IGEM:Cambridge/2008/Notebook/Voltage

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=Aim=
 
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To create a system which responds to ligand binding with a detectable voltage caused by a K<sup>+</sup> flux.
 
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[[IGEM:Cambridge/2008/Notebook/Voltage/Progress |Progress]]
 
=Background=
=Background=
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The voltage output part of our project aims to mimic the signal transduction that occurs at a neural synapse. We are engineering E.coli to create a voltage output on detection of glutamate. This imitates the creation of a postsynaptic potential in a dendrite when a neurotransmitter (such as glutamate) is present at the synapse.
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The mechanism we have designed is similar to that used in the brain – relying on ion movement across the membrane, and gated ion channels. To simplify the concept, we are only regulating and measuring the flux of potassium (K+) ions, and we are using a directly glutamate-gated K+ ion channel. This means that on the binding of glutamate, the channels will open, allowing a K+ flux, which will change the voltage of the medium enough to be detected with a very sensitive electrode.
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In order to set up a large enough K+ concentration gradient across the membrane for ions to flow down when the channels open, an ion pump is necessary. E.coli has a transmembrane P-type ATPase called Kdp, which pumps K+ into the cell. We have isolated this gene to overexpress it, therefore causing the cells to pump in a large number of K+ ions.
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However, E.coli also has a number of osmoregulatory systems which use relative K+ ion concentrations to control turgor. There are K+ leak channels (Kch and Kef) in the membrane, so we have ordered E.coli strains with mutations in these genes to allow K+ to remain sequestered inside the cells until the glutamate-gated channels open.
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[[Media:Voltage_project.ppt |Presentation]]
[[Media:Voltage_project.ppt |Presentation]]
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=Experiments=
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Progress
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[[IGEM:Cambridge/2008/Notebook/Voltage/Progress |Progress]]
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=Experiment Summaries=
[[IGEM:Cambridge/2008/Notebook/Voltage/Mutant Strains |Mutant Strains]]
[[IGEM:Cambridge/2008/Notebook/Voltage/Mutant Strains |Mutant Strains]]
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[[IGEM:Cambridge/2008/Notebook/Voltage/OD600 Calibration|OD600 Calibration]]
[[IGEM:Cambridge/2008/Notebook/Voltage/OD600 Calibration|OD600 Calibration]]
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=Next Steps=
 
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1. Ligate OsmY, RBS, and KDP in sequence into a vector. Possible difficulty due to the size of the KDP gene.
 
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2. Determine the correct mutant to use:
 
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::* Determine transformation efficiency of mutants.
 
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::* Plot growth curves of mutants in normal/varying K+ conditions.
 
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::* Estimate K+ uptake in varying K+ conditions.
 
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3. Acquire ligand gated ion channel (GluR0)
 
=Useful Links=
=Useful Links=

Revision as of 04:39, 3 September 2008



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Background

The voltage output part of our project aims to mimic the signal transduction that occurs at a neural synapse. We are engineering E.coli to create a voltage output on detection of glutamate. This imitates the creation of a postsynaptic potential in a dendrite when a neurotransmitter (such as glutamate) is present at the synapse. The mechanism we have designed is similar to that used in the brain – relying on ion movement across the membrane, and gated ion channels. To simplify the concept, we are only regulating and measuring the flux of potassium (K+) ions, and we are using a directly glutamate-gated K+ ion channel. This means that on the binding of glutamate, the channels will open, allowing a K+ flux, which will change the voltage of the medium enough to be detected with a very sensitive electrode. In order to set up a large enough K+ concentration gradient across the membrane for ions to flow down when the channels open, an ion pump is necessary. E.coli has a transmembrane P-type ATPase called Kdp, which pumps K+ into the cell. We have isolated this gene to overexpress it, therefore causing the cells to pump in a large number of K+ ions. However, E.coli also has a number of osmoregulatory systems which use relative K+ ion concentrations to control turgor. There are K+ leak channels (Kch and Kef) in the membrane, so we have ordered E.coli strains with mutations in these genes to allow K+ to remain sequestered inside the cells until the glutamate-gated channels open.

Presentation

Progress Progress

Experiment Summaries

Mutant Strains

Flame Photometer Calibration

K+ Concentrations

BioBrick Manipulation

OD600 Calibration


Useful Links

Protein prediction tools

Uniprot database

Literature

Kdp operon diagram

plasmid

The Kdp-ATPase system and its regulation

Potential Chassis: |Strain JW1242-1 Strain JW0710-1

Kdp mutant - paper from 1971

Worldwide E.coli Databases

Characterisation of kdpD - 2005

Investigations on Kdp Operon exp. & flux

Very interesting 2001 paper concerning Glutamate Channels

1999 paper on functional characterization of prokaryote Glu Channels

Sequenced Synechocystis PCC 6803 genome

Glutamate-gated K+ channel GluR0

Link to E.coli statistics page (CCDB Database)

Recent changes



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