Agr quroum sensing sensor/generator, FepA pore, with P2 + reporter
This part includes the S.aureus Agr quorum sensing sensor and generator which recognizes the oligopeptide. The FepA pore allows for the oligopeptide to diffuse through the outer-memrabe.
Derived from the TE terminator of T7 bacteriophage between Genes 1.3 and 1.4 <genbank>V01146</genbank>.
Transcription terminator for the E.coli RNA polymerase.
Suggested by Sri Kosuri and Drew Endy as a high efficiency terminator. The 5' end cutoff was placed immediately after the TAA stop codon and the 3' end cutoff was placed just prior to the RBS of Gene 1.4 (before AAGGAG). Use anywhere transcription should be stopped when the gene of interest is upstream of this terminator.
Transcriptional terminator consisting of a 64 bp stem-loop.
AIP sensor infrastructure (agrC + agrA from S. aureus with RBSes)
The original sequences for agrC and agrA were taken from the ''S. aureus'' Sequencing Project (results for strain NCTC8325) at Oklahoma University (http://www.genome.ou.edu/staph.html). The sequences were then codon-optimised for ''E. coli'' using GeneDesigner from DNA2.0 (http://www.dna20.com), and then synthesised by that company.
This part was made in order to transfer the S. aureus oligopeptide-based quorum sensing system into a BioBrick-compatible signalling mechanism. In the natural system, the signalling oligopeptide (termed AIP) is made from AgrD by the membrane-located enzyme AgrB. It is then detected by the membrane-located AgrC, which phosphorylates AgrA which then has DNA-binding activity and upregulates transcription of the promoters termed P2 and P3 in the agr locus. There are four known variants of AIP with different molecular structures and cross-inhibitory activity; this BioBrick generates group I AIP. This part contains the coding regions for group I agrC and agrA (in that order) from ''S. aureus'' strain NCTC8325, prefixed by the B0034 RBS and separated by a BamHI site.
P2 promoter in agr operon from S. aureus
The section of the sequence constituting the P2 promoter is subject to rather more debate than the sequences of the coding regions. [http://jb.asm.org/cgi/content/full/186/22/7549 Koenig, Robbin L., Ray, Jessica L., Maleki, Soheila J., Smeltzer, Mark S., Hurlburt, Barry K. Staphylococcus aureus AgrA Binding to the RNAIII-agr Regulatory Region J. Bacteriol. 2004 186: 7549-7555] shows the areas of the sequence to which phosphorylated AgrA binds while L Rao, R K Karls, and M J Betley: In vitro transcription of pathogenesis-related genes by purified RNA polymerase from Staphylococcus aureus. Bacteriol. 1995 May; 177(10): 2609???2614. shows the transcription start sites; we took the overall extent of the promoter region from that labelled in Morfeldt, E., K. Tegmark, and S. Arvidson. 1996. Transcriptional control of the agr-dependent virulence gene regulator, RNAIII, in Staphylococcus aureus. Mol. Microbiol. 21:1227???1237. (not available online).
The agr P2 operon is an autocatalytic sensory transduction system in Staphylococcus aureus. P2 promoter regulates the synthesis of AgrB (a transmembrane protein) and AgrD (precursor of AIP autoinducing peptide). AIP binds to AgrC, a membrane binding receptor and it phosphorylates AgrA. The phosphorylated AgrA increases the expression level from P2 promoter.
Codon usage has been optimised for E. coli (which also seems to give good results for B. subtilis
Built by Josh as an intermediate in screening plasmid construction.
RBS (Elowitz 1999) -- defines RBS efficiency
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix. No secondary structures are formed in the given RBS region. Users should check for secondary structures induced in the RBS by upstream and downstream elements in the +50 to -50 region, as such structures will greatly affect the strength of the RBS. Contact info for this part: Brian Chow
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003.
**highly** engineered mutant of red fluorescent protein from Discosoma striata (coral)
Campbell et al., PNAS v99 p7877 URL
monomeric RFP: Red Fluorescent Protein. Excitation peak: 584 nm Emission peak: 607 nm
TAATAA double stop codon added (DE). Four silent mutations made to remove three EcoRI sites and one PstI site: A28G, A76G, A349G, G337A.
Conversion of the iGEM parts registry to SBOL2.1