IGEM:Cambridge/2008/Notebook/Voltage

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
Line 45: Line 45:
[[IGEM:Cambridge/2008/Notebook/Voltage/Flame Photometer Calibration|Flame Photometer Calibration]]
[[IGEM:Cambridge/2008/Notebook/Voltage/Flame Photometer Calibration|Flame Photometer Calibration]]
-
[[IGEM:Cambridge/2008/Notebook/Voltage/K+ Concentrations|K+ Concentrations]]
+
[[IGEM:Cambridge/2008/Notebook/Voltage/OD600 Calibration|OD600 (Cell Density) Calibration]]
 +
 
 +
[[IGEM:Cambridge/2008/Notebook/Voltage/K+ Growth|Mutant Growth Rates]]
 +
 
 +
[[IGEM:Cambridge/2008/Notebook/Voltage/K+ Concentrations|Induced Cytoplasmic K+ Concentrations]]
[[IGEM:Cambridge/2008/Notebook/Voltage/BioBrick Manipulation|BioBrick Manipulation]]
[[IGEM:Cambridge/2008/Notebook/Voltage/BioBrick Manipulation|BioBrick Manipulation]]
-
[[IGEM:Cambridge/2008/Notebook/Voltage/OD600 Calibration|OD600 Calibration]]
+
 

Revision as of 08:44, 3 September 2008



Search this project

Customize your entry pages

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 Information

Flame Photometer Calibration

OD600 (Cell Density) Calibration

Mutant Growth Rates

Induced Cytoplasmic K+ Concentrations

BioBrick Manipulation



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



Personal tools