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Team:EPF-Lausanne/Our Project/Data
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'''Selection System''': We cloned the Berkeley Lysis cassette (BBa_K112808) under control of the T7 promoter into a low copy number plasmid (pSB3K1, formerly BBa_I739202). | '''Selection System''': We cloned the Berkeley Lysis cassette (BBa_K112808) under control of the T7 promoter into a low copy number plasmid (pSB3K1, formerly BBa_I739202). | ||
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'''In Vivo Characterization''': | '''In Vivo Characterization''': | ||
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'''In Vitro Characterization''': | '''In Vitro Characterization''': | ||
Revision as of 02:25, 22 September 2011
Data
team, please check this page: [1]
Contents |
Overview
Summary
There are three pillars that underlie our transcription factor development pipeline. The first pillar is a selection system which lyses cells containing strong matches between a transcription factor and its promoter and allows for the speedy recovery of the relevant DNA. The second pillar is an in vivo characterization system that uses two different reporter systems as a way to document the binding affinities of the TetR transcription factor and its promoter pTet. The third pillar is an in-vitro characterization system called MITOMI that allows for high-throughput analysis of DNA-protein interaction strengths.
In developing these three pillars, a number of parts were made. We have listed all of them and have highlighted our favorites.
Systems
Selection System: We cloned the Berkeley Lysis cassette (BBa_K112808) under control of the T7 promoter into a low copy number plasmid (pSB3K1, formerly BBa_I739202).
In Vivo Characterization:
In Vitro Characterization:
Favourite parts
Our three favourite parts are TetR mutants, characterised in-vivo using our reporter systems, and in-vitro by MITOMI.
- BBa_K613015 TetR E37A W43S T141A mutant
- BBa_K613016 TetR P39K mutant
- BBa_K613017 TetR Y42F mutant
Pre-existing parts characterised
- Experience - The Berkeley Lysis Device, BBa_K112808: In developing our Lysis selection device, we characterised induction, and ran proof-of-principle experiments.
All other parts submitted
TetR Mutants
In addition to our three favourite tetR mutants, we created these four:
- K613013 V36F mutant
- K613014 V36F W43S double mutant
- K613018 Y42F K108E double mutant
- K613019 P39Q Y42M double mutant
T7 Promoter variants
Our lysis selection device uses what we refer to as the wild-type T7 Promoter:
In addition, we submitted the following mutants:
Variants without additional lac operator (constitutive):
Variants with additional lac operator (LacI repressed):
Medium-strength Plac
