Team:Cornell/project/wetlab/metallothionein

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<h1 style="margin-top: 0px;">Construct Design</h1>
<h1 style="margin-top: 0px;">Construct Design</h1>
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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<sup>[1]</sup>. 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 <i>E. coli</i> had problems with stability, leading to studies conducted with stabilizing systems <sup>[2]</sup>. The system we ultimately cloned into a BioBrick was GST-YMT, a gene that codes for <i>Saccharomyces cerevisiae</i> metallothionein with a glutathione <i>S</i>-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 <i>E. coli</i> <sup>[3]</sup>.
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Our first metallothionein BioBrick <a href="http://parts.igem.org/Part:BBa_K1460001"> (BBa_K1460001)</a> 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 <a href="http://parts.igem.org/Part:BBa_K1460002">(BBa_K1460002)</a> 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.
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Revision as of 15:13, 11 October 2014

Cornell iGEM

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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



References


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