Difference between revisions of "Part:BBa K2942703"

Latest revision as of 01:32, 22 October 2019

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 effect 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.

Fig.2 Survival rate of cells. A549 and HCT116 cells were seeded 104 per well in the 96 well plate, and CB1954(100 uM, see the reaction principle in Fig.7 in composite part BBa_K2942707) and nitroreductase (NR,400ug/ml) were added 8h after transfection. The survival rate of cells was examined using MTT Cell Proliferation and Cytotoxicity Assay Kit.

[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

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal PstI site found at 100
12
INCOMPATIBLE WITH RFC[12]
Illegal PstI site found at 100
21
COMPATIBLE WITH RFC[21]
23
INCOMPATIBLE WITH RFC[23]
Illegal PstI site found at 100
25
INCOMPATIBLE WITH RFC[25]
Illegal PstI site found at 100
Illegal AgeI site found at 655
1000
COMPATIBLE WITH RFC[1000]

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-[[file:27032.png|720px]]
+[[file:WYH2703-3.png|720px]]
-Fig.2 Survival rate of cells. A549 and HCT8 cells were seeded 104 per well in the 96 well plate, and CB1954(100 Um, see the reaction principle in Fig.6) and theophylline(4 mM) were added 8h after transfection. The survival rate of cells was examined using MTT Cell Proliferation and Cytotoxicity Assay Kit.
+Fig.2 Survival rate of cells. A549 and HCT116 cells were seeded 104 per well in the 96 well plate, and CB1954(100 uM, see the reaction principle in Fig.7 in composite part BBa_K2942707) and nitroreductase (NR,400ug/ml) were added 8h after transfection. The survival rate of cells was examined using MTT Cell Proliferation and Cytotoxicity Assay Kit.
 [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)