This logical gate is based on amber stop-codon suppression via the non-canonical tRNA supD. The blue light sensor induces the expression of a mRNA coding for a T7-polymerase with an amber mutation, that can only be translated by ribosomes if the correct supD tRNA is present. The supD tRNA is expressed when the red light sensor is induced. Thus, only cells which receive both signals produce the desired substances.
We used YcgF/E as blue light sensor and Cph8 as red light sensor. We expect that this assembly will be the one with the lowest amount of unspecific gene expression which could occur due to overlapping of the absorption spectrums of the sensory domains.
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)
Double terminator consisting of BBa_B0010 and BBa_B0012
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.
Reverse RBS (RBSrev) -- corresponds to BBa_B0030
Repeats in oligos caused unusual products during cloning
Exact promoter of the blue light receptor
ycgF/E regulated promoter region ycgf is a cytoplasmic receptor which responds to blue light. it contains a Blue light Using FAD-Domain (BLUF) and an EAL domain. when blue light strikes, the receptor changes conformation and dimerizes. This allows it to bind to ycgE repressor through its EAL-domain releasing the repressor from this promoter region. See reference for more info: Natalia Tschowri, Susan Busse and Regine Hengge, The BLUF-EAL protein YcgF acts as a direct anti-repressor in a blue-light response of Escherichia coli. Genes and development23: 522-534 (2009)
T7ptag(T7polymerase with amber mutation)
The T7ptag is standardlized from the plasmid kindly provided by Voigt Lab.
T7ptag is a coding sequence of T7 polymerase with 2 Amber mutations. The transcription of T7ptag gene can only lead to the generation of its mRNA, further translation into T7 RNA polymerase is blocked because of the amber mutation. Only in the presence of the Amber mutation suppressor BBa_K228001 SupD-tRNA, it can be translated into functional T7 polymerase. T7ptag is a component of the AND Gate constructed by the Voigt Lab(UCSF)
SupD-tRNA again is from Voigt Lab(UCSF), We standardlize this part by PCR strategy.
The SupD-tRNA part functions as a tRNA, it suppresses the TAG amber stop codon and translate it into a Ser. There is no need to assembly SupD-tRNA with a rbs, since it is not translated. This part, together with the T7ptag part, make an AND Gate, that is, in the presence of both the 2 parts, T7 polymerase is tranlated into a functional polymerase that transcrips a downstream element constructed after a T7 promoter. Which is the output of the AND Gate.
There is a PstI site in the original sequence, but luckily it is near the 3' end of this part, so we designed a mutation in our primer and mutated the PstI site.
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.
Conversion of the iGEM parts registry to SBOL2.1