This part, BBa_K2578511, is the Saccharomyces cerevisiae metallothionein synthesizing gene (CUP1). The genomic sequence is referenced from NCBI (Gene ID 856450) [1].

Metallothioneins are small cysteine-rich proteins made of 61-68 amino acids which can be found in a broad range of organisms, including both eukaryotes and prokaryotes. The metallothioneins are expressed as intracellular protein. [2]

Metallothioneins are mainly responsible for metalloregulation in cells of living organisms. The rich cysteine domains in metallothioneins allow the non-covalent binding of trace metals such as cadmium(Cd2+), lead(Pb2+), copper(Cu1+), (Cu2+) and mercury(Hg2+), etc. [3][4]

Studies show that the transcription of CUP1 is only induced by copper, instead of other heavy metals. [5] It also shows that the copper-binding property of CUP1 contributes to the metal detoxification in Saccharomyces cerevisiae and leads to its high tolerance in copper-contaminated environment [6]. Early studies have also demonstrated that the CUP1 gene can be efficiently expressed in which made it a promising target of our study. [7]

The part is the gene of interest of the composite part BBa_K2578510. However, we failed to submit it to the iGEM community as IDT failed to synthesise the plasmid due to an insertion mutation. Therefore, there is an incompletion in the subproject on the CUP1 gene.

BBa_K2578611

The part BBa_K2578611 is the basic part of the Elsholtzia haichowensis metallothionein 1 synthesizing gene (EhMT1). Elsholtzia haichowensis is a plant belongs to the family Labiatae. It is an indicator plant of Copper mines and widely distributed in Copper-mining wastes and Copper-contaminated soils along the middle and lower streams of Yangtze River, China. [8]

Metallothioneins are small cysteine-rich proteins made of 61-68 amino acids which can be found in a broad range of organisms, including both eukaryotes and prokaryotes. The metallothioneins are expressed as intracellular protein. [9]

Metallothioneins are mainly responsible for metalloregulation in cells of living organisms. The rich Cysteine domains in metallothioneins allow the non-covalent binding of trace metals such as cadmium(Cd2+), lead(Pb2+), copper(Cu1+), (Cu2+) and mercury(Hg2+), etc. [10][11]

The genomic sequence of the EhMT1 gene is obtained by reversing the mRNA sequence of the gene referenced from an academic paper by Yan Xia et. al.[12] into DNA sequence.

The basic part is ordered and synthesized by IDT in the form of composite part BBa_K2578610. The ordered plasmid is transformed in E.coli(TOP10) and are used to perform a copper absorption test.The plasmid is also cloned into pSB1C3 backbone for part submission.

BBa_K2578711

This is the basic part of the Mus musculus metallothionein 1 synthesizing gene (Mt1). The genomic sequence of the Mt1 gene is referenced from NCBI (Gene ID 17748) [13].

Metallothioneins are small cysteine-rich proteins made of 61-68 amino acids which can be found in a broad range of organisms, including both eukaryotes and prokaryotes. The metallothioneins are expressed as intracellular protein. [2]

Metallothioneins are mainly responsible for metalloregulation in cells of living organisms. The rich Cysteine domains in metallothioneins allow the non-covalent binding of trace metals such as cadmium(Cd2+), lead(Pb2+), copper(Cu1+), (Cu2+) and mercury(Hg2+), etc. [3][4]

The Mt1 gene’s strong binding affinity with heavy metals including copper, cadmium, mercury and zinc [14], has been proven. It was also shown that Mt1 serves functions as metal homeostasis, metabolism as well as protect cells from oxidative stress of zinc ion in mouse liver [14][15]. Therefore, it is highly possible that MT1 chelates heavy metal ions.

The part is the gene of interest of the composite part BBa_K2578710.However, we failed to submit the part to the iGEM community as there are multiple restriction enzyme site and introns in the gene of interest. Therefore, there is an incompletion in the subproject on the Mt1 gene.

BBa_K2578721

This is the basic part of the Mus musculus synthesising gene (Mt2). The genomic sequence of the Mt2 gene is referenced from NCBI (Gene ID 17750)[16].

