IGEM:Cambridge/2008/Notebook/Voltage

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=Next Steps=
=Next Steps=
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<u>Characterise promoters</u>
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1. Ligate OsmY, RBS, and KDP in sequence into a vector. Possible difficulty due to the size of the KDP gene.
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1. Simulate and design    ‘reporter plasmids’ with correct biobricks restriction enzyme sites
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2. Determine the correct mutant to use:
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2. For each strain:   2 plasmid backbones
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::* Determine transformation efficiency of mutants.
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::* Plot growth curves of mutants in normal/varying K+ conditions.
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::* Estimate K+ uptake in varying K+ conditions.
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::* PSC101 (A) with    OsmY (promoter) + RFP + Stop
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3. Acquire ligand gated ion channel (GluR0)
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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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4. Quantify promoter    strength (light intensity, expression levels)
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=Useful Links=
=Useful Links=

Revision as of 11:55, 5 August 2008



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Aim

To create a system which responds to ligand binding with a detectable voltage caused by a K+ flux.

Progress

Background

Presentation

Experiments

Mutant Strains

Flame Photometer Calibration

K+ Concentrations

BioBrick Manipulation

OD600 Calibration

Next Steps

1. Ligate OsmY, RBS, and KDP in sequence into a vector. Possible difficulty due to the size of the KDP gene.

2. Determine the correct mutant to use:

  • Determine transformation efficiency of mutants.
  • Plot growth curves of mutants in normal/varying K+ conditions.
  • Estimate K+ uptake in varying K+ conditions.

3. Acquire ligand gated ion channel (GluR0)

Useful Links

Protein prediction tools

Uniprot database

Literature

Kdp operon diagram

Kdp 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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