Pyear + RBS + GFP + TT + TT + hyBb + ompA + AOX. It comes from the combination of two other existing Biobricks. For more information consult both other parts: http://partsregistry.org/Part:BBa_K381001 http://partsregistry.org/Part:BBa_K410000
It's a combination of the biobricks BBa_k381001 (Pyear + RBS + GFP + TT + TT) and BBa_k410000 (hyBb + ompA + AOX). We are expecting our part to be able to sense nitrate and nitrites even after experiencing a cold shock at 20?? C, making our BB employable in other places where the temperatures can be as low as 15?? C. If you are interested in sensing nitrate at low temperatures
We wanted to make sure that both parts worked in our specific conditions, and for that we did tests in the lab and by the moment everything is going good enough.
Combination of two existing biobricks from the parts registry
Nitrate and Nitrite sensitive promoter PyeaR with a GFP coding device and strong RBS to create a nitrate-sensitive system which signals through expression of GFP. Optimum use appears to be when sensing nitrate within a range of 0 - 20 mM
The part consists of three sub-parts with varying origins. The cold-shock promoter, HybB, was amplified using PCR from the biobrick BBa_J43001 in the Spring 2010 Distribution. OmpA was amplified from a commercial vector pFLAGS-CTS (Sigma), a plasmid that contains the 81 bp signal peptide. Aox1a, the alternative oxidase from Sacred Lotus, was synthesized commercially by Mr. Gene, Co. The sequence was taken from Genbank (GenBank: AB491175.1) and is from the Sacred Lotus (Nelumbo Nucifera).
What it is: This part consists of hybB-ompA-AOX1A. HybB is a cold shock promoter. OmpA is a periplasmic targeting sequence, and AOX1A is a gene responsible for thermogenesis in the sacred lotus, a plant. What it does: Induces AOX1A expression in response to a cold shock. Here, we describe a "cold shock" as incubating E. coli between 20-30C for 10-12 hours. The part should produce a thermogenic response raising the temperature of the E. coli's immediate environment (plate or liquid cultures) above ambient temperature.
Design considerations revolved primarily around targeting the AOX to the periplasm in order to localize AOX near its proper substrates.
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.
green fluorescent protein derived from jellyfish Aequeora victoria wild-type GFP (SwissProt: P42212
GFP (mut3b) [note that this part does not have a barcode]
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