Team:Aberdeen Scotland/WetLab/quorumsensing

Quorum Sensing Intro

An important design requirement for our project was that our picoplumber must possess the ability to detect a quorum; our e.coli should be able sense other cells in the surrounding environment, so that self-lysis is triggered only once a threshold population density is reached. This is important as it ensures that lysis only occurs at a breach point, and hence, avoids possible obstruction due to release of sticky proteins into the main pipe area. To satisfy this remit, we designed a quorum sensing module, based upon the lux system from the marine bacterium vibrio fischeri.

Quorum sensing is a method of bacterial communication and allows the co-ordination of gene expression in response to fluctuations in cell density. This enables populations of cells to control a diverse array of biological processes in synchrony, from biofilm formation to bioluminescence (Miller et al, 2001). In order to quorum sense individual cells produce and respond to small signalling molecules, termed autoinducers, that accumulate in the cell’s external environment (Surette et al, 1999). The concentration of autoinducer increases, both intra and extra-cellularly, with increasing cell density and once a minimal threshold concentration is reached, gene expression is altered (Miller et al, 2001).

Many bacteria monitor population density in this way, including the marine bacterium vibrio fischeri. This particular bacterium possesses two quorum sensing systems, ain and lux; both systems use acyl homoserine lactones (AHLs) as signalling molecules (Lupp et al, 2005). In the lux system, synthesis of AHL is directed by the product of the LuxI gene, using the substrates S-adenosylmethionine and acylated acyl protein (SAM and ACP) (Schaeffer et al, 1996). Once a critical concentration of AHL is reached, it associates with the transcriptional activator, LuxR, to regulate transcription of the Luminescence (lux) operon (Callahan et al, 2000). This can be seen below:

Figure 1: The Vibrio fischeri Lux quorum sensing circuit, consisting of five luciferase genes (luxCDABE) and two regulatory genes (luxI and luxR).

As can be seen from the diagram above, the luxR-autoinducer complex binds to both the left and right promoters of the lux system. This represses expression of luxR and activates expression of the LuxICDABE genes, respectively. The result is an exponential increase in autoinducer, through increased expression of luxI, and and an exponential increase in light emission, through increased transcription of LuxCDABE (Miller et al, 2001).

Aim

As aforementioned, our aim was to design a quorum sensing module based upon the lux system from vibrio fischeri. We utilised the genes LuxI, encoding an AHL synthase, and LuxR, encoding a transcriptional activator, using the biobricks BBa_K081009 and BBa_I0462 respectively. This can be seen below:

Method

1. Recipient Plasmid Choice

In order to clone our construct, we required a recipient vector. For this we chose the plasmid pSB4C5.

As can be seen from the diagram above, this plasmid contains a chloramphenicol resistance gene; this is important since our biobrick parts, BBa_k081008 and BBa_I0462, are contained within an Ampicillin resistant vector (pSB1A2). In order to select for our recombinant clones, we needed to simply grow on a medium containing the antibiotic chloramphenicol.

The part BBa_152002 encodes a ccdB selection marker. This is a lethal gene that interferes with DNA gyrase to prevent propagation of plasmids containing it. This allowed us to further select for recombinant clones as only those cells containing chloramphenicol resistant plasmids, but lacking the ccdB gene, should be viable on chloramphenicol-containing medium.

A further point to note is that psB4C5 is a low copy plasmid (~5 copies per cell). This is an important feature of our module; our modellers informed us that expressing LuxI/LuxR at higher copies risked self-induction of quorum sensing, and hence lysis events.

2. Cloning

To construct our quorum sensing module, we employed the BioBrick Standard Assembly method [RFC 10], using the restriction enzymes EcoRI, XbaI, PstI and SpeI. Our cloning strategy can be seen below:

[insert cloning diagram]

As can be seen from the diagram above, our first cloning step was to join the biobricks BBa_K081008 (LuxI) and BBa_I0462 (LuxR) together, into the plasmid psB4C5. In order to do this the upstream piece of DNA (LuxI) was cut using the restriction enzymes EcoRI and SpeI; the downstream piece of DNA (LuxR) was cut with the restriction enzymes XbaI and PstI and the recipient vector was cut using the restriction enzymes EcoRI and PstI. This allowed us to combine all three Biobrick parts in a single ligation step.

Our second cloning step involved combining the intermediate LuxI/LuxR construct with a J-series promoter (BBa_J23107): The LuxI/LuxR/plasmid construct was cut using the enzymes EcoRI and XbaI; the promoter was excised from its plasmid using the enzymes EcoRI and SpeI. Ligation of the Promoter to the LuxI/LuxR construct provided us with our final Quorum Sensing module (BBa_K182200).

3. Selection of clones of interest - luxI/LuxR intermediate

Propagation of all intermediate constructs was in the E.coli strain Xl1-Blue. Growth on Chloramphenicol containing medium indicated recombinant plasmids. Colony PCR was also performed, using standard biobrick primers (G00100 and G00101) allowing us to further identify clones of interest. This allowed us to directly compare the lengths of inserts obtained against the length expected (~1.6 kb) and controls (psB4C5 - ~ 1kb).

Once identified, clones were further diagnosed using the restriction enzyme HindIII; the HindIII site is located within the I0462 biobrick, as shown below:

{insert map of restriction sites}

Positive cut sites therefore, indicated the presence of LuxR. Furthermore, performing a double digestion with EcoRI, revealed the presence of LuxI; the expected insert length was ~733bp.

Results and Discussion

References

Surette M.G., Miller M.B. and Bassler B.L. (1999). Quorum Sensing in Escherichia coli, Salmonella typhimurium and vibrio harveyi: a new family of genes responsible for autoinducer production. Proc Natl Acad Sci USA. 96(4):1639-16144

Miller M.B and Bassler B.L (2001). Quorum Sensing in bacteria. Annu. Rev. Microbiol. 55: 165-199

Schaefer A.L, Val D.L, Hanzelka B.L, Cronan J.E, Jr, and Greenberg E.P (1996). Generation of cell-to-cell signals in quorum sensing: acyl homoserine lactone synthase activity of a purified vibrio.fischeri LuxI protein. Proc Natl Acad Sci USA. 93(18): 9505-9509

Callahan S.M and Dunlap P.V. (2000) LuxR- and Acyl-Homoserine-Lactone-Controlled Non-lux Genes Define a Quorum-Sensing Regulon in Vibrio fischeri. Journal of Bacteriology. 182(10): 2811-2822 Lupp C. and Ruby E.G. (2005) Vibrio fischeri Uses Two Quorum-Sensing Systems for the Regulation of Early and Late Colonization Factors. Journal of Bacteriology. 187(11): 3620-3629.

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