This plasmid contains code for pBAD promoter, AIP receiver Agr A and Agr C, GFP along with double terminator.
Sequence was cloned using BBa_E0240 and BBa_I746103 from iGEM parts registry.
I746101 (AIP sensor infrastructure - agrC + agrA with RBSes + Terminator) + I746104 (Inducible P2)
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 is the AIP sensor infrastructure ([http://partsregistry.org/wiki/index.php/Part:BBa_I746101 I746101]) from the agr system with an AIP-inducible promoter P2 ([http://partsregistry.org/wiki/index.php/Part:BBa_I746104 I746104]) downstream. A constitutive/inducible promoter may be added upstream of I746101, which will cause production of the components of the signal transduction pathway (AgrC and AgrA); when phosphorylated AgrA is present, the AIP-sensitive promoter (P2) is activated and PoPS is output. In an application, this PoPS output could feed into a downstream device. The original 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. 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 activity of agr P2 promoter about 50-fold.
The sequence was obtained by applying all mutations that upregulated AraC binding and subsequent promoter activity listed in reference [1].
pBAD is an E.coli promoter that is induced by L-arabinose. K206000 is a mutagenized pBAD promoter that is responsive to lower concentrations of arabinose than wild type (<partinfo>I13453</partinfo>) and, additionally, exhibits a higher maximum of protein expression as measured by coupling to a fluorescent reporter.
There were no special design considerations.
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.
RBS.3 (medium) -- derivative of BBa_0030
Weak1 RBS based on Ron Weiss thesis. Strength is considered relative to <bb_part>BBa_B0030</bb_part>, <bb_part>BBa_B0031</bb_part>, <bb_part>BBa_B0033</bb_part>.
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix ("RBS-2" in figure 4-14 of thesis). Contact info for this part: Brian Chow
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003.
Transcriptional terminator consisting of a 64 bp stem-loop.
green fluorescent protein derived from jellyfish Aequeora victoria wild-type GFP (SwissProt: P42212
GFP (mut3b) [note that this part does not have a barcode]
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
Conversion of the iGEM parts registry to SBOL2.1