This part is a combination of DPD input and Green light input. The subparts of this part is from iGEM Distribution Kits.
The circuit to receive input signals is composed of AND GATE, quorum sensing promoter and light-controlled promoter. After being induced by DPD, plsrA promoter activates the expression of T7 RNA polymerase which contains two amber mutations (T7ptag) . After being induced by green light, PcpcG2 promoter activates the expression of a special tRNA (supD), which is used to identify amber mutation. In the wild-type E.coli, amber mutation will inhibit the translation of T7 RNA polymerase. Therefore, when DPD and green light exist together, T7 RNA polymerase will be expressed, and then T7 promoter will be activated to express the downstream gene.
PT7(BBa_K1321338) contains T7 promoter and RBS(B0034, which enables easy addition to any coding part which does not already have these parts.
Double terminator consisting of BBa_B0010 and BBa_B0012
RBS.4 (weaker) -- derivative of BBa_0030
Weaker RBS based on Ron Weiss thesis. Strengths relative to <bb_part>BBa_B0030</bb_part>, <bb_part>BBa_B0031</bb_part>, <bb_part>BBa_B0032</bb_part>.
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix ("RBS-3" in figure 4-14 of thesis). 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.
lsr promoter of LuxS/AI-2 signaling pathway in Salmonalla
We extracted the genomic DNA of Salmonella enterica subsp. enterica serovar Typhimurium str. LT2, and used PCR to isolate the promoter plsr.
Quorum sensing is a process of bacterial cell-to-cell communication involving the production and detection of extracellular signaling molecules called autoinducers. And autoinducer-2 (AI-2) has been proposed to serve as a 'universal signal' for interspecies communication. In the LuxS/AI-2 signaling system of Salmonella Typhimurium, AI-2 response involves ATP binding cassette transporter encoded by genes named Lsr (LuxS regulated). And plsr is member of lsr operon which could be repressed by LsrR. In our project, we set this promoter part in front of a blue pigment as a reporter monitoring the presence of AI-2.
The lsrD gene was isolated from the genome of Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 using PCR. We added restriction enzyme sites when designing primers, and the assembly is done through the use of restriction sites (cutting and ligating).
This sequence can be amplified from the genome of Synechocystis PCC6803. Reference: Hirose Y, Shimada T, Narikawa R, Katayama M, Ikeuchi M (2008) Cyanobacteriochrome CcaS is the green light receptor that induces the expression of phycobilisome linker protein. Proc Natl Acad Sci U S A 105: 9528-9533. The 238 bp sequence can be directly taken from the pJT122 plasmid designed by Jeffrey Tabor. Reference: Tabor, J. J., Levskaya, A., and Voigt, C. A. (2011). Multichromatic control of gene expression in Escherichia coli. J. Mol. Biol. 405, 2, 315???324.
PcpcG2 is the green-light activated promoter from the genome of Synechocystis PCC6803. This 238 bp sequence is used by Jeffrey Tabor in pJT122 plasmid and contains the entire region upstream of cpcG2 and downstream of ccaR. PcpcG2 is regulated by the light-activation of ccaS/ccaR. The gene downstream to this promoter is transcribed upon activation by ccaS/ccaR, via green light.
This sequence was PCR-amplified directly from the pJT122 plasmid provided by Chris Voigt, the co-author of Tabor's paper on multichromatic light-sensing (Tabor et al. 2011). Due to its nature, optimizing primers to extract this sequence from the source plasmid was difficult. Recommend touchdown-PCR.
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