Biotricity

Cambridge iGEM2008

Project Proposal

Concept

K+

+ve relative to medium

Inducible pump

E.coli K+ K+

neutral relative to medium

Glutamate

E.coli

Constitutive channel

Steps

• Step 1 – Chassis selection

• Step 2 – Setting up a potential difference between culture and external constant potential/ground

• Step 3 – Detection of ligand and change in potential

• Step 4 – Measurement of PD change

Step 1 - Chassis

Escherichia coli mutant:• Insensitive to glutamate• No Kch channels expressed

Testing:• Glutamate on semi-solid agar plate• Immunoglobulin antibody precipitate

test for Kch protein

Step 2 - Setting up a PD

Over-express Kdp (potassium pump):• Kdp operon native to E.coli• Adjust regulation

Testing:• ELISA assay to measure Kdp protein

production• Or measure K+ concentration/ PD• Or mass spectrometry, comparing peak

intensities• Or 2D PAGE

New

Step 3 - Detection of ligand

Glutamate-gated K+ channel:

• Already sequenced, from Granulobacter bethesdensis

• Synthesise using

• Transform into chassis

New

Ttgacaccgtttttttctaacgctttcagacgccctctccgttctgtgtttaggctctgtttccttctgttctccatactggctcaggcaggcttgatgggagtcgtcctccct

cctgcccatgctatggcagcggataaaaataccgccgagcatggtgtaatcaatgcaggctattttatcgatccgcctttcgtgctgccgcatcagaagaatgat

catccggccgggctggccattgatctatgggacaagacgtcacagatgcagggctggatcacccattatcaccgctatgaaacgatagccgatcttctctccgc

tcttgagcgcggggagatcgatgtcggtacggtcggcctgacaatcagcagccagcgtatggagaaagtgatcttcagccagccctggtttcagaccgggctg

cggatgatgatcgtcaaacacaatagtaccggcctttccagacttctgagcgaactggctgcttccggccatctgcgtaactatttgttgatttttctggcgatcctgg

tggcga xxxxxxxxxxxxxxxcactgatcacagccatcatgcaccggcatgtgttgaaggattcatcaccccactggagttcctctctgtcgggcgccttc

xxxxxxxxxxxxxxxxxxxxxxxtatgatatgatggcgatgctgaccggcaatcaaacgtccggcaccgtgccagagcgagccggtgcacg

xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxgatcattgccgccatctggctgctctgtggcgttggcattgtggcctatgtcacatccagcat

xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxtaccagcgtgatgacggcttcagaaatcaatcagcgcgtggacaatatcactg

xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxaactaggccatcgcccggtcagtgcgatgaaaggcagcatcgccatga

xxxxxxxxxxxxxxxxxxxxxxxxcttttgccttggataccggcctgaacgttcacggatatacgctcctgtccgatgctgtagatgaactgatcc

xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxagggcaaaacatccgccattctgggagatgcatcacagctcgattattatatccaacaga

xxxxxxxxxxxxxxxxxxxxxxxxxaccccacccaatcactgatcgtaacaggcgctacactacgtccggaaaatctcggcttcgtcatgcggcccggtttcc

ctatgcgttatcagatcgacaggaccattctcaagttgcaggaagaaaaaatcctgtcccagatggagtctgcctatttttcacatcgataa

Step 4 - Measurement

• Sensitive electrode in culture

• Other electrode at known potential (could be ground)

Look, Engineering

!

Further Developments

1) Amplify response:

• Separate receptor and gated K+ channel

• Connect via intracellular signalling pathway

2) Detect other or multiple ligands