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Cambridge iGEM2008 Project Proposal

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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


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