1. Cambridge iGEM2008 Project Proposal
Biotricity
Cambridge iGEM2008
Project Proposal

2. Concept Inducible pump K+ Constitutive channel Glutamate
neutral relative to medium
Glutamate
E.coli
+ve relative to medium
E.coli

3. 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

4. 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

5. Step 2 - Setting up a PD New 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

6. Step 3 - Detection of ligand
Glutamate-gated K+ channel:
Already sequenced, from Granulobacter bethesdensis
Synthesise using
Transform into chassis
New
Ttgacaccgtttttttctaacgctttcagacgccctctccgttctgtgtttaggctctgtttccttctgttctccatactggctcaggcaggcttgatgggagtcgtcctccctcctgcccatgctatggcagcggataaaaataccgccgagcatggtgtaatcaatgcaggctattttatcgatccgcctttcgtgctgccgcatcagaagaatgatcatccggccgggctggccattgatctatgggacaagacgtcacagatgcagggctggatcacccattatcaccgctatgaaacgatagccgatcttctctccgctcttgagcgcggggagatcgatgtcggtacggtcggcctgacaatcagcagccagcgtatggagaaagtgatcttcagccagccctggtttcagaccgggctgcggatgatgatcgtcaaacacaatagtaccggcctttccagacttctgagcgaactggctgcttccggccatctgcgtaactatttgttgatttttctggcgatcctggtggcga xxxxxxxxxxxxxxxcactgatcacagccatcatgcaccggcatgtgttgaaggattcatcaccccactggagttcctctctgtcgggcgccttc xxxxxxxxxxxxxxxxxxxxxxxtatgatatgatggcgatgctgaccggcaatcaaacgtccggcaccgtgccagagcgagccggtgcacg xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxgatcattgccgccatctggctgctctgtggcgttggcattgtggcctatgtcacatccagcat xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxtaccagcgtgatgacggcttcagaaatcaatcagcgcgtggacaatatcactg xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxaactaggccatcgcccggtcagtgcgatgaaaggcagcatcgccatga xxxxxxxxxxxxxxxxxxxxxxxxcttttgccttggataccggcctgaacgttcacggatatacgctcctgtccgatgctgtagatgaactgatcc xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxagggcaaaacatccgccattctgggagatgcatcacagctcgattattatatccaacaga xxxxxxxxxxxxxxxxxxxxxxxxxaccccacccaatcactgatcgtaacaggcgctacactacgtccggaaaatctcggcttcgtcatgcggcccggtttccctatgcgttatcagatcgacaggaccattctcaagttgcaggaagaaaaaatcctgtcccagatggagtctgcctatttttcacatcgataa

7. Step 4 - Measurement Sensitive electrode in culture
Look, Engineering!
Sensitive electrode in culture
Other electrode at known potential (could be ground)

8. Further Developments 1) Amplify response:
Separate receptor and gated K+ channel
Connect via intracellular signalling pathway
2) Detect other or multiple ligands