Low Oxygen Level Self-induction Genetic Circuit. NarK promoter
Initially during a fermentation run, DOT levels in the batch fermenter are observed with a gradual decreasing as a result of the E.Coli cells populating in growth. Over time, the DOT in the vessel will fall below 20% in which a critical point in the fermentation is reached as such low concentrations of oxygen will result in cellular death and loss of batch material. This rectified by adding the reagent IPTG (Isopropyl β-D-1-thiogalactopyranoside) in order to induce protein expression. This momentary measurement of low oxygen levels results in what is known in industry as an "Oxygen Spike" and marks the transition of the process from Batch to Fed-Batch in which there is a consistent addition of glycerol to sustain the cells during protein expression. The resultant shift from cellular growth to protein expression can be observed in the immediate increase in OD values subsequent to inoculation. The NarK promoter (BBa_K239005) can detect the low oxygen level right before oxygen spike during fermentation. Hence initiate transcription of downstream promoter activator, T7 RNA Polymerase, which will then active the T7 promoter. The T7 promoter is followed by another T7 RNAP to enhance the production of promoter activator and the final product, which is represented as RFP in our system.
Built by Josh as an intermediate in screening plasmid construction.
T7 promoter is used as part of the "positive loop" for the self-inducing system in HYPOXON project. The constitutive promoter regulate the expression of the promoter activator, T7 RNA Polymerase, in order to produce the downstream product continuously.
Part was obtained by oligonucleotide synthesis.
T7 RNA polymerase from the T7 genome. Used as promoter activator in our HYPOXON genetic circuit.
Adding prefix&suffix sequence in front of and after the original T7 RNAP sequence by PCR is a bit difficult.
NarK promoter, Fnr activated under anaerobic conditions
Genomic sequence from biocyc, Escherichia coli K-12. Native unmodified sequence.
Sequence contains 2 Fnr, 1 Fis, 4 NarL and 1 IHF binding sites and initiates transcription by RNAP sigma-70. Function: Switched on during anaerobic conditions (upregulated 100-fold). Area of application: Detection of when the cell is switching to anaerobic metabolism.
Detect and remove possible restriction sites: XbaI, EcoRI, PstI, SpeI, AgeI, BamHI, BglII, NogMIV, NotI and XhoI. No restriction site had to be removed. Sequence starts 3 bp before the IHF binding site and ends 6 pb after the -10 box.
RBS (Elowitz 1999) -- defines RBS efficiency
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix. 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.
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.
**highly** engineered mutant of red fluorescent protein from Discosoma striata (coral)
Campbell et al., PNAS v99 p7877 URL
monomeric RFP: Red Fluorescent Protein. Excitation peak: 584 nm Emission peak: 607 nm
TAATAA double stop codon added (DE). Four silent mutations made to remove three EcoRI sites and one PstI site: A28G, A76G, A349G, G337A.
Conversion of the iGEM parts registry to SBOL2.1