Team:York/Cake

NRAMP

Gene: mntH
Organism: Escherichia Coli
Protein: NRAMP
Function: NRAMP is a membrane divalent metal-ion transporter. In addition to iron and manganese, it also transports Cd ions into the cell.
Aim: We want to use the endogenous transporter NRAMP to take up the cadmium from the environment. The cadmium will then activate pYoda and set off the phytochelatin synthesis process.
Expression: We can run assays measuring the concentration of cadmium in the environment. If NRAMP is active, the concentration of cadmium should decrease.

CysP

Gene: cysP (AKA YlnA)
Organism: Bacillus Subtilis
Protein: CysP
Function: This gene encodes for CysP, a sulfate/thiosulfate ABC transporter found in the periplasmic space.
Aim: We want to overexpress this gene in E.coli. This would lead to increased sulfate uptake, which could be used to produce cysteine and eventually phytochelatins that would bind cadmium.
Expression: We could run assays measuring the concentration of sulfate in the environment containing our bacteria.

CysE*

Original Gene: cysE
Mutant Gene: cysE*
Organism: Escherichia Coli
Protein: CysE(SAT)
Normal function: To catalyse the acetylation of L-serine (the first step in cysteine biosynthesis)
Function: Serine acetyltransferase (CysE) carries out the first step in cysteine biosynthesis; it catalyses the acetylation of L-serine which generates O-acetyl-L-serine. Cysteine itself strongly inhibits the activity of serine acetyltransferase by binding to the serine-binding site. This inhibition depends on the protein's carboxy terminus, and has been localized to Met-256 specifically. Due to cysteine production being both energetically demanding and toxic at high concentrations, the cell does not want to produce cysteine constitutively.
Aim: To over-produce cysteine.To fulfill this aim, we need to remove negative feedback from cysteine biosynthesis. To do this, we are using a mutant CysE gene (CysE*). Our mutant CysE gene will produce a protein that has a single amino acid substitution: Met-256 will be replaced by Ile by changing the corresponding AUG codon to AUC. This single amino-acid substitution will alter the three dimensional shape of the serine-binding site in our acetyltransferase. Changing the shape of the serine-binding site prevents cysteine from binding to it. Thus, our acetyltransferase will not be inhibited by cysteine. As a result, we will be able to over-produce cysteine in our cell.
Method: We had the cysE* gene synthesised to produce the mutant CysE* protein.
Literature:

1. http://aem.asm.org/content/66/10/4497.full
2. Denk D., Bock A. J. Gen. Microbiol, 1987

Gsh1*

Original Gene: gsh1/gshA
Organism: Saccharomyces cerevisiae
Protein: Glutamate-Cysteine Ligase (previously known as gamma-glutamylcysteine synthetase)
Function: Gamma glutamylcysteine synthetase catalyzes the first step in glutathione (GSH) biosynthesis;
L-glutamate + L-cysteine + ATP -> gamma-glutamyl cysteine + ADP + Pi
Expression is induced by oxidants, cadmium, and mercury. Protein abundance increases in response to DNA replication stress.
Aim: We can use the overproduced cysteine to make gamma-glutamyl cysteine, which is the monomer that forms phytochelatins (n=10-20). In order to do this, we need glutamate-cysteine ligase to catalyse the reaction. We can overexpress GSH1* using the pYodA promoter.
Literature:

1. http://biocyc.org/YEAST/NEW-IMAGE?type=GENE-IN-MAP-IN-PWY&object=YJL101C

spPCS

Gene: spPCS
Organism: Schizosaccharomyces pombe
Function: The phytochelatin synthase in S. pombe uses glutathione with a blocked thiol group to synthesise phytochelatins.
Aim: Overexpression of this gene, together with Gsh1*, in E.coli has been shown to increase phytochelatin production and lead to a 7.5-times-higher Cd accumulation. We want to use this gene to make our bacteria more efficient in taking up cadmium.
Literature

1. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2075016/