Aim

To create a system which responds to ligand binding with a detectable voltage caused by a K⁺ flux. iGEM:cambridge/2008/Notebook/Voltage/Progress Roadmap

Background

Presentation

Experiments

Mutant Strains

Flame Photometer Calibration

K+ Concentrations

BioBrick Manipulation

OD600 Calibration

Next Steps

Characterise promoters

1. Simulate and design ‘reporter plasmids’ with correct biobricks restriction enzyme sites

2. For each strain: 2 plasmid backbones

• PSC101 (A) with OsmY (promoter) + RFP + Stop

• Another (B), not yet defined, with Bba_J23100 + YFP + Stop

Tests for each strain : plasmid A, plasmid B, plasmid A + plasmid B (so 15 tests)

3. Quantify transformation efficiency (colony counter)

4. Quantify promoter strength (light intensity, expression levels)

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