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]]
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=Background=
=Background=
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[[Media:Voltage_project.ppt | Presentation]]
 
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=Experiments=
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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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*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.
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*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.
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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 chosen E.coli strains with mutations in these genes as our chassis.
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[[IGEM:Cambridge/2008/Notebook/Voltage/Mutant Strains | Mutant Strains]]
 
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[[IGEM:Cambridge/2008/Notebook/Voltage/Flame Photometer Calibration|Flame Photometer Calibration]]
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=Experiment Summaries=
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[[IGEM:Cambridge/2008/Notebook/Voltage/K+ Concentrations|K+ Concentrations]]
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[[IGEM:Cambridge/2008/Notebook/Voltage/Output| Electrical Output]]
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[[IGEM:Cambridge/2008/Notebook/Voltage/BioBrick Manipulation|BioBrick Manipulation]]
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[[IGEM:Cambridge/2008/Notebook/Voltage/K+ Growth|Mutant Growth Rates]]
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[[IGEM:Cambridge/2008/Notebook/Voltage/OD600 Calibration|OD600 Calibration]]
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[[IGEM:Cambridge/2008/Notebook/Voltage/K+ Concentrations|Cytoplasmic K+ Concentrations]]
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=Next Steps=
 
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<u>Characterise promoters</u>
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Parts Construction:
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1. Simulate and design    ‘reporter plasmids’ with correct biobricks restriction enzyme sites
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*[[IGEM:Cambridge/2008/Notebook/Voltage/BioBrick Manipulation|KDP]]
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2. For each strain:   2 plasmid backbones
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*[[IGEM:Cambridge/2008/Notebook/Voltage/GluR0 Manipulation|GluR0]]
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::* PSC101 (A) with    OsmY (promoter) + RFP + Stop
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*[[IGEM:Cambridge/2008/Extracted_Parts | Extracted Biobrick Parts]]
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::* Another (B), not    yet defined, with Bba_J23100 + YFP + Stop
 
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: Tests for each strain :  plasmid A, plasmid B, plasmid A + plasmid B (so 15 tests)
 
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3. Quantify transformation    efficiency (colony counter)
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=Progress=
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[[IGEM:Cambridge/2008/Notebook/Voltage/Progress |Progress]]
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==Technical Information==
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[[IGEM:Cambridge/2008/Notebook/Voltage/Gene Design|Gene Design]]
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[[IGEM:Cambridge/2008/Notebook/Voltage/Flame Photometer Calibration|Flame Photometer Calibration]]
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[[IGEM:Cambridge/2008/Notebook/Voltage/OD600 Calibration|OD600 (Cell Density) Calibration]]
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[[IGEM:Cambridge/2008/Notebook/Voltage/Mutant Strains |Mutant Strains Information]]
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4. Quantify promoter    strength (light intensity, expression levels)
 
=Useful Links=
=Useful Links=
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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=
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[http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1214631 Kdp operon diagram]
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[http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1214631| Kdp operon diagram]
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[http://www.jbc.org/cgi/content/abstract/276/13/9590 Kdp plasmid]
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[http://www.jbc.org/cgi/content/abstract/276/13/9590|Kdp plasmid]
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[http://www.springerlink.com/content/6042632827845551/ The Kdp-ATPase system and its regulation]
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[http://www.springerlink.com/content/6042632827845551/ The Kdp-ATPase system and its regulation]
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Potential Chassis: [http://cgsc.biology.yale.edu/Strain.php?ID=107402 Strain JW1242-1]
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Potential Chassis: [http://cgsc.biology.yale.edu/Strain.php?ID=107402 |Strain JW1242-1]
[http://cgsc.biology.yale.edu/Strain.php?ID=107065 Strain JW0710-1]
[http://cgsc.biology.yale.edu/Strain.php?ID=107065 Strain JW0710-1]
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[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=BA000022 Sequenced Synechocystis PCC 6803 genome]
[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=BA000022 Sequenced Synechocystis PCC 6803 genome]
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[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=47118304&from=1401809&to=1403002&view=gbwithparts Glutamate-gated K+ channel GluR0]
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[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=47118304&from=1401809&to=1403002&view=gbwithparts   Glutamate-gated K+ channel GluR0]
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[http://redpoll.pharmacy.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi Link to E.coli statistics page (CCDB Database)]
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[http://redpoll.pharmacy.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi| Link to E.coli statistics page (CCDB Database)]
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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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