Team:York/Constructs

Constructs

Our constructs are shown diagramatically below:

pYodA

Name: pYodA (ZinTp)
Organism: E.coli
Normal Function: pYodA is a cadmium-induced promoter that activates the yodA(zinT) gene, leading to the production of ZinT metal-binding protein.
Our aim: We are using pYodA in an alternative way; to regulate the expression of the CysP gene (sulfate transporter) and the NRAMP gene (Cadmium transporter). Due to using pYodA, the expression of these two genes will be regulated by the concentration of Cadmium in the extracellular environment. The over-production of Cysteine will only occur when the concentration of Cadmium reaches the sensitivity threshold of pYodA. Due to cysteine production being both energetically demanding and toxic at high concentrations, we do not want these genes to be expressed constitutively.
Characterisation: In order to characterise pYodA, we will couple it with a GFP gene. This will allow us to calculate the sensitivity threshold of pYodA and measure the amount of cadmium that can be chelated at various concentrations.
Literature:

1. http://mic.sgmjournals.org/content/148/12/3801.long
2. http://www.uniprot.org/uniprot/F4VWH2
3. http://sbkb.org/uid/F4VWH2/uniprot#structures
4. http://www.jbc.org/content/278/44/43728
5. http://biocyc.org/ECOLI/NEW-IMAGE?type=GENE&object=G7061

NRAMP

Gene: MntH
Organism: Ecoli
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.

CysE*

Original Gene: CysE
Mutant Gene: CysE*
Organism: E.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 AUC codon to AUG. 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/