Metallothioneins are small cysteine-rich proteins made of 61-68 amino acids which can be found in a broad range of organisms, including both eukaryotes and prokaryotes. The metallothioneins are expressed as intracellular protein. [2]

Metallothioneins are mainly responsible for metalloregulation in cells of living organisms. The rich Cysteine domains in metallothioneins allow the non-covalent binding of trace metals such as cadmium(Cd2+), lead(Pb2+), copper(Cu1+), (Cu2+) and mercury(Hg2+), etc. [3][4]

The Mt2 gene has similar ability to Mt1 gene as they are equivalent proteins. They have strong binding affinity to heavy metals[17] and serve severe function like metal homeostasis and metabolism and protect cell from oxidative stress of zinc ion in mouse liver[17][18]. But when compared to the metal binding ability to metal ions, results shown that Mt2 has better binding ability of Zinc ions (Zn2+) while Mt1 gene has better binding ability of copper ions(Cu1+) and cadmium ions (Cd2+).[19]

The part is the gene of interest of the composite part BBa_K2578720.However, we failed to submit the part to the iGEM community as there are introns in the gene of interest. Therefore, there is an incompletion of the Mt2 subproject.

BBa_K2578811

This is the basic part of the Homo sapiens metallothionein 1 synthesizing gene (MT1A). The genomic sequence of the MT1A gene is referenced from NCBI (Gene ID 4489)[20].

Metallothioneins are small cysteine-rich proteins made of 61-68 amino acids which can be found in a broad range of organisms, including both eukaryotes and prokaryotes. The metallothioneins are expressed as intracellular protein. [2]

Metallothioneins are mainly responsible for metalloregulation in cells of living organisms. The rich Cysteine domains in metallothioneins allow the non-covalent binding of trace metals such as cadmium(Cd2+), lead(Pb2+), copper(Cu1+), (Cu2+) and mercury(Hg2+), etc. [3][4]

MT mammalian family contains four members, MT1 - MT4. Each member shares similar properties but slightly different affinity in binding different metals. Among most studies, it was found that cadmium, lead and mercury, which are some most toxic metal ions displayed the highest binding affinity with MTs. [21] These results show that MTs are capable in the use of bioremediation of toxic heavy metals.

As what has been mentioned above, this part is the protein coding sequence of human MT1A protein. This isoform of MT1 in human has been well reported to bind with copper and their expressions will be regulated directly by copper concentration in the cells. [22][23]

MT1A is involved in human metalloregulation (Including copper, cadmium etc.). In our studies, the human MT1A gene is being expressed in e. coli to enhance the ability of removing copper of their surrounding medium.

The basic part is ordered and synthesized by IDT in the form of composite part BBa_K2578810. The ordered plasmid is transformed in E.coli(TOP10) by heat shock. Transformed E.coli are used to perform a copper absorption test.

The plasmid is also cloned into pSB1C3 backbone for part submission.

BBa_K2578821

This is the basic part of the Homo sapiens metallothionein 2 synthesising gene (MT2A). The genomic sequence of the MT2A gene is referenced from NCBI (NCBI Gene ID 4502)[24].

Metallothioneins are small cysteine-rich proteins made of 61-68 amino acids which can be found in a broad range of organisms, including both eukaryotes and prokaryotes. The metallothioneins are expressed as intracellular protein. [2]

Metallothioneins are mainly responsible for metalloregulation in cells of living organisms. The rich cysteine domains in metallothioneins allow the non-covalent binding of trace metals such as cadmium(Cd2+), lead(Pb2+), copper(Cu1+), (Cu2+) and mercury(Hg2+), etc. [3][4]

MT2A has similar ability with MT2A gene. They are also involved in zinc homeostasis. But MT2A gene is found in liver, where MT1A was found in human kidney. The zinc binding ability of both gene are similar.[25]

We did not order the MT2A gene because we do not have enough time and we are out of budget of the 20kb range. Nonetheless, there may be a risk of cross species expression.

Reference

[1] https://www.ncbi.nlm.nih.gov/gene/856450

[2] N Thirumoorthy, KT Manisenthil Kumar, A Shyam Sundar, L Panayappan, Malay Chatterjee (2007). Metallothionein: An overview. World journal of gastroenterology, ISSN 1007-9327

[3] Almaguer-Cantú V1, Morales-Ramos LH, Balderas-Rentería I. (2011) Biosorption of lead (II) and cadmium (II) using Escherichia coli genetically engineered with mice metallothionein I. Water Sci Technol. 2011;63(8):1607-13.

