Part:BBa_K3746006
T7-LacO-tvLac-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 laccase from Trametes versicolor (tvLac) (BBa_K3746002).
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 laccase protein is tagged with a 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.
Laccase is a multicopper oxidase found in various organisms, such as plants and fungi. Laccase catalyzes the oxidation of phenol groups in aromatic organic substances.
Laccase has a wide range of applications, such as melanin production, lignin degradation, and lacquer synthesis. In recent years, laccase produced by some fungal species has also been reported to be involved in the bio-degradation of polyvinyl chloride (PVC) plastic in laboratory conditions [1]. Thus, it may also be a solution to the problem of micro-plastic waste pollution.
In our project, we focused on the aflatoxin B1 degrading ability of laccase.
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. [2] Among all AF, aflatoxin B1 (AFB1) is considered the most potent and chronic. [3]
Different groups have demonstrated the effectiveness of AFB1 degradation by using native and recombinant laccase [4]. Among all the laccase produced by different species reviewed, laccase produced by Trametes versicolor (white rot) was found to be the most effective in AFB1 degradation. [5] Thus, our team proposes to heterogeneously express this tvLac in probiotic E. coli and to see if it can be a plausible measurement to degrade AFB1 in food and act as a food preservative for Aspergillus sp. infection.
References
[1] Sumathi T, Viswanath B, Sri Lakshmi A, SaiGopal DV. Production of Laccase by Cochliobolus sp. Isolated from Plastic Dumped Soils and Their Ability to Degrade Low Molecular Weight PVC. Biochem Res Int. 2016;2016:9519527. doi: 10.1155/2016/9519527. Epub 2016 May 12. PMID: 27293894; PMCID: PMC4880699.
[2] Organization WH. aflatoxin. Manuf Comput Solut. 2000;6(8):20-3
[3] 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
[4] Alberts, J. F., Gelderblom, W. C. A., Botha, A., & van Zyl, W. H. (2009). Degradation of aflatoxin B1 by fungal laccase enzymes. International Journal of Food Microbiology, 135(1), 47–52. https://doi.org/10.1016/j.ijfoodmicro.2009.07.022
[5] 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
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 78
Illegal PstI site found at 1188
Illegal PstI site found at 1231 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 78
Illegal PstI site found at 1188
Illegal PstI site found at 1231 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1586
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 78
Illegal PstI site found at 1188
Illegal PstI site found at 1231 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 78
Illegal PstI site found at 1188
Illegal PstI site found at 1231
Illegal AgeI site found at 170
Illegal AgeI site found at 983
Illegal AgeI site found at 1413 - 1000COMPATIBLE WITH RFC[1000]
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