http://2008.igem.org/wiki/index.php?title=IGEM:Cambridge/2008/Notebook/Voltage/K%2B_Growth&feed=atom&action=historyIGEM:Cambridge/2008/Notebook/Voltage/K+ Growth - Revision history2024-03-28T09:56:05ZRevision history for this page on the wikiMediaWiki 1.16.5http://2008.igem.org/wiki/index.php?title=IGEM:Cambridge/2008/Notebook/Voltage/K%2B_Growth&diff=92602&oldid=prevEllis at 17:49, 29 October 20082008-10-29T17:49:56Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:RelGrowth200mmK.JPG |800px]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:RelGrowth200mmK.JPG |800px]]</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">presynaptic plasma membrane. The neurotransmitter diffuses through the synaptic cleft and binds to chemical receptor molecules on the membrane of the postsynaptic cell. These receptors cause ion channels to open so that ions rush out, changing the transmembrane potential. Attempting to mimic this in a prokaryotic system is particularly attractive as, in a more general sense, it provides an interface between chemical or biological and electrical systems.'''</del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">[[Image:GluR0.PNG|right|370px|GluR0 Plasmid]]</del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">Using the iGEM synthetic biology concept, we identified genes which could contribute to a synapse-like response. We also used a number of existing </del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">[[IGEM:Cambridge/2008/Extracted_Parts | parts from the registry]]. To keep the system as simple and therefore feasible as possible the aim was to engineer E.coli to respond to a specific ligand by allowing a flux of ions which would be measurable as a small change in the voltage of the medium the cells are suspended in. The first step was to find prokaryotic ion channels which open in response to a particular molecule or small set of molecules. We searched the [http://www.uniprot.org/ Uniprot database] and found that the organism [http://www.ebi.ac.uk/2can/genomes/bacteria/Synechocystis.html Synechocystis PCC 6803] has a transmembrane protein, GluR0 which is glutamate-gated, and allows flux of potassium ions when opened. We decided to try using the protein coding sequence of the gene for this channel. However, as the sequence came from a different organism, it was necessary to optimise the codons for expression in E.coli. We therefore decided to have the [[IGEM:Cambridge/2008/Notebook/Voltage/GluR0 Manipulation| GluRo Biobrick plasmid]] we had designed synthesised by DNA2.0. [[IGEM:Cambridge/2008/Notebook/Voltage/Gene Design|''Gene Design'']]</del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">Using the GluR0 channel defined our ion of interest as potassium. As potassium is a naturally present ion in organisms, E. coli has its own regulatory systems to control its concentration. However, our review of the literature suggested that E. coli only varies potassium concentration as a mechanism of osmoregulation. It has a number of proteins involved in this process including a potassium influx pump (Kdp), a pressure sensitive potassium channel (Kch), and a potassium efflux channel (Kef).</del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">The idea of being able to measure a change in voltage of the medium brings with it a number of challenges. The number of ions flowing across the membrane should be as high as possible, to produce a signal significantly higher than the noise. This could be achieved by first packing the cells with potassium, so that a large gradient was set up between the inside and the outside of the cell. Then when the GluR0 channels open, potassium ions will suddenly flow down the concentration gradient. In order to build up this gradient in the first place, we planned to over-express the Kdp-ATPase, which would pump potassium into the cell. However, to stop the balance being restored, the potassium channels would need to be absent or disabled.</del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">[[Image:Kallstrains.JPG|left|350px|K+ Concentrations]]</del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">We ordered a variety of [[IGEM:Cambridge/2008/Notebook/Voltage/Mutant Strains |mutant E.coli strains]] with mutations in each of the genes coding for the potassium flux membrane proteins Kdp, Kch and Kef. To examine the behaviour of these mutant strains we grew them in a variety of potassium concentrations and used [[IGEM:Cambridge/2008/Notebook/Voltage/OD600 Calibration| OD600 readings]] to measure their [[IGEM:Cambridge/2008/Notebook/Voltage/K+ Growth|growth rates]]. We also measured [[IGEM:Cambridge/2008/Notebook/Voltage/K+ Concentrations| internal potassium concentrations]] using [[IGEM:Cambridge/2008/Notebook/Voltage/Flame Photometer Calibration|flame photometry]]. However there was little difference seen between most of the mutants and the control strain although there was highly variable poasium concentration seen in the different growth conditions.