NOx(Oxides of Nitrogen) sensor device (pYeaR-Yellow 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 second oxides of nitrogen (NOx) 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 NsrR(K2056001) a NOx sensitive repressor protein and single terminator(B0010) followed by NsrR repressed promoter pYeaR(K216005) with RBS(B0030) and 'Yellow chromoprotein'(K1033910) with terminator(BBa_B0012). If NOx are present, they will bind to the constitutive expressed NsrR that will relieve repression on the pYeaR promoter activating transcription of the yellow chromoprotein. Sequence and Features
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.
This chromoprotein naturally exhibits strong color when expressed.
This construct is synthetic. It was synthesized from a template where the consensus sequence for the "color dependancy" in order to obtain a chromoprotein with a unique yellow color.
NsrR - NO(x) sensitive pYear, pNirk repressor protein
This part has been designed after the part Bba_K1682011 in the Registry of Standard Biological Parts.
This part contains the NsrR (NO(x) sensitive pYear, pNirk repressor protein) encoding region. Escherichia coli (E. coli) detects environmental nitrate by the yeaR-yoaG operon. PyeaR is regulated by the NsrR repressor protein. Nitric oxide binds to the NsrR protein and relieves the repression on PyeaR. As a result, any genes that are downstream of PyeaR are expressed. This part is also related with BBa_K2056002, the promoter region of the NirK protein, because NsrR activates the transcription of NirK under high nitrite conditions. Due to these properties, we can use this part as a sensor for oxides of nitrogen.
Because the part Bba_K1682011 was not available from the registry, we had to get it synthesized. We had to codon optimized this part to conform to assembly standards and the requirements for synthesis.
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
PyeaR promoter, responsive to nitrate, nitrite and nitric oxide
BioBricked from E. coli K-12 genomic DNA by the Edinburgh 2009 iGEM team.
PyeaR promoter. This is the promoter of the Escherichia coli yeaR/yoaG operon (see Lin, H.-Y., Bledsoe, P.J., and Stewart, V. 2007. Activation of yeaR-yoaG operon transcription by the nitrate-responsive regulator NarL is independent of oxygen-responsive regulator Fnr in ''Escherichia coli'' K-12. J. Bacteriol. 189, 7539-7548). Unlike other E. coli promoters responding to nitrate and nitrite, this promoter is not repressed under aerobic conditions.
Conversion of the iGEM parts registry to SBOL2.1