IGEM:cambridge/2008/Notebook/Voltage/Progress

		Personnel	Progress
Research
	Potassium intake
	Preventing K+ efflux
	Bacterial tolerance for high K+ and turgor Osmolites (“inert” sugars)
	Ligand gated channels
	Media
Preliminary wet work
	Extract promoter, RBS and terminator BioBricks from registry Refine protocol for paper-bound DNA extraction Use PCR and transformations to confirm presence of DNA
Internal K+ build-up
	PCR Kdp K+ pump gene from E.coli MG1655 Design and order primers, including BioBrick prefix and suffix
	Put Kdp gene under control of stationary phase promoter (osmY, used by MIT 2006 team) Obtain primer sequences and order, for PCR from BioBricks containing osmY (J45992) Ligate to RBS, Kdp gene and terminators in plasmid
	Transform into wildtype and mutant E.coli strains
	Test Measure internal K+ concentration using flame photometry
Chassis
	Order from Yale Kch- mutant, preventing uncontrolled K+ efflux Kef- mutants, preventing uncontrolled K+ efflux Kdp- mutants, preventing regulation of K+ intake		√ √ √
	Test Check competence using YFP BioBrick plasmid Measure internal K+ concentration using flame photometry Quantify growth relative to wildtype E.coli strain
Controlled K+ efflux
	Design sequence based on GluR0 glutamate-gated K+ channel from Synechocystis PCC 6803
	Send to DNA 2.0 for synthesis
	Backup Obtain Synechocystis PCC 6803 strain (from Imperial College London) Design and order primers for GluR0, PCR Include rare tRNA plasmid in transformation
	Ligate gene into BioBrick plasmid
	Transform into chosen chassis
	Test Measure internal K+ concentration with and without presence of glutamate, using flame photometry
Measuring voltage
	Quantify output using oxygen electrode or glass capillary microelectrode
Medium optimisation
	Vary K+ concentrations, using KCl
	Vary nutrient levels
Output optimisation
	Vary strength of promoters/RBS