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| ||Design sequence based on GluR0 glutamate-gated K+ channel from Synechocystis PCC 6803 || ||
| ||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 || ||
|-
|-
|}
|}
o
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

Revision as of 15:27, 4 August 2008

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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
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
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