Team:Cornell/project/wetlab/metallothionein

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Wet Lab

Construct Design


Metallothionein is a low molecular weight, cysteine-rich family of proteins that provides protection against metal toxicity to a wide range of taxonomic groups. The thiols clustered at the core of the protein tightly chelates the metal ions by forming strong coordinate bonds[1]. Cloned and overexpressed metallothioneins can sequester metal ions transported by a metal transport system, but simultaneously inhibit growth in microorganisms. A number of metallothioneins expressed in E. coli had problems with stability, leading to studies conducted with stabilizing systems [2]. The system we ultimately cloned into a BioBrick was GST-YMT, a gene that codes for Saccharomyces cerevisiae metallothionein with a glutathione S-transferase carboxy-terminal fusion system. In previous research, this gene had proven to have higher stability and was approximated to be 25% of the total protein in transformed E. coli [3]. Our first metallothionein BioBrick (BBa_K1460001) consists of GST-YMT synthesized with a T7 promoter in pSC1C3. This is part of an inducible system consisting of an arabinose activating pathway in which the araBAD promoter turns on the highly active T7 polymerase that in turns reads the metallothionein gene. Our second metallothionein BioBrick (BBa_K1460002) consists of GST-YMT without the T7 promoter for other promoters to clone into the backbone and better interweave metallothionein’s functions with novel systems.

Results


Because successfully expressed metallothionein inhibits growth in microorganisms, we can use growth tests as a tool for determining successful expression of our metallothionein constructs. We transformed BBa_K1460001 into E.coli BL21-AI and grew it and unmodified BL21-AI in LB+.1% L-Arabinose for 24 hours in an incubated plate reader at 37 degrees Celsius. Plotted below is the average OD for three biological triplicates of BL21 and BL21 BBa_K1460001. Plotted OD is corrected for OD of media.
This graph displays statistically significant (student’s two-tailed t-test, p<.05) differences between unengineered BL21 and BL21 engineered to express metallothionein. This data suggests that GST-YMT is being successfully expressed in this engineered strain. Additionally, when working with these cultures for subsequent metal sequestration tests, final culture ODs were consistently observed to be less than that of wild type cells.

References


  1. Ref 1
  2. Ref 2
  3. Ref 3