Part:BBa_M36667:Design

The objective of our vector was to express ß-galactosidase within E. Coli cells with an inducible transcription factor activated by absence of oxygen. A constitutive promoter initiates transcription of FNR(fumarate nitrate reductase), which is deactivated in aerated conditions and activated in hypoxic conditions with a glutathione supplement and cysteine desulphurase. For this construct, we elected to omit cysteine desulphurase as it is expressed within E. Coli. The sensor also consists of a pepT7 promoter driving the Gemini actuator, which produces ß-galactosidase. Additionally the system also includes kanamycin resistance gene to allow for the creation of a selectable stable cell line, and a high copy number origin of replication.

The sensor part of our construct that allows for production of FNR consists of a constitutive promoter attached to the gene for FNR, and the FNR-inducible pepT7 promoter. The promoter, BBa_J23100, was obtained from IGEM and was selected for its high success in other experiments and promoter strength (https://parts.igem.org/Part:BBa_J23100). Prokaryotic ribosome binding domain pRBS-SD1 was selected from the GeneDesigner Database, followed by the FNR gene (obtained from NCBI gene database). Furthermore, the FNR gene was codon optimized to remove a restriction site while preserving the amino acid sequence. The terminator for FNR, part BBa_B0015, was obtained from iGEM parts registry for its high forward efficiency (0.984)( https://parts.igem.org/Part:BBa_B0015). The second part of the sensor, the FNR-inducible pepT7 promoter was selected primarily for its activation by active-FNR and obtained from Lombardo et al.

(Lombardo, Mary-Jane, Angela A. Lee, Tina M. Knox, and Charles G. Miller. "Regulation of the Salmonella Typhimurium PepT Gene by Cyclic AMP Receptor Protein (CRP) and FNR Acting at a Hybrid CRP-FNR Site."Journal Of Bacteriology 179.6 (1997): 1909-917. American Society for Microbiology. Web. 04 May 2014)