</del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">We used PCR to copy the Kdp sequence from the E.coli MG1655 genome. As the pump is an ATPase, we did not want it to be expressed during exponential phase as it could stunt growth. We therefore also used PCR to extract the OsmY promoter from Biobricks developed by MIT in 2006. It is a stationary phase promoter, so by putting Kdp under its control, we could postpone its expression until the cells have stopped needing ATP for growth and division. </del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">Unfortunately the ligation and expression of the [[IGEM:Cambridge/2008/Notebook/Voltage/BioBrick Manipulation| osmY-Kdp construct]] proved impossible and flame photometry data showed it was unecessary. Instead, we simply grew the E.coli in high K+ concentrations (100mM) and re-suspended them in low [K+] before adding the agonist, glutamate.</del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">Transformation of the GluR0 plasmid into the mutant strains mentioned earlier did not work. Instead, we tried transforming into the more traditional expression strain DH5 alpha Z1, with success. Using very[[IGEM:Cambridge/2008/Notebook/Voltage/Output| sensitive electrodes connected to an amplifier and oscilloscope]] we have been able to see voltage changes on application of glutamate to the medium.</del></div></td><td colspan="2"> </td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">=Literature=</del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">[http://expasy.org/tools/ Protein prediction tools]</del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">[http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1214631| Kdp operon diagram]</del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">[http://www.jbc.org/cgi/content/abstract/276/13/9590|Kdp plasmid]</del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">[http://www.springerlink.com/content/6042632827845551/ The Kdp-ATPase system and its regulation]</del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">[http://cgsc.biology.yale.edu/Strain.php?ID=107402 Potential Chassis: Strain JW1242-1]</del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">[http://cgsc.biology.yale.edu/Strain.php?ID=107065 Strain JW0710-1]</del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">[http://www.ncbi.nlm.nih.gov/pubmed/4942756 Kdp mutant - paper from 1971]</del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">[http://www.shigen.nig.ac.jp/WGR/link/link_E.coli_e.html Worldwide E.coli Databases]</del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">[http://jb.asm.org/cgi/content/abstract/188/5/1950 Characterisation of kdpD - 2005]</del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">[http://jb.asm.org/cgi/content/full/180/19/5102 Investigations on Kdp Operon exp. & flux]</del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">[http://dx.doi.org/10.1006/jmbi.2001.4884 Very interesting 2001 paper concerning Glutamate Channels]</del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">[http://www.nature.com/nature/journal/v402/n6763/full/402817a0.html 1999 paper on functional characterization of prokaryote Glu Channels]</del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=BA000022 Sequenced Synechocystis PCC 6803 genome]</del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=47118304&from=1401809&to=1403002&view=gbwithparts Glutamate-gated K+ channel GluR0]</del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">[http://redpoll.pharmacy.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi| Link to E.coli statistics page (CCDB Database)]</del></div></td><td colspan="2"> </td></tr>
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</table>Ellishttp://2008.igem.org/wiki/index.php?title=IGEM:Cambridge/2008/Notebook/Voltage/K%2B_Growth&diff=92571&oldid=prevEllis at 17:48, 29 October 20082008-10-29T17:48:06Z<p></p>
