Difference between revisions of "Part:BBa K2278001"

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<h3 id="RT">2. C8-CAI-1 production assay</h3>
 
<h3 id="RT">2. C8-CAI-1 production assay</h3>
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We used a Nuclear Magnetic Resonance (NMR) analysis approach to assay if we were able to produce C8-CAI-1 using <i>E. coli</i>. Strain <i>E. coli</i>-VhCqsA was grown in M9 medium and sampling was perform 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 (800 MHz spectrometer from Bruker, Germany).
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We used a Nuclear Magnetic Resonance (NMR) analysis approach to assay if we were able to produce C8-CAI-1 using <i>E. coli</i> (800 MHz spectrometer from Bruker, Germany). Strain <i>E. coli</i>-VhCqsA was grown in M9 medium and sampling was perform 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).
  
  
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<figure><p style="text-align:center;"><img src="https://static.igem.org/mediawiki/2017/9/9b/Solid.png" width = "500"/><figcaption> Figure 5: <b>Detection of C8-CAI-1 in E.coli (cqsA) culture supernatant by NMR analysis. </b>Overlaid NMR spectra of E. coli (cqsA) culture supernatant (green), V. harveyi WT culture supernatant (positive control, red), E. coli (pSB1C3) culture supernatant (negative control, blue). A zoom is performed to focus the view on the smallest peaks. 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). </figcaption></figure>
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<figure><p style="text-align:center;"><img src="https://static.igem.org/mediawiki/2017/9/9b/Solid.png" width = "500"/><figcaption> Figure 4: <b>Detection of C8-CAI-1 by NMR analysis in culture supernatant from <i>E. coli</i>-pSB1C3 (negative control) in green, from <i>E. coli</i>-cqsA (assay) in red, and from wild type <i>V. harveyi</i> (positive control) in blue. Characteristic pics of C8-CAI-1 are expected at XXX and XXX ppm. </figcaption></figure>
  
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<p>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).</p>
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<p>There are differences between the sample profiles, but it was difficult to actually conclude about the production of C8-CAI-1 by <i>E. coli</i> .</p>
  
 
<p><b>Discussion : </b> </p>
 
<p><b>Discussion : </b> </p>
 
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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.
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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).
  
 
<p><b>Perspectives :  </b> </p>
 
<p><b>Perspectives :  </b> </p>
 
<p>
 
<p>
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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.
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Culture parameter and induction by IPTG could be optimized. Alternatively, the sample treatment could be modified to increase C8-CAI-1 concentration.
 
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Revision as of 09:24, 18 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]
  • 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 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 restriction map. Digested plasmids are electrophoresed through a 0.7% agarose gel. The desired fragment lengths are in brackets. The 2029 bp band corresponds to the vector pSB1C3 while the 1300 bp corresponds to the cqsA insert)

Sequencing

Figure 3: Sequencing of pSB1C3_Vh cqsA 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

2. 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 perform 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-cqsA (assay) in red, and from wild type V. harveyi (positive control) in blue. Characteristic pics of C8-CAI-1 are expected at XXX and XXX ppm.

There are differences between the sample profiles, but it was difficult 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

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.

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: Plates used 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.


Design Notes

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.

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).

Source

This enzyme cames from Vibrio harveyi DNA genomic sequence.


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. <p>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