Difference between revisions of "Part:BBa K5136037"
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==Biology== | ==Biology== | ||
===MT3=== | ===MT3=== | ||
| − | The metallothioneins (MTs) are a class of low molecular weight and cysteine-rich metal binding proteins, and each one of them can | + | The metallothioneins (MTs) are a class of low molecular weight and cysteine-rich metal binding proteins, and each one of them can bind to 6-9 heavy metal ions. The MTs are expressed as intracellular protein and are primarily responsible for metal regulation in cells of living organisms. General MTs can widely non-covalently bind divalent heavy metal ions, such as Zn<sup>2+</sup>, Ni<sup>2+</sup>, Pb<sup>2+</sup>, Hg<sup>2+</sup>, Cd<sup>2+</sup>, as well as As<sup>3+</sup>. MT3 has an excellent adsorption effect on ordinary metal ions. (1). |
==Usage and Design== | ==Usage and Design== | ||
| − | To absorb Cd<sup>2+</sup> in | + | To absorb Cd<sup>2+</sup> in wastewater, we introduced MntA membrane protein in <i>E. coli</i>, which translocates external Cd<sup>2+</sup> into the cell. To increase the strain's tolerance to Cd<sup>2+</sup>, we produce intracellular MTs in engineered bacteria, mitigating the cell damage of Cd<sup>2+</sup>. This basic part (BBa_K5136037) which codes the MT3 was constructed and then used for the construction of the composite part (<partinfo>BBa_K5136230</partinfo>) . |
==Characterization== | ==Characterization== | ||
===Agarose gel electrophoresis (AGE)=== | ===Agarose gel electrophoresis (AGE)=== | ||
| − | I0500 promoter was employed to start the expression of MT3 (<partinfo>BBa_K5136037</partinfo>) in <I>E. coli</I> BL21(DE3). The basic part (<partinfo>BBa_K5136037</partinfo>) is a component of the composite part (<partinfo>BBa_K5136230</partinfo>). The composite part (<partinfo>BBa_K5136230</partinfo>) constructed was introduced into the backbone plasmid (pSB1C3) through standard assembly and transformed into <I>E. coli</I> BL21(DE3).The positive clones were selected, and colony PCR and gene sequencing were used to verify that the clones were correct . Target bands (2106 bp) can be observed at the position around 2000bp (Figure 1). | + | I0500 promoter was employed to start the expression of MT3 (<partinfo>BBa_K5136037</partinfo>) in <I>E. coli</I> BL21(DE3). The basic part (<partinfo>BBa_K5136037</partinfo>) is a component of the composite part (<partinfo>BBa_K5136230</partinfo>). The composite part (<partinfo>BBa_K5136230</partinfo>) constructed was introduced into the backbone plasmid (pSB1C3) through standard assembly and transformed into <I>E. coli</I> BL21(DE3).The positive clones were selected, and colony PCR and gene sequencing were used to verify that the clones were correct. Target bands (2106 bp) can be observed at the position around 2000bp (Figure 1). |
<center><html><img src="https://static.igem.wiki/teams/5136/part/kyh/2301.png"width="200px"></html></center> | <center><html><img src="https://static.igem.wiki/teams/5136/part/kyh/2301.png"width="200px"></html></center> | ||
<center><b>Figure 1 Colony PCR of BBa_K5136230_pSB1C3 in <i>E. coli</i> BL21(DE3)</b></center> | <center><b>Figure 1 Colony PCR of BBa_K5136230_pSB1C3 in <i>E. coli</i> BL21(DE3)</b></center> | ||
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==Reference== | ==Reference== | ||
1. A. A. Uçkun, M. Uçkun, S. Akkurt, Efficiency of Escherichia Coli Jm109 and Genetical Engineering Strains (E. Coli MT2, E. Coli MT3) in Cadmium Removal from Aqueous Solutions. Environ. Technol. Innovation 24, 12 (2021). | 1. A. A. Uçkun, M. Uçkun, S. Akkurt, Efficiency of Escherichia Coli Jm109 and Genetical Engineering Strains (E. Coli MT2, E. Coli MT3) in Cadmium Removal from Aqueous Solutions. Environ. Technol. Innovation 24, 12 (2021). | ||
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<!-- Add more about the biology of this part here | <!-- Add more about the biology of this part here | ||
===Usage and Biology=== | ===Usage and Biology=== | ||
Latest revision as of 08:53, 2 October 2024
mt3
Biology
MT3
The metallothioneins (MTs) are a class of low molecular weight and cysteine-rich metal binding proteins, and each one of them can bind to 6-9 heavy metal ions. The MTs are expressed as intracellular protein and are primarily responsible for metal regulation in cells of living organisms. General MTs can widely non-covalently bind divalent heavy metal ions, such as Zn2+, Ni2+, Pb2+, Hg2+, Cd2+, as well as As3+. MT3 has an excellent adsorption effect on ordinary metal ions. (1).
Usage and Design
To absorb Cd2+ in wastewater, we introduced MntA membrane protein in E. coli, which translocates external Cd2+ into the cell. To increase the strain's tolerance to Cd2+, we produce intracellular MTs in engineered bacteria, mitigating the cell damage of Cd2+. This basic part (BBa_K5136037) which codes the MT3 was constructed and then used for the construction of the composite part (BBa_K5136230) .
Characterization
Agarose gel electrophoresis (AGE)
I0500 promoter was employed to start the expression of MT3 (BBa_K5136037) in E. coli BL21(DE3). The basic part (BBa_K5136037) is a component of the composite part (BBa_K5136230). The composite part (BBa_K5136230) constructed was introduced into the backbone plasmid (pSB1C3) through standard assembly and transformed into E. coli BL21(DE3).The positive clones were selected, and colony PCR and gene sequencing were used to verify that the clones were correct. Target bands (2106 bp) can be observed at the position around 2000bp (Figure 1).
Reference
1. A. A. Uçkun, M. Uçkun, S. Akkurt, Efficiency of Escherichia Coli Jm109 and Genetical Engineering Strains (E. Coli MT2, E. Coli MT3) in Cadmium Removal from Aqueous Solutions. Environ. Technol. Innovation 24, 12 (2021).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 481
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 330
Illegal XhoI site found at 1089 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 72
Illegal NgoMIV site found at 405
Illegal AgeI site found at 429 - 1000COMPATIBLE WITH RFC[1000]