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<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">presynaptic plasma membrane. The neurotransmitter diffuses through the synaptic cleft and binds to chemical receptor molecules on the membrane of the postsynaptic cell. These receptors cause ion channels to open so that ions rush out, changing the transmembrane potential. Attempting to mimic this in a prokaryotic system is particularly attractive as, in a more general sense, it provides an interface between chemical or biological and electrical systems.'''</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">[[Image:GluR0.PNG|right|370px|GluR0 Plasmid]]</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">Using the iGEM synthetic biology concept, we identified genes which could contribute to a synapse-like response. We also used a number of existing </ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">[[IGEM:Cambridge/2008/Extracted_Parts | parts from the registry]]. To keep the system as simple and therefore feasible as possible the aim was to engineer E.coli to respond to a specific ligand by allowing a flux of ions which would be measurable as a small change in the voltage of the medium the cells are suspended in. The first step was to find prokaryotic ion channels which open in response to a particular molecule or small set of molecules. We searched the [http://www.uniprot.org/ Uniprot database] and found that the organism [http://www.ebi.ac.uk/2can/genomes/bacteria/Synechocystis.html Synechocystis PCC 6803] has a transmembrane protein, GluR0 which is glutamate-gated, and allows flux of potassium ions when opened. We decided to try using the protein coding sequence of the gene for this channel. However, as the sequence came from a different organism, it was necessary to optimise the codons for expression in E.coli. We therefore decided to have the [[IGEM:Cambridge/2008/Notebook/Voltage/GluR0 Manipulation| GluRo Biobrick plasmid]] we had designed synthesised by DNA2.0. [[IGEM:Cambridge/2008/Notebook/Voltage/Gene Design|''Gene Design'']]</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">Using the GluR0 channel defined our ion of interest as potassium. As potassium is a naturally present ion in organisms, E. coli has its own regulatory systems to control its concentration. However, our review of the literature suggested that E. coli only varies potassium concentration as a mechanism of osmoregulation. It has a number of proteins involved in this process including a potassium influx pump (Kdp), a pressure sensitive potassium channel (Kch), and a potassium efflux channel (Kef).</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">The idea of being able to measure a change in voltage of the medium brings with it a number of challenges. The number of ions flowing across the membrane should be as high as possible, to produce a signal significantly higher than the noise. This could be achieved by first packing the cells with potassium, so that a large gradient was set up between the inside and the outside of the cell. Then when the GluR0 channels open, potassium ions will suddenly flow down the concentration gradient. In order to build up this gradient in the first place, we planned to over-express the Kdp-ATPase, which would pump potassium into the cell. However, to stop the balance being restored, the potassium channels would need to be absent or disabled.</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">[[Image:Kallstrains.JPG|left|350px|K+ Concentrations]]</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">We ordered a variety of [[IGEM:Cambridge/2008/Notebook/Voltage/Mutant Strains |mutant E.coli strains]] with mutations in each of the genes coding for the potassium flux membrane proteins Kdp, Kch and Kef. To examine the behaviour of these mutant strains we grew them in a variety of potassium concentrations and used [[IGEM:Cambridge/2008/Notebook/Voltage/OD600 Calibration| OD600 readings]] to measure their [[IGEM:Cambridge/2008/Notebook/Voltage/K+ Growth|growth rates]]. We also measured [[IGEM:Cambridge/2008/Notebook/Voltage/K+ Concentrations| internal potassium concentrations]] using [[IGEM:Cambridge/2008/Notebook/Voltage/Flame Photometer Calibration|flame photometry]]. However there was little difference seen between most of the mutants and the control strain although there was highly variable poasium concentration seen in the different growth conditions.</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">We used PCR to copy the Kdp sequence from the E.coli MG1655 genome. As the pump is an ATPase, we did not want it to be expressed during exponential phase as it could stunt growth. We therefore also used PCR to extract the OsmY promoter from Biobricks developed by MIT in 2006. It is a stationary phase promoter, so by putting Kdp under its control, we could postpone its expression until the cells have stopped needing ATP for growth and division. </ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">Unfortunately the ligation and expression of the [[IGEM:Cambridge/2008/Notebook/Voltage/BioBrick Manipulation| osmY-Kdp construct]] proved impossible and flame photometry data showed it was unecessary. Instead, we simply grew the E.coli in high K+ concentrations (100mM) and re-suspended them in low [K+] before adding the agonist, glutamate.