Difference between revisions of "Part:BBa K5136039"

Revision as of 17:57, 1 October 2024

mnta

Biology

MntA

MntA is a membrane protein from Lactobacillus plantarum which transports Cd²⁺ and Mn²⁺ into the cell. It is an effective Mn²⁺and Cd²⁺ uptake system that exhibits rapid accumulation of the two metal ions. It endows cells with sensitivity to Cd²⁺ and energy-dependent Cd²⁺ uptake activity. MntA falls into the family of P-type adenosine triphosphatases (ATPase). Its transport efficiency is affected by some factors such as other ion of high concentration and temperature (1,2).

Usage and Design

After deinking, heavy metal ions in the ink will be released into the wastewater. Thus, this wastewater needs harmless treatmentremove before discharging. Hence, we use metallothioneins (MTs) to treat wastewater to remove heavy metals. However，due to the poor competitiveness of Cd²⁺ in mixed metal ions, engineered bacteria expressing only MTs lack enough affinity and selectivity for Cd²⁺ to reduce Cd²⁺ in the wastewater to the levels required by strict government regulations.This basic part (BBa_K5136039) which codes MntA was constructed and then used for the construction of the composite part (BBa_K5136225) .

Characterization

Agarose gel electrophoresis (AGE)

I0500 promoter was employed to start the expression of MntA (BBa_K5136039) in E. coli DH10β. The basic part (BBa_K5136041) is a component of the composite part (BBa_K5136225). The composite part (BBa_K5136225) constructed was introduced into the backbone plasmid (pSB1C3) through standard assembly and transformed into E. coli DH10β. The positive clones were selected, and colony PCR and gene sequencing were used to verify that the clones were correct . Target bands (2584 bp) can be observed at the position between 2000 bp and 3000 bp (Figure 1).

Figure 1 Colony PCR of BBa_K5136225_pSB3K3 in E. coli BL21(DE3)

Reference

1. Z. Hao, S. Chen, D. B. Wilson, Cloning, Expression, and Characterization of Cadmium and Manganese Uptake Genes from Lactobacillus Plantarum. Appl Environ Microbiol 65, 4746-4752 (1999).
2. S. K. Kim, B. S. Lee, D. B. Wilson, E. K. Kim, Selective Cadmium Accumulation Using Recombinant Escherichia Coli. J Biosci Bioeng 99, 109-114 (2005).

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 128
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 436
Illegal AgeI site found at 1028
Illegal AgeI site found at 1537
1000
COMPATIBLE WITH RFC[1000]

@@ Line 5: / Line 5: @@
 ===Biology===
 ===MntA===
-MntA is a membrane protein from <i>Lactobacillus plantarumATCC 14917</i> which transports Cd<sup>2+</sup> and Mn<sup>2+</sup> into the cell. It is an effective Mn<sup>2+</sup>and Cd<sup>2+</sup> uptake system that exhibits rapid accumulation of the two metal ions. It endows cells with sensitivity to Cd<sup>2+</sup> and energy-dependent Cd<sup>2+</sup> uptake activity. MntA falls into the family of P-type adenosine triphosphatases(ATPase). Its transport efficiency is affected by some factors such as other ion of high concentration and temperature (1,2).
+MntA is a membrane protein from <i>Lactobacillus plantarum</i> which transports Cd<sup>2+</sup> and Mn<sup>2+</sup> into the cell. It is an effective Mn<sup>2+</sup>and Cd<sup>2+</sup> uptake system that exhibits rapid accumulation of the two metal ions. It endows cells with sensitivity to Cd<sup>2+</sup> and energy-dependent Cd<sup>2+</sup> uptake activity. MntA falls into the family of P-type adenosine triphosphatases (ATPase). Its transport efficiency is affected by some factors such as other ion of high concentration and temperature (1,2).
 ===Usage and Design===
@@ Line 12: / Line 12: @@
 ===Characterization===
 ===Agarose gel electrophoresis (AGE)===
-I0500 promoter was employed to start the expression of MntA (BBa_K5136039) in <I>E. coli</I> DH10β. The basic part (BBa_K5136041) is a component of the composite part (<partinfo>BBa_K5136225</partinfo>). The composite part  (<partinfo>BBa_K5136225</partinfo>) constructed was introduced into the backbone plasmid (pSB1C3) through standard assembly and transformed into <I>E. coli</I> DH10β. The positive clones were selected, and colony PCR and gene sequencing were used to verify that the clones were correct . Target bands (2584 bp) can be observed at the position between 2000bp and 3000bp (Figure 1).
+I0500 promoter was employed to start the expression of MntA (BBa_K5136039) in <I>E. coli</I> DH10β. The basic part (BBa_K5136041) is a component of the composite part (<partinfo>BBa_K5136225</partinfo>). The composite part  (<partinfo>BBa_K5136225</partinfo>) constructed was introduced into the backbone plasmid (pSB1C3) through standard assembly and transformed into <I>E. coli</I> DH10β. The positive clones were selected, and colony PCR and gene sequencing were used to verify that the clones were correct . Target bands (2584 bp) can be observed at the position between 2000 bp and 3000 bp (Figure 1).
 <center><html><img src="https://static.igem.wiki/teams/5136/part/kyh/2251.png"width="200px"></html></center>
 <center><b>Figure 1 Colony PCR of BBa_K5136225_pSB3K3 in <i>E. coli</i> BL21(DE3)</b></center>