Carbon Monoxide sensor device (pCooF-Blue Chromoprotein)
All parts in this sequence come from the Registry and have been listed. However, the complete part has been synthesized from IDT using their free gBLocks offer.
This part is the carbon monoxide (CO) sensing module of the 'Inspector NOxCO' device. 'Inspector NOxCO' is a bacterial sensor for carbon monoxide (CO) and oxides of nitrogen (NOx) in vehicle emissions. This part consists of the Anderson constitutive promoter(BBa_J23110) and RBS(B0030) with the coding sequence for CooA(BBa_K352001) a CO dependent transcriptional activator and single terminator(B0010) followed by CooA activated strong promoter pCooF(K352002) with RBS(B0030) and 'amilCP, blue chromoprotein' with terminator(BBa_B0012). If CO is present, the constitutive expressed CooA will bind to it and then bind to the pCOoF promoter activating transcription of the blue chromoprotein.
The selection of the RBS and Constitutive promoter. Because of limitations of repeats that could be synthesized, we had to give up on the double terminators in the original design and go with single different terminators. A 70bp spacer(B0040) has been added for transcriptional efficiency.
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.
RBS.1 (strong) -- modified from R. Weiss
Strong RBS based on Ron Weiss thesis. Strength is considered relative to <bb_part>BBa_B0031</bb_part>, <bb_part>BBa_B0032</bb_part>, <bb_part>BBa_B0033</bb_part>.
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix ("orig" in figure 4-14 of Ron Weiss 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 Brian Chow
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003.
Hwan Youn, Robert L. Kerby, Mary Conrad, et al. 2004. Functionally Critical Elements of CooA-Related CO Sensors. J. Bacteriol. 186(5):1320-1329. doi:10.1128/JB.186.5.1320-1329.2004.
CooA is a heme-containing transcriptional activator that enables Rhodospirillum rubrum to sense and grow on CO as a sole energy source. CooA is a member of a family of transcriptional regulators similar to the cAMP receptor protein and fumavate nitrate reduction from Escherichia coli. The protein is active in sequence-specific DNA binding in the presence of CO, but not in the absence of CO. The protein to be a dimer in the absence of CO. The product, CooA, is 28% identical (51% similar) to CRP(cAMP receptor protein) and 18% identical (45% similar) to FNR(fumavate nitrate reduction) from Escherichia coli. Inactive Fe(II) CooA structure adapted from that of the strain with PDB identification no. 1FT9. The protein consists of two monomers, shaded differently, which dimerize along the central C-helices of adjacent effector-binding domains. The solved structure is asymmetric, in which one monomer contains fused C- and D-helices. Nonetheless, both F-helices that interact with DNA in a sequence-specific manner are buried from the surface in the structure. The 4/5 loop is noted and so are the Pro2 and His77 heme Fe(II) ligands.
The sequence information was acquired from NCBI and physical DNA was synthesized from GENEART. Classical cloning strategies(restriction digestion) were used to produce biobricks.
pCooF Promoter from Rhodospirillum rubrum
S.W. Singer, M.B. Hirst and P.W. Ludden, CO-dependent H2 evolution by Rhodospirillum rubrum: role of CODH:CooF complex, Biochim Biophys Acta - Bioenerg 1757 (2006), pp. 1582???1591
CooA is a CO-sensing protein that activates the transcription of genes encoding the CO-oxidation (coo) regulon, whose polypeptide products are required for utilizing CO as an energy source in Rhodospirillum rubrum. CooA binds to a position overlapping the -35 element of the P(cooF) promoter, similar to the arrangement of class II CRP (cAMP receptor protein)- and FNR (fumarate and nitrate reductase activator protein)-dependent promoters when expressed in Escherichia coli. The CO-dependent anaerobic growth of Rhodospirillum rubrum relies on a CO oxidation system encoded by two CO regulated transcriptional units, cooMKLXUH and cooFSCTJ. The key products of the coo regulon are an O2- sensitive CO dehydrogenase (CooS), a CooS-associated Fe-S protein (CooF), and a CO-tolerant hydrogenase (CooH), and the expression of the genes depends upon the activity of the CooA protein, which senses CO under anaerobic conditions. CO-independent basal level transcription of PcooF is not detectable due to the absence of active CooA.
This chromoprotein, amilCP, naturally exhibits very strong color when expressed. The color is blue/purple and is visible to naked eye, thereby requiring no instruments to observe. This DNA was provided by Jeffrey Miller at UCLA. It was made BioBrick-compatible after removal of one illegal internal restriction site (EcoRI).
Illegal internal restriction site had to be removed (EcoRI).
Randomly generated and optimized for several parameters (see Design notes).
Generic spacer for ensuring a 70 bp distance between the end of the suffix of the BioBrick part containing the double terminator and the prefix of the BioBrick part containing the promoter of the new gene. Please, use the AlignX function of Vector NT to check for homology with the components in your plasmid before using this spacer.
The size of the spacer was choosed to meet the minimum length of a sequence that can be queried using the BLAST search engine. However, subsequences of it can be used to design shorter spacers. The sequence was selected from many more sequences randomly generated using the Random DNA Generator engine; the GC% parameter used as input was 50%. The sequences were selected based on the following constraints listed in their order of importance: the absence of any putative promoter regions, a low degree of homology with the Elowitz plasmid (whose components are widely used in our designs), no homology with other E.coli sequences as shown by BLASTN search results and the presence of a number of TAA stop codons. The second constraint was the most stringent leading to the elimination of most sequences. DE made the following changes to the original sequence in order to add stop codons in the -3 frame and more in the +2 frame (note, not all of these stop codons are UAA. Thus, if used in an organism that inserts an amino acid @ UGA or UAG the obvious will occur): T->A @ 85 T->A @ 42 C->T @ 79 A->T @ 64 A->T @ 31 T->A @ 34 C->A @ 37 Also, note that the above changes further reduce (the already very weak) homology to current NCBI-stored sequences. In the process of selecting the best sequence it appeared that a good alternative sequence for a spacer would be: AGGTTCTGATATGTAACTGTGCCCAATGTCGTTAGTGACGCATACCTCTTAAGAGGCCACTGTCCTAACA. The sequence contains no putative promoters and shows moderate homology with the 5' end of the Ampicillin resistance gene. However a strong promoter sequence starts 12 bp downstream of this sequence, and therefore the sequence presented above was preferred. The sequence is compatible (does not show significant homology) with the components in the Elowitz repressilator plasmid.
Vinay S. Mahajan, Brian Chow, Peter Carr
Conversion of the iGEM parts registry to SBOL2.1