</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">Transformation of the GluR0 plasmid into the mutant strains mentioned earlier did not work. Instead, we tried transforming into the more traditional expression strain DH5 alpha Z1, with success. Using very[[IGEM:Cambridge/2008/Notebook/Voltage/Output| sensitive electrodes connected to an amplifier and oscilloscope]] we have been able to see voltage changes on application of glutamate to the medium.</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">=Literature=</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">[http://expasy.org/tools/ Protein prediction tools]</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">[http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1214631| Kdp operon diagram]</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">[http://www.jbc.org/cgi/content/abstract/276/13/9590|Kdp plasmid]</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">[http://www.springerlink.com/content/6042632827845551/ The Kdp-ATPase system and its regulation]</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">[http://cgsc.biology.yale.edu/Strain.php?ID=107402 Potential Chassis: Strain JW1242-1]</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">[http://cgsc.biology.yale.edu/Strain.php?ID=107065 Strain JW0710-1]</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">[http://www.ncbi.nlm.nih.gov/pubmed/4942756 Kdp mutant - paper from 1971]</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">[http://www.shigen.nig.ac.jp/WGR/link/link_E.coli_e.html Worldwide E.coli Databases]</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">[http://jb.asm.org/cgi/content/abstract/188/5/1950 Characterisation of kdpD - 2005]</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">[http://jb.asm.org/cgi/content/full/180/19/5102 Investigations on Kdp Operon exp. & flux]</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">[http://dx.doi.org/10.1006/jmbi.2001.4884 Very interesting 2001 paper concerning Glutamate Channels]</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">[http://www.nature.com/nature/journal/v402/n6763/full/402817a0.html 1999 paper on functional characterization of prokaryote Glu Channels]</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=BA000022 Sequenced Synechocystis PCC 6803 genome]</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=47118304&from=1401809&to=1403002&view=gbwithparts Glutamate-gated K+ channel GluR0]</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">[http://redpoll.pharmacy.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi| Link to E.coli statistics page (CCDB Database)]</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
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<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">__NOTOC__</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">__NOEDITSECTION__</ins></div></td></tr>
</table>Ellishttp://2008.igem.org/wiki/index.php?title=IGEM:Cambridge/2008/Notebook/Voltage/K%2B_Growth&diff=92525&oldid=prevEllis at 17:45, 29 October 20082008-10-29T17:45:21Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Method==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Method==</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*For each of the 9 mutants, 3 [K+] of growth medium were examined: 1mM, 50mM & 200mM</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*For each of the 9 mutants, 3 [K+] of growth medium were examined: 1mM, 50mM & 200mM</div></td></tr>
</table>Ellishttp://2008.igem.org/wiki/index.php?title=IGEM:Cambridge/2008/Notebook/Voltage/K%2B_Growth&diff=79957&oldid=prevEllis: New page: ==Method== *For each of the 9 mutants, 3 [K+] of growth medium were examined: 1mM, 50mM & 200mM '''Composition of Each Cuvette:''' * 100ul cells ...2008-10-28T17:28:14Z<p>New page: ==Method== *For each of the 9 mutants, 3 [K+] of growth medium were examined: 1mM, 50mM & 200mM '''Composition of Each Cuvette:''' * 100ul cells ...</p>
<p><b>New page</b></p><div>==Method==<br />
*For each of the 9 mutants, 3 [K+] of growth medium were examined: 1mM, 50mM & 200mM<br />
'''Composition of Each Cuvette:''' <br />
* 100ul cells<br />
* 100ul KCl (10x stocks)<br />
* 800ul LB <br />
----------------------<br />
1ml Final Volume<br />
*The spectrophotometer was zeroed using LB. <br />
*OD readings were taken every 30 minutes for 3 hours. <br />
*In between readings, cultures were stored at room temperature & covered with parafilm.<br />
<br />
==Results==<br />
[[Media:MutantGrowthData.xls |Link to raw OD600 Data in Excel]]<br />
<br />
[[Image:RelGrowth1mmK.JPG |800px]]<br />
<br />
[[Image:RelGrowth50mmK.JPG |800px]]<br />
<br />
[[Image:RelGrowth200mmK.JPG |800px]]</div>Ellis