Part:BBa_K2278001

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

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 cqsA 1300bp insert)

Sequencing

Figure 3: Sequencing of pSB1C3_Vh cqsA restriction map. 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.

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

Strain E. coli-VhCqsA was grown in M9 medium and sampling was perform at after an overnight incubation at 30°C after IPTG induction. Presence of C8-CAI1 was then investigated by Mass Spectrometry (MS) and Nuclear Magnetic Resonance (NMR) analysis.

NMR and MS analysis

The supernatant is extracted by liquid liquid extraction with dichloromethane. The organic layer is washed with NaCl saturated water and dried. The solvant is evaporated and the sample is resuspended in Chloroform for the analysis.

Bioluminescence assay

V. harveyi JMH626 strain (kind gift from Bassler laboratory) was used since it is deleted for cqsA and other quorum sensing systems (AI-2 and HAI-1) and therefore unable to produce C8-CAI-1 or bioluminescing when stimulated by QS other that dependant of the activation of CAI-1 like molecules.

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]

Figure 5: Detection of C8-CAI-1 in E.coli (cqsA) culture supernatant by NMR analysis. Characteristic pics of C8-CAI-1 in NMR appears at (On CDCl3) δ 4.13 (td, J = 6.7, 4.5 Hz, 1H), 3.51 (d, J =4.9 Hz, 1H).

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 :

Both MS and NMR approaches failed to confirm the production of C8-CAI-1 molecule in E. coli. This does not mean the tested construction is not functional since quorum sensing mechanisms are very sensitive and our production could be insufficient.

Perspectives :

Culture parameter and induction by IPTG could be optimized. Alternatively, the sample treatment could be modified to increase C8-CAI-1, for example, by using Vibrio harveyi supernatant as a positive control. It will also be interesting to challenge the VcCqsA construction.

2. Validation of C8-CAI-1 bioactivity

A property of V. harveyi is to become bioluminescent in response to its quorum sensing molecule. To test our engineered E. coli-VhCqsA strain, we assessed if it was able to trigger luminescence in V. harveyi.

We had to optimize a protocol before we obtained luminescence from our controls (V. harveyi wild type strain and JMH626 strain with wild type V. harveyi supernatant, which contains C8-CAI1). A very low basal luminescence level was observed for the JMH626 strain with or without E. coli MG1655 supernatant. Very nicely, JMH626 strain with E. coli-VhCqsA supernatant appeared as luminescent as the positive controls. This results was reproducible (n=4).

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.

Initial picture of the plate used for the experiment.

Figure 7: Plate use for the solid bioluminescence assay (A) was obtained at day light and (B) was revealed under dark conditions. The positive control, JMH626 culture supplemented with V. harveyi WT supernatant (2) is as bright as the Vibrio harveyi WT drop (1). Thus, supernatant from Vibrio harveyi WT can restore bioluminesence to its normal state, as far as human eyes can tell. The negative control (3) shows faint bioluminscence, corresponding to a basal expression of quorum sensing. The supernatant of clones A and T show a bioluminescence as strong as the negative control (4 and 5), no real activity of C8-CAI-1 can be observed. The supernatant of clone M (6) seems to restore bioluminescence to normal state, as for the positive control.

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. Even if the presence of the molecule with MS + NMR was not detected mainly because of detection threshold issue, 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 a sufficient concentration for the further experiments i.e. enough to trigger the detection module.