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, P2 activity will be measured based on the production of green fluorescent protein (GFP e.g. BBa_E0040) over time. A negative control of GFP under the control of a Pbad promoter (BBa_I13453) will be used. A positive control of GFP under the control of a constitutive promoter (J23100) will be used. All other parts will be kept the same: RBS, terminator and GFP. FACS will be used to detect fluorescence.
The reason why AIP production is not used to measure promoter activity is because (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. Furthermore, available S. aureus strains that lack AgrB are pathogenic, which presents an obstacle to their usage.
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.
E. coli DNA)
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.
"
