Biotricity
description
Transcript of Biotricity
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Biotricity
Cambridge iGEM2008
Project Proposal
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Concept
K+
+ve relative to medium
Inducible pump
E.coli K+ K+
neutral relative to medium
Glutamate
E.coli
Constitutive channel
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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
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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
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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
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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
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Step 4 - Measurement
• Sensitive electrode in culture
• Other electrode at known potential (could be ground)
Look, Engineering
!
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Further Developments
1) Amplify response:
• Separate receptor and gated K+ channel
• Connect via intracellular signalling pathway
2) Detect other or multiple ligands