[4] Dziegiel, P., Pula, B., Kobierzycki, C., Stasiolek, M., Podhorska-Okolow, M. (2016), Metallothioneins in Normal and Cancer Cells, Springer International Publishing, DOI: 10.1007/978-3-319-27472-0

[5] Brenes-Pomales, A., Lindegren, G., & Lindegren, C. C. (1955). Gene Control of Copper-Sensitivity in Saccharomyces. Nature, 176(4487), 841-842. doi:10.1038/176841a0

[6] Adamo, G., Brocca, S., Passolunghi, S., Salvato, B., & Lotti, M. (2012). Laboratory evolution of copper tolerant yeast strains. Microbial Cell Factories, 11(1), 1. doi:10.1186/1475-2859-11-1

[7] Berka, T., Shatzman, A., Zimmerman, J., Strickler, J., & Rosenberg, M. (1988). Efficient expression of the yeast metallothionein gene in Escherichia coli. Journal of Bacteriology, 170(1), 21-26. doi:10.1128/jb.170.1.21-26.1988

[8] Tang et al. 1999; Lou et al. 2004; Qian et al. 2005 Adaptive Copper Tolerance in Elsholtzia haichowensis Involves Production of Cu-induced Thiol Peptides
https://www.researchgate.net/publication/225518069/download

[9] N Thirumoorthy, KT Manisenthil Kumar, A Shyam Sundar, L Panayappan, Malay Chatterjee (2007). Metallothionein: An overview. World journal of gastroenterology, ISSN 1007-9327

[10] Almaguer-Cantú V1, Morales-Ramos LH, Balderas-Rentería I. (2011) Biosorption of lead (II) and cadmium (II) using Escherichia coli genetically engineered with mice metallothionein I. Water Sci Technol. 2011;63(8):1607-13.

[11] Dziegiel, P., Pula, B., Kobierzycki, C., Stasiolek, M., Podhorska-Okolow, M. (2016), Metallothioneins in Normal and Cancer Cells, Springer International Publishing, DOI: 10.1007/978-3-319-27472-0

[12] Xia, Y., Lv, Y., Yuan, Y. et al. Acta Physiol Plant (2012) 34: 1819. https://doi.org/10.1007/s11738-012-0980-4

[13] https://www.ncbi.nlm.nih.gov/gene/17748

[14] Almaguer-Cantú, V., Morales-Ramos, L. H., & Balderas-Rentería, I. (2011). Biosorption of lead (II) and cadmium (II) using Escherichia coli genetically engineered with mice metallothionein I. Water Science and Technology, 63(8), 1607-1613. doi:10.2166/wst.2011.225

[15] Jiang, Y., & Kang, Y. J. (2004). Metallothionein Gene Therapy for Chemical-Induced Liver Fibrosis in Mice. Molecular Therapy, 10(6), 1130-1139. doi:10.1016/j.ymthe.2004.08.011

[16] https://www.ncbi.nlm.nih.gov/gene/17750

[17] Almaguer-Cantú, V., Morales-Ramos, L. H., & Balderas-Rentería, I. (2011). Biosorption of lead (II) and cadmium (II) using Escherichia coli genetically engineered with mice metallothionein I. Water Science and Technology, 63(8), 1607-1613. doi:10.2166/wst.2011.225

[18] Jiang, Y., & Kang, Y. J. (2004). Metallothionein Gene Therapy for Chemical-Induced Liver Fibrosis in Mice. Molecular Therapy, 10(6), 1130-1139. doi:10.1016/j.ymthe.2004.08.011

[19] Mammalian MT1 and MT2 metallothioneins differ in their metal binding abilities. Artells E1, Palacios Ò, Capdevila M, Atrian S.

[20]https://www.ncbi.nlm.nih.gov/gene/4489

[21] Dziegiel, P., Pula, B., Kobierzycki, C., Stasiolek, M., Podhorska-Okolow, M. (2016), Metallothioneins in Normal and Cancer Cells, Springer International Publishing, DOI: 10.1007/978-3-319-27472-0

[22] M Karin and R I Richards (1984). The human metallothionein gene family: structure and expression. Environ Health Perspect. 1984 Mar; 54: 111–115.

[23] Scheller JS, Irvine GW, Wong DL, Hartwig A, Stillman MJ.(2017) Stepwise copper(i) binding to metallothionein: a mixed cooperative and non-cooperative mechanism for all 20 copper ions. Metallomics. 2017 May 24;9(5):447-462. doi: 10.1039/c7mt00041c.

[24]https://www.ncbi.nlm.nih.gov/gene/4502

[25] Zinc binds non-cooperatively to human liver metallothionein 2a at physiological pH. Jayawardena DP1, Heinemann IU2, Stillman MJ3

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