Part:BBa_K3746010
T7-LacO-FDRA-6His
Description
This is a part design for the Phase I project (2021 iGEM) of Team Hong Kong JSS.
The IPTG inducible expression construct of F420H2-dependent reductase (FDR)-A from Mycobacterium smegmatis (BBa_K3746003).
The coding sequence of tvLac is driven by T7 promoter (BBa_I719005), a lac operon (BBa_K3076802), strong RBS (BBa_K3076801), and a T7 terminator (BBa_K395601).
The FDR-A protein is tagged with a C-terminal 6-His Tag (CATCATCATCATCATCAT) for protein purification.
In our Phase I iGEM Project, we planned to transform this construct into E. coli and use the E. coli to act as a biological control of Aflatoxin B1 (AFB1) contamination.
F420H2-dependent reductase (FDR) is a family of enzymes produced by microorganisms that catalyze the reduction of aflatoxin and lead to their spontaneous degradation and produce a non-toxic product. [1]
This enzyme can be found in bacterial strains such as Mycobacterium sp., Arthrobacter sp., and Pseudomonas sp.. The native function of the enzymes in these organisms is for methanogenesis [2], antibiotic resistance [3], and other redox-related metabolic activities. Nevertheless, it was believed that FDR’s native functions do not involve aflatoxin degradation since the above bacterial species seldom exist near any source of aflatoxin. Thus, it was a novel finding that FDRs are involved in the degradation of aflatoxin and there was considerable interest in the study of FDRs properties recently. [1]
In our project, we aim to investigate the AFB-1 degrading activity of FDR-A.
Aflatoxin (AF) is a family of carcinogenic toxins produced by Aspergillus sp.. According to the World Health Organization (WHO), 25% of food crops are destroyed due to aflatoxin contamination each year. About 5 billion people are at risk of chronic AF exposure and more than 80% of them will develop AF-related diseases such as hepatocellular carcinoma and liver failure. [4] Among all AF, aflatoxin B1 (AFB1) is considered the most potent and chronic. [5]
Among all species reviewed, Mycobacterium smegmatis was found to have the FDRs with the highest activity in AFB-1 degradation [6].
And from all the loci of the two FDR families (FDR-A and FDR-B) in M. smegmatis, FDR-A at locus MSMEG_5998 was found to have the highest enzymatic activity in degrading AFB1, AFB2, and AFG1. [1]
References
[1] Taylor, M. C., Jackson, C. J., Tattersall, D. B., French, N., Peat, T. S., Newman, J., Briggs, L. J., Lapalikar, G. V., Campbell, P. M., Scott, C., Russell, R. J., & Oakeshott, J. G. (2010). Identification and characterization of two families of F420H2-dependent reductases from mycobacteria that catalyse aflatoxin degradation. Molecular Microbiology, 78(3), 561–575. https://doi.org/10.1111/j.1365-2958.2010.07356.x
[2] Graham, D.E., and White, R.H. (2002) Elucidation of methanogenic coenzyme biosyntheses: from spectroscopy to genomics. Nat Prod Rep 19: 133–147.
[3] Hasan, M.R., Rahman, M., Jaques, S., Purwantini, E., and Daniels, L. (2010) Glucose-6-phosphate accumulation in mycobacteria: implications for a novel F420-dependent anti-oxidant defense system. J Biol Chem 285: 19135– 19144.
[4] Organization WH. aflatoxin. Manuf Comput Solut. 2000;6(8):20-3
[5] Okwara, P. C., Afolabi, I. S., & Ahuekwe, E. F. (2021). Application of laccase in aflatoxin B1 degradation: A Review. IOP Conference Series: Materials Science and Engineering, 1107(1), 012178. https://doi.org/10.1088/1757-899x/1107/1/012178
[6] Verheecke, C., Liboz, T., & Mathieu, F. (2016). Microbial degradation of aflatoxin B1: Current status and future advances. International Journal of Food Microbiology, 237, 1–9. https://doi.org/10.1016/j.ijfoodmicro.2016.07.028
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 132
- 1000COMPATIBLE WITH RFC[1000]
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