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Team:British Columbia/Project QS
From 2010.igem.org
Introduction
The goal of the Quorum Sensing sub-team 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. AgrC is a transmembrane protein that detects auto-inducing peptides (AIP) and then phosphorylates AgrA. The phosphorylated AgrA can then induce P2 promoter activity, leading to transcription of the agr operon. The precursor of AIP, AgrD is a straight-chain polypeptide that is circularized and exported out of the cell by AgrB. The agr operon is therefore an auto-catalytic 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 and prediction of P2-regulated viral and DspB production in the presence of a S. aureus biofilm.
Approach
As is common in other iGEM projects, we aimed to measure P2 activity based on the production of green fluorescent protein (GFP) over time. Our negative control was the arabinose-inducible Pbad promoter (BBa_I13453) and our positive control was a constitutive promoter (J23100). Our promoter-reporter constructs consisted of their respective promoters and the following parts: RBS (BBa_B0034), terminator (BBa_B0015) and GFP (BBa_E0040 and BBa_I145015). See parts BBa_K391009 and BBa_K391001 for more information. Fluorescence could be analysed via FACS.
There were several difficulties in trying to characterize the P2 promoter:(i) the agr operon is already present in most S. aureus strains and (ii) the auto-catalytic agr system makes it difficult to relate P2 activity directly to AIP concentration.
Therefore, we decided to use an agr operon null strain SH1001 (Thoendel and Horswill, 2009) that lacks the ability to synthesize AIP since it is agrB- and agrD-. To allow the host to detect AIP, we tried to clone the genes encoding AgrA and AgrC onto a plasmid and transform them into SH1001. To remove auto-catalytic activity, these genes would be placed under the control of a constitutive promoter to obtain a steady state of AgrA and AgrC. The previously described promoter-reporter constructs would be cloned onto the same plasmid as the genes for AgrA and AgrC and transformed into SH1001. AIP concentration could then be controllably varied and P2 activity could be directly related to AIP concentration. Primers (BBa_K391002 and BBa_K391003) were designed to PCR the genes for AgrA and AgrC including their associated ribosomal binding sites. The PCR was successful as shown by the correct band size after gel electrophoresis. However, we were not able to clone these onto biobrick plasmids pSB1C3 and psB13 for unknown reasons.
Therefore, to complete the project, we decided to reduce the scope of the track to showing if the Biobrick P2 part works. The promoter-reporter constructs were electroporated into S. aureus strain RN4220 (derived from NCTC 8325; mutated and selected for compatibility with restriction sites and E. coli DNA). It was expected that the host containing the P2 construct would show increased fluorescence over time due to its natural agr system. This also presented an opportunity to characterize Pbad and the constitutive promoter in S. aureus. Biobrick plasmids were not used as they lack the S. aureus replicon and replication genes. The Novick lab’s pCN33 plasmid (Charpentier, et al. , 2004) was used. pCN33 contains both E. coli and S. aureus replicons alongside erythromycin and ampicillin resistance genes. Its multiple cloning site region 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.
From this work, we realized the utility of obtaining different bacterial plasmid replicons as Biobrick parts to facilitate the characterization of parts across an increasing range of hosts. Thus, as a side project, primers (BBa_K391004 and BBa_K391005)were designed to PCR and clone the replicon of pCN33 as a BioBrick part. However, due to time constraints and insufficient manpower, this potential new part has not been made.
Results & Discussion
The main problem this summer was the lack of success in cloning PCR products and BioBrick parts. With valuable experience gained from having tried different protocols, it should be possible to accomplish the following tasks:
*1. Clone agrCA from S. aureus to a BioBrick plasmid and ligate also reporter constructs on the same plasmid.
*2. Clone S. aureus plasmid replicon to a BioBrick plasmid or otherwise modify a Biobrick plasmid to include S. aureus plasmid replicon.
*3. Transform S. aureus.
*4. Measure GFP levels over time and hence measure P2 activity via PoPs.
*5. Alternatively, knock out agrB or agr D in a strain of S. aureus (this removes the autocatalytic activity of the agr operon) so 1. is not necessary.
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.
Matthew Thoendel and Alexander R. Horswill. Identification of Staphylococcus aureus AgrD Residues Required for Autoinducing Peptide Biosynthesis. The Journal of Biological Chemistry. 2009. 284, 21828-21838.
