Composite Parts

BBa_K2578410

The part BBa_K2578410[1] is the composite part of the Mycobacterium tuberculosis metallothionein synthesizing gene (MymT). The genomic sequence of the MymT gene is reference from Team Oxford in 2016. The MymT gene, registered by team Oxford in 2016 as BBa_K1980002, is designed by our teammates to be composite part BBa_K2578410 by adding consecutive promoter J23100[2], strong RBS B0032[3] and double terminator B0015[4].

Metallothioneins (MTs) 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 MTs are expressed as intracellular protein. [5]

MTs are mainly responsible for metalloregulation in cells of living organisms. The rich Cys domains in MTs allow the non-covalent binding of trace metals such as cadmium, lead, copper and mercury, etc. [6][7]

The plasmid containing composite part BBa_K2578410 is ordered and synthesized by IDT. The part cannot be submitted to the IGEM community due to lacking of time and resources.

BBa_K2578510

The part BBa_K2578510 is the composite part of the Saccharomyces cerevisiae metallothionein synthesizing gene (CUP1). The genomic sequence of the CUP1 gene is referenced from NCBI (Gene ID 856450)[8]. The CUP1 gene, registered as BBa_K2578511, is designed to be the composite part BBa_K2578510 by adding consecutive promoter J23100[2], strong RBS B0032[3] and double terminator B0015[4].

Metallothioneins (MTs) 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 MTs are expressed as intracellular protein. [5]

MTs are mainly responsible for metalloregulation in cells of living organisms. The rich Cys domains in MTs allow the non-covalent binding of trace metals such as cadmium, lead, copper and mercury, etc. [6][7]

Studies showed that the transcription of CUP1 is only induced by copper, instead of other heavy metals. [9] It was also shown that copper-binding property of yeast MT1 contributes to the metal detoxification in Saccharomyces cerevisiae and leads to its high tolerance in copper-contaminated environment [10]. Early studies have also demonstrated that CUP1 gene can be efficiently expressed in which made it a promising target of our study. [11]

The designed part is synthesized by IDT, but the order failed as IDT failed to synthesis the plasmid and insertion mutation occurs. Therefore, there is an incompletion in the subproject on the CUP1 gene.

BBa_K2578610

The part BBa_K2578610 is the composite part of the Elsholtzia haichowensis metallothionein 1 synthesizing gene (EhMT1).

Elsholtzia haichowensis is plant belonging to the family Labiatae. It is an indicator plant of copper mines and widely distributed in Cu-mining wastes and Cu-contaminated soils along the middle and lower streams of Yangtze River, China. [12]

Metallothioneins (MTs) 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 MTs are expressed as intracellular protein. [5]

MTs are mainly responsible for metalloregulation in cells of living organisms. The rich Cys domains in MTs allow the non-covalent binding of trace metals such as cadmium, lead, copper and mercury, etc. [6][7]

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.[13] into DNA sequence. The EhMT1 gene, registered as BBa_K2578611, is designed to be the composite part BBa_K2578610 by adding consecutive promoter J23100[2], Strong RBS B0032[3] and double terminator B0015[4].

The plasmid containing composite part BBa_K2578610 is ordered and synthesized by IDT. 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_K2578710

The part BBa_K2578710 is the composite part of the Mus musculus metallothionein 1 synthesizing gene (Mt1). The genomic sequence of the Mt1 gene is referenced from NCBI (Gene ID 17748)[14]. The Mt1 gene, registered as BBa_K2578711, is designed to be the composite part BBa_K2578710 by adding consecutive promoter J23100[2], strong RBS B0032[3] and double terminator B0015[4].

Metallothioneins (MTs) 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 MTs are expressed as intracellular protein. [5]

MTs are mainly responsible for metalloregulation in cells of living organisms. The rich Cys domains in MTs allow the non-covalent binding of trace metals such as cadmium, lead, copper and mercury, etc. [6][7]

The Mt1 gene has a strong binding affinity of Mt1 with heavy metals including copper, cadmium, mercury and zinc [15], 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 [15][16]. Therefore, it is highly possible that MT1 chelates heavy metal ions.

The designed part is synthesized by IDT, but the order failed as there are multiple restriction enzyme site and introns in the Mt1 gene BBa_K2578711. Therefore, there is an incompletion in the subproject on the Mt1 gene.

BBa_K2578720

The part BBa_K2578720 is the composite part of the Mus musculus metallothionein 2 synthesising gene(Mt2). The genomic sequence of the Mt2 gene is referenced from NCBI (Gene ID 17750)[17].

The Mt2 gene, registered as BBa_K2578721, is designed to be the composite part BBa_K2578720 by adding consecutive promoter J23100 [2], strong RBS B0032 [3] and double terminator B0015 [4].

Metallothioneins (MTs) 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 MTs are expressed as intracellular protein. [5]

MTs are mainly responsible for metalloregulation in cells of living organisms. The rich Cys domains in MTs allow the non-covalent binding of trace metals such as cadmium, lead, copper and mercury, etc. [6,7]

The Mt2 gene has similar ability to Mt1 gene as they are equivalent proteins. They have strong binding affinity to heavy metals[18] and serve severe function like metal homeostasis and metabolism and protect cell from oxidative stress of zinc ion in mouse liver [18][19]. But when compared to the metal binding ability to metal ions, results shown that Mt2 has better binding ability of Zinc 2+ ions while Mt1 gene has better binding ability of copper 1+ ions and cadmium 2+ ions.[20]

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_K2578810

The part BBa_K2578810 is the composite part of the Homo sapiens metallothionein 1 synthesizing gene (MT1A). The genomic sequence of the MT1A gene is referenced from NCBI (Gene ID 4489)[21]. The MT1A gene, registered as BBa_K2578811, is designed to be the composite part BBa_K2578710 by adding consecutive promoter J23100[2], strong RBS B0032[3] and double terminator B0015[4].

Metallothioneins (MTs) 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 MTs are expressed as intracellular protein. [5]

MTs are mainly responsible for metalloregulation in cells of living organisms. The rich Cys domains in MTs allow the non-covalent binding of trace metals such as cadmium, lead, copper and mercury, etc. [6,7]

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. [22] 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 MT1A has been well reported to bind with copper and their expressions will be regulated directly by copper concentration in the cells. [23][24]

The plasmid containing composite part BBa_K2578810 is ordered and synthesized by IDT. 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_K2578820

The part BBa_K2578820 is the composite part of the Homo sapiens. metallothionein 2 synthesising gene (MT2A). The genomic sequence of the MT2A gene is referenced from NCBI (Gene ID 4502) [25].The MT1A gene, registered as BBa_K2578821, is designed to be the composite part BBa_K2578710 by adding consecutive promoter J23100[2], strong RBS B0032[3] and double terminator B0015[4].

Metallothioneins (MTs) 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 MTs are expressed as intracellular protein. [5]

MTs are mainly responsible for metalloregulation in cells of living organisms. The rich Cys domains in MTs allow the non-covalent binding of trace metals such as cadmium, lead, copper and mercury, etc. [6,7]

MT2A has similar ability with MT1A 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.[26]

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]http://parts.igem.org/Part:BBa_K1980002

[2]http://parts.igem.org/Part:BBa_J23100

[3]http://parts.igem.org/Part:BBa_B0032

[4]http://parts.igem.org/Part:BBa_B0015

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

[6] 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.

[7] 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

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

[9] 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

[10] 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

[11] 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

[12] 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

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

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

[15] 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

[16] 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

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

[18] 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

[19] 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

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

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

[22] 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

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

[24] 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.

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

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