IGEM:Cambridge/2008/Notebook/Voltage

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=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 voltage output part of our project aims to mimic the signal transduction that occurs at a neural 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.
+
*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.
-
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.
+
*The mechanism we have designed is similar to that used in the brain – relying on ion movement across the membrane, and gated ion channels.  
-
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.
+
*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.
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[[Media:Voltage_project.ppt |Presentation]]
 
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=Progress=
 
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[[IGEM:Cambridge/2008/Notebook/Voltage/Progress |Progress]]
 
=Experiment Summaries=
=Experiment Summaries=
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[[IGEM:Cambridge/2008/Notebook/Voltage/Output| Electrical Output]]
[[IGEM:Cambridge/2008/Notebook/Voltage/K+ Growth|Mutant Growth Rates]]
[[IGEM:Cambridge/2008/Notebook/Voltage/K+ Growth|Mutant Growth Rates]]
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[[IGEM:Cambridge/2008/Notebook/Voltage/K+ Concentrations|Cytoplasmic K+ Concentrations]]
[[IGEM:Cambridge/2008/Notebook/Voltage/K+ Concentrations|Cytoplasmic K+ Concentrations]]
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[[IGEM:Cambridge/2008/Notebook/Voltage/BioBrick Manipulation|BioBrick Manipulation]]
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Parts Construction:
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*[[IGEM:Cambridge/2008/Notebook/Voltage/BioBrick Manipulation|KDP]]
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*[[IGEM:Cambridge/2008/Notebook/Voltage/GluR0 Manipulation|GluR0]]
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*[[IGEM:Cambridge/2008/Extracted_Parts | Extracted Biobrick Parts]]
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=Progress=
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[[IGEM:Cambridge/2008/Notebook/Voltage/Progress |Progress]]
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[http://www.uniprot.org/ Uniprot database]
[http://www.uniprot.org/ Uniprot database]
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[[Media:Voltage_project.ppt |Presentation]]
=Literature=
=Literature=

Current revision


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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:


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)

Recent changes



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