Red light sensing system with mRFP reporter
This part consists of a red light sensing system and an RFP reporter system. When bacterial colonies are exposed to red light, the OmpR promoter is not activated, preventing the expression of RFP. When colonies are kept in dark, the corresponding transcription factor of mpR promoter is activated and RFP is expressed, making the colonies red.
Positively regulated, OmpR-controlled promoter from OmpC gene (R0082) directing transcription of mRFP (I13507). Constructed with standard assembly method except R0082 was isolated using VF and VR-directed PCR. Consequently, there may be silent mutations that were inadvertently introduced. An alternative clone is M30012. Cells bearing this construct are pale pink.
This part is mainly composed of Cph8(BBa_K592018), ho1(BBa_I15008) and PcyA(BBa_I15009), which form the red light sensing system in E.coli. Cph8 is a synthetic red light sensor, consisting of Cph1, a natural red light sensor, and EnvZ, a histidine kinase. The most important part of the light sensor is PCB(phycocyanobilin), without which the red light sensor cannot work. However, PCB is not generated by E.coli naturally. So we need to introduce ho1 and PcyA into E.coli. These two enzymes can change heme into PCB. Here we combine the three main parts together to facilitate parts users.
We need to add a promoter and three RBSs.
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.
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.
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)
Transcriptional terminator consisting of a 64 bp stem-loop.
**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.
Overlap extension of synthetic oligonucleotides
One of two requisite genes required for the biosynthesis of phycocyanobilin from heme.
phycocyanobilin:ferredoxin oxidoreductase (PcyA) from synechocystis
Second of two required phycocyanobilin biosynthetic genes.
The cph8 was taken from Tabor's pJT122 plasmid.
This is the red light sensor Cph8 with RBS.
Since Cph8 was considerably longer than the RBS added, sequencing was necessary to confirm the construct.
This part is identical to BBa_I15010, the cph8 red light sensor. We were unable to transform BBa_I15010 from the the 2011 iGEM distribution kit. PCR attempts using VF2 and VR failed to amplify any DNA from the well sample. In the end, we chose to amplify cph8 from Jeffrey Tabor's pJT122 plasmid provided by Chris Voigt. The cph8 in pJT122 contained an internal PstI site, which was removed by point-mutagenesis. The resultant DNA sequence is identical to that of BBa_I15010.
The source DNA (pJT122) contained illegal restriction site (PstI) that had to be removed.
Conversion of the iGEM parts registry to SBOL2.1