IGEM:Cambridge/2008/Notebook/Voltage
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=Background= | =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 voltage output part of our project aims to mimic the signal transduction that occurs at a neural 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. | + | *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, | + | *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 | + | *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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| + | =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= | =Progress= | ||
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[[IGEM:Cambridge/2008/Notebook/Voltage/Progress |Progress]] | [[IGEM:Cambridge/2008/Notebook/Voltage/Progress |Progress]] | ||
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| - | [[IGEM:Cambridge/2008/Notebook/Voltage/ | + | ==Technical Information== |
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| + | [[IGEM:Cambridge/2008/Notebook/Voltage/Gene Design|Gene Design]] | ||
[[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/ | + | [[IGEM:Cambridge/2008/Notebook/Voltage/OD600 Calibration|OD600 (Cell Density) Calibration]] |
| - | [[IGEM:Cambridge/2008/Notebook/Voltage/ | + | [[IGEM:Cambridge/2008/Notebook/Voltage/Mutant Strains |Mutant Strains Information]] |
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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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