Difference between revisions of "Part:BBa K2384007"

Revision as of 19:01, 1 November 2017

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.

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