Difference between revisions of "Part:BBa K5136229"

Revision as of 13:30, 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 at the position 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

@@ Line 14: / Line 14: @@
 ===Characterization===
 ===Agarose gel electrophoresis (AGE)===
-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).
+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 at the position between 2000 bp and 3000 bp (Figure 1).
 <br>
 <center><html><img src="https://static.igem.wiki/teams/5136/part/kyh/229.png"width="200px"></html></center>
 <br>
 <center>Figure 1 colony PCR of BBa_K5136229_pSB1C3 in <i>E. coli</i> DH10β</center>
 ===Reference===