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<td class="tdboxes"><a href="http://openwetware.org/wiki/iGEM:Cambridge/2008/Notebook/Voltage"><img border="0" src="http://openwetware.org/images/f/fe/Tur_logo.JPG" width="201" height="201"></a></td>
<td class="tdboxes"><a href="http://openwetware.org/wiki/iGEM:Cambridge/2008/Notebook/Voltage"><img border="0" src="http://openwetware.org/images/f/fe/Tur_logo.JPG" width="201" height="201"></a></td>
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{| {{table}}
| align="center" style="background:#f0f0f0;"|''' '''
| align="center" style="background:#f0f0f0;"|''' '''
| align="center" style="background:#f0f0f0;"|'''Personnel'''
| align="center" style="background:#f0f0f0;"|'''Progress'''
|-
| '''Research'''|| ||40 μL||O
|-
| ||Potassium intake || ||O
|-
| ||Preventing K+ efflux || ||O
|-
| ||Ligand gated channels || ||O
|-
| 10X NEB Buffer||buffer 3, 2.5 μL||buffer 2, 2.5 μL ||buffer 3, 2.5 μL||buffer 2, 2.5 μL||buffer 2, 2.5 μL||buffer 3, 2.5 μL||buffer 3, 2.5 μL||buffer 3, 2.5 μL
|-
| 25X BSA||1 μL||1 μL||1 μL||1 μL||1 μL||1 μL||1 μL||1 μL
|}
o
o
o
o Bacterial tolerance for high K+ and turgor
 Osmolites (“inert” sugars)
o Media
 Preliminary wet work
o 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
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

Revision as of 08:04, 4 August 2008

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<html> <td class="tdboxes"><a href="http://openwetware.org/wiki/iGEM:Cambridge/2008/Notebook/Voltage"><img border="0" src="http://openwetware.org/images/0/00/Vol_logo.JPG" width="256" height="201"></a></td> <td class="tdboxes"><a href="http://openwetware.org/wiki/iGEM:Cambridge/2008/Notebook/Voltage"><img border="0" src="http://openwetware.org/images/d/d1/Mag_logo.JPG" width="210" height="201"></a></td> <td class="tdboxes"><a href="http://openwetware.org/wiki/iGEM:Cambridge/2008/Notebook/Voltage"><img border="0" src="http://openwetware.org/images/f/fe/Tur_logo.JPG" width="201" height="201"></a></td> </html>

Personnel Progress
Research 40 μL O
Potassium intake O
Preventing K+ efflux O
Ligand gated channels O
10X NEB Buffer buffer 3, 2.5 μL buffer 2, 2.5 μL buffer 3, 2.5 μL buffer 2, 2.5 μL buffer 2, 2.5 μL buffer 3, 2.5 μL buffer 3, 2.5 μL buffer 3, 2.5 μL
25X BSA 1 μL 1 μL 1 μL 1 μL 1 μL 1 μL 1 μL 1 μL

 o o o o Bacterial tolerance for high K+ and turgor  Osmolites (“inert” sugars) o Media


 Preliminary wet work o 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 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

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