Phycocyanobilin synthesis genes + Red light detection system (EYFP as reporter)
PCR of BBa_M30109 (which is truncated in the 2010 igem distribution).
There are some problems with the original coliroid parts that are in the 2010 distribution. We resubmitted this in pSB1C3, with the reporter system attached. This part contains the phycocyanobilin synthesis genes. PCB binds to cph8 and is required for light detection.
EYFP is used as a reporter system for precise characterisation.
Chris French and Richard Partridge Hicks
Positively regulated, OmpR-controlled promoter. This promoter is taken from the upstream region of ompC. Phosphorylated OmpR binds to the three operator sites and activates transcription.
The promoter includes the three OmpR binding sites: C1, C2, and C3. The promoter starts at -107 and goes up to the transcriptional start site at +1. An alternate version, BBa_R0083, cuts out the C2 and C3 sites. The efficacy of this promoter versus the truncated ompC upstream region (BBa_R0083) or the ompF upstream region (BBa_R0084) is currently unknown.
Stephen Lee, Roshan Kumar, Joe Levine, Ziyan Chu (Polkadorks, IAP 2004)
enhanced yellow fluorescent protein derived from A. victoria GFP
Modificaitons to Clontech EYFP by Reshma Shetty
Phycobilin synthesis operon without terminator
This is derived from K098010 but PCR was used to remove the terminator, so that cph8 could be added at the end of the operon.
Phycobilin is chromophores (light-capturing molecules) found in cyanobacteria and in the chloroplasts of red algae, glaucophytes and some cryptomonads (though not in green algae and higher plants)， used in light sensor.
The actual forward sequence has the same changes at the start as seen in clone H11 described above. The reverse sequence is as expected. Features same as for the one above apart from the terminator.
Chris French and Richard Edward Partridge-Hicks
Red light sensor Cph8 (from Coliroid) with RBS
BBa_I15010 (Photosensing Chimaera (Cph8) (not working, but amplified out of the whole construct))
Cph8 is a combination of cph1 and envZ which, together with PCB, form a transmembrane, histidine kinase-based light sensing system. It contains the photoreceptor from Cph1 and the histidine kinase and response-regulator from EnvZ???OmpR, producing a particularly strongresponse to light. Features: cph8 coding sequence 16 to 2250, RBS 6 to 10.
Notes: we were unable to revive BioBrick I15010 with the coding sequence of the cph8 red light sensor fusion protein, so we sought to amplify it from BioBrick BBa_M30109. This led to numerous problems. It is clear from the Registry sequence data that M30109 is not what it is supposed to be. It does include cph8 with a ribosome binding site, but the RBS is not joined to cph8 by a standard scar; promoter R0040 may be present, but there appears to be an EcoRI site a little upstream of the ribosome binding site so that, when we cloned our PCR product, we failed to recover the promoter region; also the PstI site in cph8, which was mutated out in I15010, is still present in the version in M30109. We therefore performed corrective PCR to obtain a clone with the correct prefix, a ribosome binding site, and the cph8 coding sequence with the PstI site properly removed, which we present here, cloned in pSB1C3.
The DNA was derived by PCR from E. coli BL21, which possesses an intact lacZ gene. Primers were designed based on sequence from GenBank accession J01636, GI:146575. A stop codon was artificially introduced to replace codon 78. The sequence reported here is derived by sequencing of the Biobrick construct.
This gene encodes the N-terminal 77 amino acid residues of LacZ. When expressed in an E. coli host carrying the lacZ-delta-M15 mutation, common in laboratory strains, it complements the delection resulting in the production of active LacZ, which can be detected by various means, including chromogenic substrates such as Xgal (5-bromo-4-chloro-3-indolyl-beta-D-galactoside) and ONPG (o-nitrophenyl galactoside).
Note that a stop codon was introduced to replace codon 78 of lacZ, resulting in production only of the N-terminal 77 amino acid residues of LacZ. This is sufficient to complement the lacZ-delta-M15 mutation commonly found in laboratory strains of E. col used for alpha-complementation.
Conversion of the iGEM parts registry to SBOL2.1