IGEM:Cambridge/2008/Notebook/Voltage/Progress: Difference between revisions
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:*Refine protocol for paper-bound DNA extraction | :*Refine protocol for paper-bound DNA extraction | ||
:*Use PCR and transformations to confirm presence of DNA | :*Use PCR and transformations to confirm presence of DNA | ||
|| || | || || √ | ||
|- | |- | ||
|'''Internal K+ build-up''' || || || | |'''Internal K+ build-up''' || || || | ||
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| ||PCR Kdp K+ pump gene from E.coli MG1655 | | ||PCR Kdp K+ pump gene from E.coli MG1655 | ||
:*Design and order primers, including BioBrick prefix and suffix | :*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) | | ||Put Kdp gene under control of stationary phase promoter (osmY, used by MIT 2006 team) | ||
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:*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''' || || || | ||
|- | |- | ||
| ||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 ||James ||√ | ||
|- | |- | ||
| ||Send to DNA 2.0 for synthesis || || | | ||Send to DNA 2.0 for synthesis ||James ||√ | ||
|- | |- | ||
| ||Backup | | ||Backup | ||
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|'''Medium optimisation''' || || || | |'''Medium optimisation''' || || || | ||
|- | |- | ||
| ||Vary K+ concentrations, using KCl || || | | ||Vary K+ concentrations, using KCl ||Chris || | ||
|- | |- | ||
| ||Vary nutrient levels || || | | ||Vary nutrient levels || || |
Latest revision as of 08:15, 5 August 2008
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 | |||
Extract promoter, RBS and terminator BioBricks from registry
|
√ | ||
Internal K+ build-up | |||
PCR Kdp K+ pump gene from E.coli MG1655
|
James | √ | |
Put Kdp gene under control of stationary phase promoter (osmY, used by MIT 2006 team)
|
|||
Transform into wildtype and mutant E.coli strains | |||
Test
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|||
Chassis | |||
Order from Yale
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√ √ | ||
Test
|
√ | ||
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
|
|||
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 | Chris | ||
Vary nutrient levels | |||
Output optimisation | |||
Vary strength of promoters/RBS |