Difference between revisions of "Part:BBa K5136228"

 
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<partinfo>BBa_K5136228 short</partinfo>
 
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===Biology===
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===GST tag===
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Glutathione-S-transferase (GST) tag is a peptide tag derived from <i>Schistosoma japonicum</i>. GST tag has a large relative molecular mass of about 26 KDa and is often inserted at the N-terminus of target proteins. It facilitates the separation of target proteins from cell extracts by its affinity for glutathione. In addition, most of these fusion proteins are stable and water-soluble (1).
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===MT2A===
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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 proteins 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 is a metallothioneins (MT) found in <i>Homo sapiens</i>. 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> (2).
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===Usage and Design===
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We decided to use MTs to treat wastewater. In order to increase the stability of MTs, we added a GST tag to its N-terminal. Thus, this composite part BBa_K5136228 was constructed to express fused protein GST-linker-MT2A.
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==Characterization==
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===Agarose gel electrophoresis (AGE)===
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The composite part (BBa_K5136228) 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 (2560 bp) can be observed at the position between 2000 bp and 3000 bp (Figure 1).
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<center><html><img src="https://static.igem.wiki/teams/5136/part/kyh/2281.png"width="200px"></html></center>
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<center><b>Figure 1 Colony PCR of BBa_K5136230_pSB1C3 in <i>E. coli</i> DH10β</b></center>
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===Reference===
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1.D. B. Smith et al., Mr 26,000 Antigen of Schistosoma Japonicum Recognized by Resistant WEHI 129/J Mice is a Parasite Glutathione S-Transferase. Proc. Natl. Acad. Sci. U. S. A. 83, 8703-8707 (1986).
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<br>2. 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

Latest revision as of 07:24, 2 October 2024


I0500-B0034-gst-linker-mt2a-B0015

Biology

GST tag

Glutathione-S-transferase (GST) tag is a peptide tag derived from Schistosoma japonicum. GST tag has a large relative molecular mass of about 26 KDa and is often inserted at the N-terminus of target proteins. It facilitates the separation of target proteins from cell extracts by its affinity for glutathione. In addition, most of these fusion proteins are stable and water-soluble (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 proteins 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 Cr2O72- is not satisfactory. MT2a is a metallothioneins (MT) found in Homo sapiens. MT2A not only has efficient adsorption capacity for ordinary metal ions, but also exhibits efficient processing capacity for Cr2O72- (2).

Usage and Design

We decided to use MTs to treat wastewater. In order to increase the stability of MTs, we added a GST tag to its N-terminal. Thus, this composite part BBa_K5136228 was constructed to express fused protein GST-linker-MT2A.

Characterization

Agarose gel electrophoresis (AGE)

The composite part (BBa_K5136228) 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 (2560 bp) can be observed at the position between 2000 bp and 3000 bp (Figure 1).

Figure 1 Colony PCR of BBa_K5136230_pSB1C3 in E. coli DH10β

Reference

1.D. B. Smith et al., Mr 26,000 Antigen of Schistosoma Japonicum Recognized by Resistant WEHI 129/J Mice is a Parasite Glutathione S-Transferase. Proc. Natl. Acad. Sci. U. S. A. 83, 8703-8707 (1986).
2. 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
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1205
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1144
    Illegal BamHI site found at 1909
    Illegal BamHI site found at 1926
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 979
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 961
    Illegal SapI.rc site found at 1321