Difference between revisions of "Part:BBa K5136038"

Revision as of 21:53, 1 October 2024

mt2a

Biology

MT2A

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⁺, but their effectiveness in treating Cr₂O₇^2- is not satisfactory. MT2A and MT3 are metallothioneins(MTs) found in Homo sapiens. MT2A not only has efficient adsorption capacity for ordinary metal ions, but also exhibits efficient processing capacity for Cr₂O₇^2-.

Usage and Design

To absorb Cd²⁺ in waste water, 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 MT2A 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 MT2A (BBa_K5136038) in E. coli BL21(DE3). The basic part (BBa_K5136038) 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 2000 bp (Figure 1).

Figure 1 Colony PCR of BBa_K5136230_pSB1C3 in E. coli BL21(DE3)

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

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 3
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

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 ==Biology==
-===MT2a===
-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 a excellent adsorption effect on ordinary metal ions. (1).
+===MT2A===
+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>, but their effectiveness in treating Cr<sub>2</sub>O<sub>7</sub><sup>2-</sup> is not satisfactory. MT2A and MT3 are metallothioneins(MTs) found in Homo sapiens. MT2A not only has efficient adsorption capacity for ordinary metal ions, but also exhibits efficient processing capacity for Cr<sub>2</sub>O<sub>7</sub><sup>2-</sup>.
 ==Usage and Design==
-To absorb Cd<sup>2+</sup> in waste water, 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 MT2a was constructed and then used for the construction of the composite part (<partinfo>BBa_K5136230</partinfo>) .
+To absorb Cd<sup>2+</sup> in waste water, 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 (<partinfo>BBa_K5136037</partinfo>) which codes the MT2A was constructed and then used for the construction of the composite part (<partinfo>BBa_K5136230</partinfo>) .
 ==Characterization==
 ===Agarose gel electrophoresis (AGE)===
-I0500 promoter was employed to start the expression of mt2a (BBa_K5136038) in <I>E. coli</I> BL21(DE3). The basic part (BBa_K5136038) 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 MT2A (<partinfo>BBa_K5136038</partinfo>) in <I>E. coli</I> BL21(DE3). The basic part (<partinfo>BBa_K5136038</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 2000 bp (Figure 1).
 <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>