Team:Cambridge/Voltage

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|colspan="2" style="background-color: #F2F2F2;" align="right"|[[/Entry_Base|Customize your entry pages]] [[Help:Notebook/Project_Base/Customize_entry_page|<html><img src="/images/a/aa/Help.png" border="0" /></html>]]
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=Background=
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This year, the Cambridge iGEM team is working towards creating an integrated Bacterial Recombinant Artificial Intelligence Network (iBRAIN). Our concept is to model eukaryotic neural behaviour using populations of bacteria. We are looking at two main aspects of this concept: self-organisation using Turing pattern formation, and synaptic signal transduction using voltage output glutamate detection
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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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[[iGEM:Cambridge/2008/Concept |<font style="color:#cccccc">(read more...)]]
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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.
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*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.
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*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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=Experiment Summaries=
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[[IGEM:Cambridge/2008/Notebook/Voltage/Output| Electrical Output]]
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[[IGEM:Cambridge/2008/Notebook/Voltage/K+ Growth|Mutant Growth Rates]]
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[[IGEM:Cambridge/2008/Notebook/Voltage/K+ Concentrations|Cytoplasmic K+ Concentrations]]
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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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==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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=Useful Links=
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[http://expasy.org/tools/ Protein prediction tools]
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[http://www.uniprot.org/ Uniprot database]
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[[Media:Voltage_project.ppt |Presentation]]
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=Literature=
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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.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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[http://cgsc.biology.yale.edu/Strain.php?ID=107065 Strain JW0710-1]
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[http://www.ncbi.nlm.nih.gov/pubmed/4942756 Kdp mutant - paper from 1971]
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[http://www.shigen.nig.ac.jp/WGR/link/link_E.coli_e.html Worldwide E.coli Databases]
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[http://jb.asm.org/cgi/content/abstract/188/5/1950 Characterisation of kdpD - 2005]
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[http://jb.asm.org/cgi/content/full/180/19/5102 Investigations on Kdp Operon exp. & flux]
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[http://dx.doi.org/10.1006/jmbi.2001.4884 Very interesting 2001 paper concerning Glutamate Channels]
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[http://www.nature.com/nature/journal/v402/n6763/full/402817a0.html 1999 paper on functional characterization of prokaryote Glu Channels]
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[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://redpoll.pharmacy.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi| Link to E.coli statistics page (CCDB Database)]
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Revision as of 17:26, 28 October 2008

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Home   Signalling   Bacillus   Voltage   Modelling   Protocols   People   </center>

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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, 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

[http://expasy.org/tools/ Protein prediction tools]

[http://www.uniprot.org/ Uniprot database]

Presentation

Literature

[http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1214631| Kdp operon diagram]

[http://www.jbc.org/cgi/content/abstract/276/13/9590|Kdp plasmid]

[http://www.springerlink.com/content/6042632827845551/ The Kdp-ATPase system and its regulation]

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://www.ncbi.nlm.nih.gov/pubmed/4942756 Kdp mutant - paper from 1971]

[http://www.shigen.nig.ac.jp/WGR/link/link_E.coli_e.html Worldwide E.coli Databases]

[http://jb.asm.org/cgi/content/abstract/188/5/1950 Characterisation of kdpD - 2005]

[http://jb.asm.org/cgi/content/full/180/19/5102 Investigations on Kdp Operon exp. & flux]

[http://dx.doi.org/10.1006/jmbi.2001.4884 Very interesting 2001 paper concerning Glutamate Channels]

[http://www.nature.com/nature/journal/v402/n6763/full/402817a0.html 1999 paper on functional characterization of prokaryote Glu Channels]

[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?val=47118304&from=1401809&to=1403002&view=gbwithparts Glutamate-gated K+ channel GluR0]

[http://redpoll.pharmacy.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi| Link to E.coli statistics page (CCDB Database)]

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