DNA

Part:BBa_K2384007

Designed by: Size Cai   Group: iGEM17_FAFU-CHINA   (2017-10-23)


GST-CRS5

Metallothionein is a low molecular weight, cysteine-rich protein family that provides metal toxicity for a wide range of taxonomic groups. The thiols that accumulate at the core of the protein tightly chelate the metal ions by forming a strong coordination bond. Cloned and overexpressed metallothionein can chelate the metal ions transported by the metal transport system while inhibiting the growth of microorganisms. Many of the metallothioneins expressed in E. coli have stability problems that lead to studies with stable systems. The system that we finally cloned into BioBrick is MBP, which is a maltose binding protein, with a glutathione S-transferase carboxyl terminal fusion system (GST-MT). In previous studies, the fusion protein has been shown to have higher stability and is approximately about 25% by mass of the total protein expressed for transformation of E. coli.Our first metallothionein BioBrick consists of GST-MT synthesized in pSC1C3 with the T7 promoter. This is part of the induction system consisting of arabinose activation pathway, in which the araBAD promoter initiates a highly active T7 polymerase, which in turn reads the metallothionein gene. Our second metallothionein, BioBrick, is composed of GST-MT without the T7 promoter, cloned into the plasmid backbone of the xylose-containing promoter, and is better interwoven with the new system for the function of metallothionein. </br></br> This part is originally derived from 2014 Cornell.(BBa_K1460001)

The optimization of this sequence is more suitable for the expression of Bacillus megaterium


Usage and Biology

Optimization Report

1.Codon Used Adjustment The best value is 1 for sequence optimization.

Figure 1:After Codon Adjustment


2.GC Content: The comparison of GC content between original sequence and optimized sequence

Figure 2:After Optimization
Figure 3:The expression of GST-CRS5 detected using SDS-PAGE
Figure 4:Plasmid digested by EcoRI and HindIII

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 673
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 85


[edit]
Categories
Parameters
None