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| Personnel | Progress | ||
| Research | 40 μL | ||
| Potassium intake | |||
| Preventing K+ efflux | |||
| Bacterial tolerance for high K+ and turgor | |||
| Ligand gated channels | |||
| Osmolites (“inert” sugars) | |||
| Media | |||
| Preliminary wet work | |||
Extract promoter, RBS and terminator BioBricks from registry
| |||
| Ligand gated channels |
Internal K+ build-up o PCR Kdp K+ pump gene from E.coli MG1655 Design and order primers, including BioBrick prefix and suffix o 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 o Transform into wildtype and mutant E.coli strains o Test Measure internal K+ concentration using flame photometry
Chassis
o Order from Yale
Kch- mutant, preventing uncontrolled K+ efflux
Kdp- mutants, preventing regulation of K+ intake
Kef- mutants, preventing uncontrolled K+ efflux
o 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
o Design sequence based on GluR0 glutamate-gated K+ channel from Synechocystis PCC 6803
o Send to DNA 2.0 for synthesis
o Backup
Obtain Synechocystis PCC 6803 strain (from Imperial College London)
Design and order primers for GluR0, PCR
Include rare tRNA plasmid in transformation
o Ligate gene into BioBrick plasmid
o Transform into chosen chassis
o Test
Measure internal K+ concentration with and without presence of glutamate, using flame photometry
Measuring voltage
o Quantify output using oxygen electrode or glass capillary microelectrode
Medium optimisation
o Vary K+ concentrations, using KCl
o Vary nutrient levels
Output optimisation
o Vary strength of promoters/RBS
