Difference between revisions of "Part:BBa K2942703"

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Nitroreductase

Nitroreductase

Nitroreductase is an enzyme that can catalyze the reduction of nitro aromatic compounds to aromatic amines, and it plays an important role in oxygen return systems in which NADPH or NADH is the reducing agent. Nitroreductase increases the sensitivity of living organisms to nitrogen-containing drugs, such as metronidazole, which converts nitrogen groups into toxic nitrogen free radicals. Thus it is studied to promote prodrug activation [1]. The following picture shows how nitroreductase works.

Elsie M. Williams, et al. Nitroreductase gene-directed enzyme prodrug therapy: insights and advances toward clinical utility. Biochem J 15 October 2015; 471 (2): 131–153.

In our project we optimized the nitroreductase that we carried out human codon optimization on the original nitro reductase and added flag tag for labeling, which Increases the expression of protein as well as detection . This part was mainly used for the construction of composite part. We artificially synthesized the sequence, and then we design the primers (see details in the composite part BBa_2942707) to gain and recycle the PCR product (Fig.1) for the multiple fragment homologous recombination next. Its characterization can be seen in the composite part BBa_2942707 in details. The characterization of protein expressed and extracted in E. coli shows as flollows (Fig.2).

Fig.1 PCR result. We followed the protocol of NEB Q5® High-Fidelity 2X Master Mix to perform the PCR, and the PCR product was identified by 1% agarose gel electrophoresis.

[1] Nillius, D. J, et al. Nitroreductase (GlNR1) increases susceptibility of Giardia lamblia and Escherichia coli to nitro drugs. Antimicrobial Agents and Chemotherapy 66, 1029-1035, (2011)

Sequence and Features