IGEM:Cambridge/2008/Notebook/Voltage/Progress

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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
James
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 James
Send to DNA 2.0 for synthesis James
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 Chris
Vary nutrient levels
Output optimisation
Vary strength of promoters/RBS
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