RBS (Elowitz 1999) -- defines RBS efficiency
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003.
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
In order to work,Cph8 has to form chromophore with PCB biosynthetic genes, where the formation of PCB requires the gene pcyA and hol to function as accounted below. Hol is a sort of Iron red pigment oxidase which can oxidize the heme group using a ferredoxin cofactor, generating biliverdin IXalpha. And then, PcyA, a kind of ferredoxin oxidase from Synechocystis Sauv, functions to turn biliverdin IXalpha (BV) into PCB. We connected gene pcyA（BBa_I15009）and ho1（BBa_I15008）together along with the constitutive promoter(BBa_J23100) to ensure a continuous work of the photoinduction system in our E.coli.
We used oe-pcr to assemble these parts together seamlessly.
pcyA gene and ho1 gene both come from Synechocystis.
LacZ alpha fragment; complements matching N-terminal deletion mutant (lacZ-omega)
to greatly reduce the number of bases, this is only a portion of the LacZ gene
Restriction sites (modified) 76-79 (gcc to gca) 79-81 (gct to gca) 85-87 (gaa to gag) 106-108 (tgc to tgt) 109-111(agg to aga)
PompR is a OmpR-controlled promoter which can be positively regulated by phosphorylated OmpR. This promoter is taken from the upstream region of ompC. Phosphorylated OmpR binds to the three operator sites and activates transcription. We insert PompR(BBa_R0082) sequences into the upstream region of our target gene gp35 through restriction-ligation method, which together is connected to vector PSB1C3 afterwards, and therefore we are able to regulate the expression of in through the control of light. Bxb1 gp35 is a serine integrase in Mycobacterium phage Bxb1. This intergrase can exclusively catalyze site-specific recombination. The dismiss of light singal can drive the expression of integrase, which alone can set a DNA sequence(there it means the promoter) flanked by oppositional attB and attP sites, thereby produce an inverted sequence flanked by attL and attR sites. As a consequence, the promotor moves on to express the toxic protein instead of the attractant. We insert the gp35 gene into the downstream region of the PompR promotor by means of 3A synthesis so that the expression of integrase can be regulated by light. In addition, we add a 6N promotor between PompR and gp35 to decrease basal integrase expression above flipping threshold level which could lower the efficiency of int translation. Meanwhile, we insert the recombined sites of attB and attP into the flanking sequences of our constitutive promotor (BBa_J23110 ) and so far we accomplish our reverse promotor system.
we add a 6N promotor between PompR and gp35 to decrease basal integrase expression above flipping threshold level which could lower the efficiency of int translation.
Chimeric Cph1 light receptor/EnvZ protein.
Silent mutation at base 108 (G-A) to remove PstI site
This part is just for upload test. It is meaningless
Strong promoter BBa_J23119. Chimeric Red light receptor cph8 BBa_I15010 (cph1-envZ) and proteins involved in phycobilin biosynthesis ho1 (BBa_I15008), pcyA (BBa_I15009).
Sequences are from Cyanobacteria : - Cph1 domain - ho1 BBa_I15008 - pcyA BBa_I15009
PompR is a OmpR-controlled promoter which can be positively regulated by phosphorylated OmpR. This promoter is taken from the upstream region of ompC. Phosphorylated OmpR binds to the three operator sites and activates transcription. We insert PompR(BBa_R0082) sequences into the upstream region of rfp gene, which together is connected to vector PSB1C3 afterwards, and therefore we are able to observe the OmpR promoter efficiency through the control of light.
We constructed this biobrick through pcr.
Light responsive system, dual regulation
Pbad regulated phycobilins (ho1 and pcyA) followed by Ptet regulated light sensor (Cph8). This should allow repressed Pbad to give low levels of accessory proteins and derepressed Ptet to crank out lots of light sensor protein
Stephen Lee, Roshan Kumar, Joe Levine, Ziyan Chu (Polkadorks, IAP 2004)
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.
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.
One of two requisite genes required for the biosynthesis of phycocyanobilin from heme.
This is the light sensor Cph8 with a promoter and RBS.
An intermediate part designed to be used with the AND gate designed by Caltech iGEM '10: [http://2010.igem.org/Team:Caltech/Project Project Details].
Designed to be placed upstream of positive feedback mechanisms and desired output genes.
The Registry of Standard Biological Parts
phycocyanobilin:ferredoxin oxidoreductase (PcyA) from synechocystis
Second of two required phycocyanobilin biosynthetic genes.
*ompR, E. coli two-component signal transduction system used along with EnvZ *controls expression of genes under ompC and ompF promoters *doi:10.1016/j.physletb.2003.10.071
*Heme oxygenase and phycocyanobilin:ferredoxin oxidoreductase *Used for light sensing system