IGEM:Cambridge/2008/Notebook/Voltage

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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, cells are grown in high K+ medium (100mM) and resuspended in low K+ medium.
 * 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 chosen E.coli strains with mutations in these genes as our chassis.

=Experiment Summaries=

Electrical Output

Mutant Growth Rates

Cytoplasmic K+ Concentrations

Parts Construction:


 * KDP


 * GluR0


 * Extracted Biobrick Parts

=Progress=

Progress

Technical Information
Gene Design

Flame Photometer Calibration

OD600 (Cell Density) Calibration

Mutant Strains Information

=Useful Links=

Protein prediction tools

Uniprot database

[[Media:Voltage_project.ppt |Presentation]]

=Literature=

| Kdp operon diagram

|Kdp 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)


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