 comparé a coli plasm vide).

Figure 4: **Detection of C8-CAI-1 in E.coli (cqsA) culture supernatant by MS analysis**

Characteristic m/z of C8-CAI-1 appears on the MS spectra of (concentrated) supernatant from of production of CqsA with E.coli MG1655. Compared to the control (fig 1A) where no signal from C8-CAI-1 has been detected, the fact that a signal at XXX appears from our production (fig 1B) mean that we were able to produce C8-CAI-1

NMR analysis :

[C8-CAI-1 + deplacement chimique des C d’interets + Image NMR qui correspond aux ppm caractéristiques de C8-CAI-1 comparé a coli plasm vide]

Those integrations are characteristics of CAI-1 like molecule because of the ketone + hydroxyle environment of carbon a and carbon b. Those two signals are presents in our production using E.coli MG1655 (blue) compared to the controle of E.coli bearing no plasmid (red).

Discussion :

Combined approach of MS + NMR allows us to be confident in the fact that we were able to produce CAI-1 like molecule into E.coli. Data of the exact mass of the molecule added to characteristic signals in NMR give strengh to this assertion and allow us to realise further experiment such as testing if our produced molecul is bioactive.

2. Validation of C8-CAI-1 bioactivity

Figure 6: **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).

Discussion :

Here we can be confident in the production of C8-CAI-1 bioactive. Addition of data assessing the presence of the molecule with MS + NMR and successful bioactivity test means that C8-CAI-1 is produced with E.coli system, directly with the use of the novel iGEM part that we produced. Moreover, we design a bioluminescence assay on plate for detecting C8-CAI-1 molecule, without using any device for reading bioluminescence. Finally, we are able, thanks to those results to mimic Vibrio cholerae at high concentration for the further experiments.

@@ Line 47: / Line 47: @@
 <figure><p style="text-align:center;"><img src="https://static.igem.org/mediawiki/parts/1/12/SeqVhcqsA.png" width = "500"/><figcaption> Figure 3: <b>Sequencing  of pSB1C3_Vh cqsA restriction map.  </b> 1500 ng of plasmid are sequenced. 3 oligos were used to perform the sequencing. The obtained sequence were blast on the BBa_K2278001 sequence with the iGEM sequencing online tools. </figcaption></figure>
-The sequencing show a deletion at the end of cqsA gene. The last nine amino acid of the protein are probably changed. However this mutation does not seem to affect the catalytic activity of the enzyme.
+Sequencing revealed that the VhCqsA construction slightly differs from the initial design, with a loss of the 9 last amino acid of the protein (position 382 to 391).
+However this mutation does not seem to affect the catalytic activity of the enzyme.
 <h3 id="RT"> 2- Expression of CqsA to produce <i>in vivo</i> C8-CAI-1 </h3>