IGEM:Cambridge/2008/Electric Output

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Aim

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

Background

Presentation

Current Progress

-Ordered mutant E.coli chassis to test: Kch^-, KdpD^-, KdpE^-, KdpF-E^-

-Designed two plasmids:

• Kdp Plasmid

• Glu Gated channel Plasmid

-Sourced V.harveyi BB170 culture to obtain glutamate-gated potassium channel gene

-Modelled protein structure

-Designed primers for extraction of Kdp Operon from E.coli and Glu Gated channel gene from V.harveyi

-Extracted BioBricks from registry to use in plasmids:

• Promoter BBa_J23100

• RBS BBa_B0030

• Terminators BBa_B1002 and BBa_B1006

-Decided on oxygen electrode for small voltage change measurements

Current Plan

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