Difference between revisions of "Part:BBa K5136229"

 
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<partinfo>BBa_K5136229 short</partinfo>
 
<partinfo>BBa_K5136229 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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===CRS5===
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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 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>. CRS5 is a metallothionein from <i>S. cerevisiae</i>. It is mainly responsible for copper ion homeostasis and detoxification in yeast cells (1).
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===Usage and Design===
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We decided to use MTs to treat wastewaters. In order to increase the stability of MTs, we added a GST tag to its N-terminal. Thus, this composite part BBa_K5136229 was constructed to express fused protein GST-linker-CRS5.
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===Characterization===
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===Agarose gel electrophoresis (AGE)===
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The composite part (BBa_K5136229) constructed was introduced into the backboneplasmid (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 between 2000 bp and 3000 bp (Figure 1).
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<center><html><img src="https://static.igem.wiki/teams/5136/part/kyh/229.png"width="200px"></html></center>
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<center>Figure 1 colony PCR of BBa_K5136229_pSB1C3 in <i>E. coli</i> DH10β</center>
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===Reference===
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1.V. C. Culotta, W. R. Howard, X. F. Liu, CRS5 encodes a metallothionein-like protein in Saccharomyces cerevisiae. J. Biol. Chem. 269, 25295-25302 (1994).
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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).
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 12:29, 1 October 2024


I0500-B0034-gst-linker-crs5-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).

CRS5

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+. CRS5 is a metallothionein from S. cerevisiae. It is mainly responsible for copper ion homeostasis and detoxification in yeast cells (1).

Usage and Design

We decided to use MTs to treat wastewaters. In order to increase the stability of MTs, we added a GST tag to its N-terminal. Thus, this composite part BBa_K5136229 was constructed to express fused protein GST-linker-CRS5.

Characterization

Agarose gel electrophoresis (AGE)

The composite part (BBa_K5136229) constructed was introduced into the backboneplasmid (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 between 2000 bp and 3000 bp (Figure 1).


Figure 1 colony PCR of BBa_K5136229_pSB1C3 in E. coli DH10β


Reference

1.V. C. Culotta, W. R. Howard, X. F. Liu, CRS5 encodes a metallothionein-like protein in Saccharomyces cerevisiae. J. Biol. Chem. 269, 25295-25302 (1994).
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
  • 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