Final Lab Handout

The Vibrio harveyi autoinducer-2 (AI-2) quorum sensing system was engineered into Escherichia coli using standard Biobrick parts.

Additions to the Registry of Standard Biological Parts: 1) A second signalling system to the registry responsive to the bacterial esperanto (the interspecies signal). 2) The LuxOD47E Mutant protein acting as a constitutive transcriptional activator. 3) A system capable of inhibiting biofilm formation and maintenance via quorum quenching coupled to AI-2 signalling.

Why is iGEM Calgary’s Quorum Sensing Project Important? Beyond the contributions to the Registry of Standard Biological Parts, the construction of a synthetic AI-2 signalling cascade in the model organism E. coli will allow us to investigate the robustness of AI-2 signalling and provide further insight into how this system works. Moreover, we provide a novel approach for the targeting of biofilms: quorum sensing-couple quorum quenching

i G E M

C a l g a r y 2 0 0 9 Sponsors of

iGEM Calgary 2009 Lab Team

Cedarlane Labs

New England Biolabs Corning Life Sciences BioAlberta VWR International

Alberta Research Council Qiagen Integrated DNA Technologies Sigma-Aldrich Talisman Energy

Carol Chan

Jamie Feng

Emily Hicks

Vicki Komisar

Jeremy Kubik

Prima Moinul

Kevin Shin iGEM Calgary 2009 Facilitators Thane Kubik

Sonja Georgijevic

Anders Nygren

Christian Jacob

1

Miller, S., Xavier, K., Campagna, S., Taga, M., Semmelhack, M., Bassler, B. & Hughson, F. 2004. Salmonella typhimurium Recognizes a Chemically Distinct Form of the Bacterial Quorum-Sensing Signal AI-2. Mol. Cell. 15: 677-687 Bassler, B., Wright, M., Showalter, R. & Silverman, M. 1993. Intercellular signaling in Vibrio harveyi: sequence and function of genes regulating expression of luminescence. Mol. Microbiol. 9: 773–786 3 Schauder, S., Shokat, K., Surette, M. & Bassler, B. The LuxS family of bacterial autoinducers: biosynthesis of a novel quorum-sensing signal molecule. Mol. Microbiol. 41(2): 463–476 4 Surette, M., Miller, M. & Bassler, B. 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: 1639–1644 Vibrio harveyi luminescent bacterial restreak courtesy of Bonnie Bassler. Obtained from http://publications.nigms.nih.gov/biobeat/05-09-20/. 2

2009.igem.org/Team:Calgary [email protected]

L a b

is a process whereby microorganisms use pheromones to monitor their own population density as well as to detect and interact with other microbial species. This process allows for the . coordination of bacterial behavior including the induction of virulence, swarming and biofilm formation. The University of Calgary's 2009 iGEM . team has engineered the Vibrio harveyi autoinducer-2 (AI-2) signalling system in . Escherichia coli.

Quorum sensing .

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Genetic circuits Signalling circuit In order to ensure the expression of appropriate amounts of LuxP and LuxQ in the periplasm and the periplasmic space, respectively, a library of synthetic sigma 70 promoters with variable strengths was constructed. The variability in promoter strength was afforded by incorporating variability at two base positions within the -10 and -35 regions of the promoter region, critical for determining the strength of the promoter.

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Mutant circuits

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Results

Purpose: To test the functionality of the Reporter and Response circuits. LuxO D47A mimics the unphosphorylated and thus inactive form of LuxO, whereas LuxO D47E mimics the phosphorylated and thus active form of LuxO.

Reporter circuit

Purpose: To test the functionality of the Signalling circuit. In the absence of AI-2, the cells glow, whereas in the presence of AI-2, the cells do not glow.

In order to test and characterize the AI-2 system, AI-2 was isolated from Vibrio harveyi. Vibrio harveyi MM32 (BB120 luxN::Tn5 luxS::Tn5)1 is an ideal reporter strain as it cannot respond to AHL nor synthesize AI-2, respectively. In addition, since E. coli produce AI-2, we need to set our system up in cells that do not produce AI-2 (i.e. DH5α cells) and thus, we need to verify that these cells do not produce AI-2.

In the absence of AI-2, the signalling system acts as a phosphorylation cascade that eventually leads to the phosphorylated form of LuxO, which then complexes with a transcription factor to initiate transcription. When AI-2 is present, it binds to LuxP and switches the system into a dephosphorylation . cascade, leading to the unphosphorylated form of LuxO. Consequently, no binding occurs at the qrr4 promoter. Element LuxP LuxQ LuxU LuxO qrr4-σ54 c1λ pc1λ aiiA AHL Biofilm

Without AI-2 No binding Kinase LuxU-P LuxO-P Bound to LuxO-P Expression Repression No expression No degradation Proliferates

With AI-2 AI-2 binds Phosphatase LuxU LuxO No binding No expression Activated Expression Degradation No proliferation

Table 1. State of important aspects of the AI-2 quorum sensing system dependent on presence of AI-2. LuxP, LuxQ, LuxU and LuxO are proteins found in the (de)phosphorylation cascade. qrr4-σ54 is a complex consisting of a promoter and a transcription factor, respectively. The c1λ protein represses the pc1λ. The AiiA enzyme which hydrolyzes acyl homoserine lactone (AHL) is a pheromone used by a gramnegative bacteria in quorum sensing. By degrading AHL (i.e. quorum quenching), biofilm formation and growth will be inhibited.

Figure 1. Luminescence readings from an autoinducer bioassay using cell-free supernatants from overnights of various bacterial strains with Vibrio harveyi MM32 as the reporter strain. MM32 is responsive (i.e. luminescent) to the AI-2 present in the supernatant of V. harveyi BB120, a wildtype strain, and BB152 (BB120 luxLM::Tn5)2, a strain deficient in AHL production. Salmonella typhimurium 14028 produces AI-2 but due to differences in boration1, specificity to the V. harveyi reporter strain, and therefore luminescence, decreases. S. typhimurium SS007 (14028 luxS::T-POP)3 is incapable of producing AI-2 and serves as a negative control. E. coli DH5α has been reported as lacking the ability to produce AI-24. The lack of a peak in luminescence for MM32 grown with DH5α supernatant validates using DH5α as the chassis for our system. Our signalling system in DH5α will be responsive solely to exogenously added AI-2 derived from BB152 as it only produces AI-2. Cell-free supernatants were made by centrifugation of overnight cultures and filtering it with a 0.2μm (indicated by an ‘F’). Controls indicate the importance of filtering the supernatant (data not shown). The graph indicates an average obtained from 3 trials.

Next Steps Refining results for reporter circuit testing with LuxO mutants Introduce synthetic AI-2 signalling system and reporter circuit into E. coli DH5α for testing and characterization