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:
Progress
Progress
Technical Information
Gene Design
Flame Photometer Calibration
OD600 (Cell Density) Calibration
Mutant Strains Information
Useful Links
Protein prediction tools
Uniprot database
Presentation
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)
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