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 in an E. coli strain. C8-CAI-1 is a quorum sensing molecule of Vibrio harveyi ((Z)-3-aminoundec-2-en-4-one), used to mimic its presence in a medium.

1- Biological background

The production of the Vibrio harveyi quorum sensing inducer C8-CAI-1 is under the control of the cqsA gene encoding the CqsA synthase. This enzyme catalyzes the production of C8-CAI-1, an analog of the CAI-1 quorum sensing molecule of Vibrio cholerae. CqsA catalyzes the following reaction (figure 1) :

Figure 1: C8-CAI-1 simplified production reaction. The CqsA synthase catalyses the reaction between (S)-adenosylmethionine (SAM) and octanoyl-coenzyme A to produce Ea-C8-CAI-1. This product 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 why Vibrio harveyi only produces C8-CAI-1. (figure is adapted from Wei et al. 2011)

2- Usage in iGEM projects

The BBa_K2278001 is issued 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 wild type Vibrio harveyi in a water sample. In this project synthetic microbial system, the C8-CAI-1 molecule is then detected by a Vibrio harveyi strain unable to produce C8-CAI-1 and presenting a modified receptor able to detect both C8-CAI-1 from Vibrio harveyi (for testing purpose as Vibrio harveyi is a BSL1 organism) and CAI-1 from Vibrio cholerae (for application purpose to detect the pathogen ).

The part includes Vibrio harveyi cqsA (sequence obtained from genomic database) 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.

Construction

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 the pSB1C3 plasmid and then transformed into E. coli Dh5-alpha strain (figure 2). Three transformants were obtained.

Analysis of the restriction map

Figure 2: Analyses of pSB1C3_Vh cqsA length and restriction map. BBa_K2278001 was cloned in pSB1C3. The plasmids of 3 obtained clones were analyzed to check their length. SacI digested plasmids are electrophoresed through a 1% agarose gel. Lane 1 is the DNA ladder (New England biolab), the 0.5kb, 1 kb and 3kb DNA fragments are annotated. Lane 2 is the linearized pSB1C3 vector containing a 700bp insert (2700bp). Lanes 3-5 are the digested plasmids resulting from DNA extraction of the 3 obtained clones. We expected two bands at 1901 and 1458 bp.

Sequencing

Figure 3: Sequencing of pSB1C3-VhCqsA restriction map. 1500 ng of plasmid were 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 (figure 3) revealed that the VhCqsA construction slightly differs from the initial design, with a loss of the 9 last amino acids of the protein (position 382 to 391 ; confirmed on two different runs). However this mutation does not seem to affect the catalytic activity of the enzyme (see below).

Characterization

1. C8-CAI-1 production assay

We used a Nuclear Magnetic Resonance (NMR) analysis approach to assay if we were able to produce C8-CAI-1 using E. coli (800 MHz spectrometer from Bruker, Germany). Strain E. coli-VhCqsA was grown in M9 medium and sampling was performed after an over-night incubation at 30°C in presence of IPTG. The supernatant was extracted by liquid/liquid extraction with dichloromethane. The organic layer was washed with NaCl saturated water and dried. The solvant was then evaporated and the sample was resuspended in chloroform for subsequent NMR analysis (figure 4).

Figure 4: Detection of C8-CAI-1 by NMR analysis in culture supernatant from E. coli-pSB1C3 (negative control) in green, from E. coli-VhCqsA (assay) in red, and from wild type V. harveyi (positive control) in blue. Characteristic peaks of C8-CAI-1 are expected at 4.14, 3.51, 2.48, 1.92, 1.63, 1.44, 1.33, 0.91, and 0.85 ppm.

We did not detect C8-CAI-1 hallmark peaks (for example, 4.14 or 2.48), neither in our VhCqsA assay, neither in the positive control. It was therefore not possible to actually conclude about the production of C8-CAI-1 by E. coli .

Discussion :

NMR approach 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 under the detection level. Mass spectrometry analyses were also used, but we we did not have enough time to optimize them (not shown).

Perspectives :

Culture parameter and induction by IPTG could be optimized. Alternatively, the sample treatment could be modified to increase C8-CAI-1 concentration.

2. Validation of C8-CAI-1 bioactivity

Wild type V. harveyi has the property to phosphoresce when submitted to its quorum sensing molecules. We used the V. harveyi JMH626 strain (kind gift from Bassler laboratory) since it is deleted for cqsA and other quorum sensing systems (AI-2 and HAI-1). This strain can only emit light if submitted to C8-CAI-1 molecules produced by other strain, such as E. coli-VhCqsA. 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).

Figure 5: bioluminescence assay. Cells from a strain were mixed with supernantant from other strains prior to plating on rich medium Petri dish and picture of the colonies was taken in the dark after 24H growth (upper part). From left to right : wild type V. harveyi with wild type V. harveyi supernatant (positive control), V. harveyi JMH626 with supernatant of wild type V. harveyi (positive control), V. harveyi JMH626 with no supernatant (negative control), V. harveyi JMH626 with supernatant of E. coli-pSB1C3 (negative control), and V. harveyi JMH626 with supernatant of E. coli-VhCqsA (assay). Lower part illustrates the results at the full plate level.

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). This demonstrates that C8-CAI-1 was produced by E. coli and is active in vivo. This also means that we successfully created synthetic communication between E. coli and V. harveyi.

Discussion :

Here, we can be confident in the facts that the production of C8-CAI-1 by E. coli is efficient and that the molecule is bioactive. NMR approach failed to detect the molecule likely because of a detection threshold issue. Moreover, we design a bioluminescence assay on plate for detecting C8-CAI-1 molecule, without using any device for reading bioluminescence. Finally, we were able to trigger the Vibrio harveyi response to quorum sensing and this opens the way toward the detection module of our project.

References

Henke, J. and Bassler, B. (2004). Three Parallel Quorum-Sensing Systems Regulate Gene Expression in Vibrio harveyi. Journal of Bacteriology,186(20), pp.6902-6914. Sequence is available at: https://www.ebi.ac.uk/ena/data/view/AAT86008&display=text

Ng W-L, Perez LJ, Wei Y, Kraml C, Semmelhack MF & Bassler BL (2011) Signal production and detection specificity in Vibrio CqsA/CqsS quorum-sensing systems: Vibrio quorum-sensing systems. Molecular Microbiology 79 1407–1417 https://www.ncbi.nlm.nih.gov/pubmed/21219472

Wei, Y., Perez, L., Ng, W., Semmelhack, M. and Bassler, B. (2011). Mechanism of Vibrio cholerae Autoinducer-1 Biosynthesis. ACS Chemical Biology, 6(4), pp.356-365.

Bolitho ME, Perez LJ, Koch MJ, Ng W-L, Bassler BL & Semmelhack MF (2011) Small molecule probes of the receptor binding site in the Vibrio cholerae CAI-1 quorum sensing circuit. Bioorganic & Medicinal Chemistry 19 6906–6918, https://www.ncbi.nlm.nih.gov/pubmed/22001326