Sandbox: Difference between revisions
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|'''Preliminary wet work ''' || || || | |'''Preliminary wet work ''' || || || | ||
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| ||Extract promoter, RBS and terminator BioBricks from registry | | ||Extract promoter, RBS and terminator BioBricks from registry |:*Refine protocol for paper-bound DNA extraction |:*Use PCR and transformations to confirm presence of DNA|| || | ||
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|'''Internal K+ build-up''' || || || | |'''Internal K+ build-up''' || || || | ||
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:*Kch- mutant, preventing uncontrolled K+ efflux | :*Kch- mutant, preventing uncontrolled K+ efflux | ||
:*Kef- mutants, preventing uncontrolled K+ efflux | :*Kef- mutants, preventing uncontrolled K+ efflux | ||
:*Kdp- mutants, preventing regulation of K+ intake|| || | :*Kdp- mutants, preventing regulation of K+ intake | ||
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|- | |- | ||
| ||Test | | ||Test | ||
:*Check competence using YFP BioBrick plasmid | :*Check competence using YFP BioBrick plasmid | ||
:*Measure internal K+ concentration using flame photometry | :*Measure internal K+ concentration using flame photometry | ||
:*Quantify growth relative to wildtype E.coli strain || || | :*Quantify growth relative to wildtype E.coli strain | ||
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|'''Controlled K+ efflux''' || || || | |'''Controlled K+ efflux''' || || || | ||
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| ||Backup | | ||Backup | ||
:* Obtain Synechocystis PCC 6803 strain (from Imperial College London) | :* Obtain Synechocystis PCC 6803 strain (from Imperial College London) | ||
:*Design and order primers for GluR0, PCR | :*Design and order primers for GluR0, PCR | ||
:*Include rare tRNA plasmid in transformation || || | :*Include rare tRNA plasmid in transformation | ||
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|- | |- | ||
| ||Ligate gene into BioBrick plasmid || || | | ||Ligate gene into BioBrick plasmid || || |
Revision as of 08:37, 4 August 2008
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Personnel | Progress | ||
Research | |||
Potassium intake | |||
Preventing K+ efflux | |||
Bacterial tolerance for high K+ and turgor
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Ligand gated channels | |||
Media | |||
Preliminary wet work | |||
:*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
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Put Kdp gene under control of stationary phase promoter (osmY, used by MIT 2006 team)
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Transform into wildtype and mutant E.coli strains | |||
Test
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Chassis | |||
Order from Yale
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Test
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Controlled K+ efflux | |||
Design sequence based on GluR0 glutamate-gated K+ channel from Synechocystis PCC 6803 | |||
Send to DNA 2.0 for synthesis | |||
Backup
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Ligate gene into BioBrick plasmid | |||
Transform into chosen chassis | |||
Test
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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 |