Team:British Columbia/Project QS

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Introduction

The goal of the Quorum Sensing sub-project is to characterize the P2 promoter (BBa_I746104). This promoter controls the transcription of the agr operon found naturally in S. aureus (Novick et al., 1995). The agr operon itself is involved in the quorum sensing activity of S. aureus . Bioflm activity is affected by this quorum sensing. Agr C is a transmembrane protein that detects auto inducing peptides (AIP) and then phosphorylates agr A. The phosphorylated agr A can then induce P2 promoter activity, leading to transcription of the agr operon. AIP is created by agr B and D. Agr D is a straight-chain polypeptide that is circularized and exported out of the cell by agr B. The agr operon is therefore an autocatalytic system: the presence of AIP initiates P2 promoter activity which leads to synthesis of more AIP (Lyon et al., 2000). A better understanding of P2 promoter activity in the presence of AIP can therefore lead to a rational design or prediction of viral reproduction or DspB production in the prescence of S. aureus biofilm should they be engineered to act under the P2 promoter.

Approach

As is common in other iGEM projects, the basis of P2 activity will be the production of fluorescent proteins (e.g. BBa_E0040) over time. The production of fluorescent proteins is under the control of the P2 promoter. In order to compare with background activity, a negative control consisting of fluorescent protein under the control of BBa_I13453 (a Pbad promoter) will be used. A positive control consisting of fluorescent protein under the control of J23100 (a constitutive promoter) will be used to compare P2 activity with high level promoter activity. All other parts will be kept the same (RBS, terminator, and of course fluorescent protein type). FACS will be used to detect fluorescence.

The agr operon is already present in most S. aureus strains (e.g. NCTC 8325) and also in the most commonly used cloning strain RN4220. which is NCTC 8325 mutated and selected for compatibility with restriction sites and E. coli DNA. As well, it is an auto-catalytic system. Therefore, it would be difficult to relate P2 activity directly to AIP concentration. The original plan was to use as host a strain of S. aureus that lacks agr B or D. If the host cannot create AIP, then P2 activity would be solely due to experimenter-introduced AIP. However, while a strain of this kind does exist, it is difficult to work with, as it is a pathogenic strain. As well, there would still be some residual auto-catalytic activity in the form of agr AC production. The production of agrAC may lead to increased AIP detection capability (and hence P2 activity) of the host cell and agrAC production is under the control of P2.

Therefore, it was decided to use an agr operon null strain (e.g. SH 1001 ). Without agr AC, it is not possible for the host to detect AIP and so it was decided that agr AC would be cloned onto a plasmid and inserted into placed into SH1001 via electroporation . To remove auto-catalytic activity, agrAC would be placed under the control of a constitutive promoter. It was hoped that a steady state of agr AC would exist after time. P2-GFP constructs (including negative and positive controls, as defined above) would be cloned onto the same plasmid and placed also into SH1001. AIP concentration could then be varied and P2 activity would be directly related to AIP concentration.

Primers were designed to PCR off agrAC including its associated ribosomal binding sites. Even though it was possible to PCR off agrAC, as shown through the correct band size after gel electrophoresis, the process of cloning agrAC onto biobrick plasmids pSB1C3 and psB13 failed. Therefore, to complete the project, it was decided to reduce the scope of the track to showing if the Biobrick P2 part works or not. The promoter-reporter constructs were electroporated into RN4220. It is expected that the host containing the P2 construct will show increased fluorescence over time as its natural agr system activates. Most of this increase should occur in the early exponential growth phase of the host, since AIP production ramps up most appreciably during this phase. Comparing the fluorescence of the P2 construct with the negative and positive controls should result in useable data that future iGEM teams will appreciate. Biobrick plasmids were not used in this case, as it lacks replication genes for replication in S. aureus. Novick Lab’s pCN33 (Charpentier, et al. , 2004) will be used instead. This plasmid contains both E. coli and S. aureus replicons and also erythromycin and ampicillin resistance genes. The multiple cloning site region of pCN33 is compatible with EcoRI and PstI sites in Biobricks. In other words, it can serve as a final expression vector for Biobrick parts with S. aureus as a host.

Results

Discussion

*temporary storage location for references *Charpentier, et al.. Novel Cassette-Based Shuttle Vector System for Gram-Positive Bacteria. Appl Environ Microbiol. 2004 October; 70(10): 6076–6085. *Novick R P; Projan S J; Kornblum J; Ross H F; Ji G; Kreiswirth B; Vandenesch F; Moghazeh S The agr P2 operon: an autocatalytic sensory transduction system in Staphylococcus aureus. Molecular & general genetics : MGG 1995;248(4):446-58. *Gholson J. Lyon, Patricia Mayville, Tom W. Muir, and Richard P. Novick. Rational design of a global inhibitor of the virulence response in Staphylococcus aureus, based in part on localization of the site of inhibition to the receptor-histidine kinase, AgrC. PNAS. 2000; 97 (24): 13330-35.