Team:EPF-Lausanne/Our Project/Data

From 2011.igem.org

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{{:Team:EPF-Lausanne/Templates/Header|title=Data}}
{{:Team:EPF-Lausanne/Templates/Header|title=Data}}
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team, please check this page: [https://igem.org/Sample_Data_Page]
 
== Overview ==
== Overview ==
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=== Summary ===
=== Summary ===
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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.  
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[[File:EPFL-Summary_drawing.png|right|thumb|250px]]
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In developing these three pillars, a number of parts were made. We have listed all of them and have highlighted our favorites.  
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Three components constitute our transcription factor development pipeline. The first is a selection system which lyses cells containing strong matches between a mutated transcription factor and its promoter and allows for the speedy recovery of the relevant DNA. The second component 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 component is an ''in vitro'' characterization system called MITOMI that allows high-throughput quantitative analysis of DNA-protein interaction strengths.
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In developing these three pillars, a number of parts were made. We have listed all of them here and have highlighted our favorites.
=== Systems ===
=== Systems ===
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Selection System: We made a T7-promoter-driven lysis device by adding our wildtype T7 promoter () upstream of the Berkeley lysis cassette () inserted in the () plasmid.  
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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).  
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In Vivo Characterization:
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'''In Vivo Characterization''': We cloned an RFP gene under control of T7 promoter variants (parts BBa_K613001 through BBa_K613012) into a low copy number plasmid (pSB3K1, formerly BBa_I739202). We also cloned TetR mutants (parts BBa_K613013 through BBa_K613019) into a  a low copy number plasmid (pSB3K1, formerly BBa_I739202). Both sets of plasmids were thoroughly characterized using IPTG platereader experiments.
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In Vitro Characterization:
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'''In Vitro Characterization''': We characterized TetR mutants (parts BBa_K613013, BBa_K613015, BBa_K613016, BBa_K613017, BBa_K613019) in the low copy number plasmid pSB3K1 (formerly BBa_I739202).
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== Biobricks ==
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== Favourite parts ==
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=== Favourite parts ===
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Our three favourite parts are TetR mutants, characterised ''in-vivo'' using our reporter systems, and ''in-vitro'' by MITOMI.
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[http://partsregistry.org/Part:BBa_K613015 BBa_K613015]
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* [http://partsregistry.org/Part:BBa_K613015 BBa_K613015] '''TetR E37A W43S T141A mutant'''
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* [http://partsregistry.org/Part:BBa_K613016 BBa_K613016] '''TetR P39K mutant'''
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* [http://partsregistry.org/Part:BBa_K613017 BBa_K613017] '''TetR Y42F mutant'''
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[http://partsregistry.org/Part:BBa_K613016 BBa_K613016]
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== Pre-existing parts characterised ==
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[http://partsregistry.org/Part:BBa_K613017 BBa_K613017]
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* [http://partsregistry.org/Part:BBa_K112808:Experience BBa_K112808 Experience Page] - '''The Berkeley Lysis Device''': In developing our Lysis selection device, we characterised induction, and ran proof-of-principle experiments.
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=== Pre-existing parts characterised ===
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== All other parts submitted ==
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* [http://partsregistry.org/Part:BBa_K112808:Experience Experience] - '''The Berkeley Lysis Device, BBa_K112808''': In developing our Lysis selection device, we characterised induction, and ran proof-of-principle experiments.
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=== TetR Mutants ===
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=== All other parts submitted ===
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In addition to our three favourite tetR mutants, we created these four:
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==== TetR Mutants ====
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* [http://partsregistry.org/Part:BBa_K613013 K613013] '''V36F mutant'''
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* [http://partsregistry.org/Part:BBa_K613014 K613014] '''V36F W43S mutant'''
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* [http://partsregistry.org/Part:BBa_K613018 K613018] '''Y42F K108E mutant'''
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* [http://partsregistry.org/Part:BBa_K613019 K613019] '''P39Q Y42M mutant'''
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* V36F mutant: [http://partsregistry.org/Part:BBa_K613013 K613013]
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=== T7 Promoter variants ===
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* V36F W43S double mutant: [http://partsregistry.org/Part:BBa_K613014 K613014]
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* E37A W43S T141A triple mutant: [http://partsregistry.org/Part:BBa_K613015 K613015]
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* P39K mutant: [http://partsregistry.org/Part:BBa_K613016 K613016]
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* Y42F K108E double mutant: [http://partsregistry.org/Part:BBa_K613018 K613018]
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* P39Q Y42M double mutant: [http://partsregistry.org/Part:BBa_K613019 K613019]
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==== T7 Promoter variants ====
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We submitted a family of T7 promoters with varying strength. Our lysis selection device uses what we refer to as the wild-type (100% reported strength) T7 Promoter:
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Our lysis selection device uses what we refer to as the wild-type T7 Promoter:
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* [http://partsregistry.org/Part:BBa_K613001 K613001] '''T7 Promoter''', 100% reported strength
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[http://partsregistry.org/Part:BBa_K613001 K613001]
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In addition, we submitted the following mutants:
In addition, we submitted the following mutants:
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Variants without additional lac operator (constitutive):
Variants without additional lac operator (constitutive):
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[http://partsregistry.org/Part:BBa_K613002 K613002]
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* [http://partsregistry.org/Part:BBa_K613002 BBa_K613002] '''T7 Promoter''', 14% reported strength
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* [http://partsregistry.org/Part:BBa_K613003 BBa_K613003] '''T7 Promoter''', 30% reported strength
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[http://partsregistry.org/Part:BBa_K613003 K613003]
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* [http://partsregistry.org/Part:BBa_K613004 BBa_K613004] '''T7 Promoter''', 54% reported strength
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* [http://partsregistry.org/Part:BBa_K613005 BBa_K613005] '''T7 Promoter''', 80% reported strength
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[http://partsregistry.org/Part:BBa_K613004 K613004]
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* [http://partsregistry.org/Part:BBa_K613006 BBa_K613006] '''T7 Promoter''', 111% reported strength
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[http://partsregistry.org/Part:BBa_K613005 K613005]
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[http://partsregistry.org/Part:BBa_K613006 K613006]
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Variants with additional lac operator (LacI repressed):
Variants with additional lac operator (LacI repressed):
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[http://partsregistry.org/Part:BBa_K613007 K613007]
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* [http://partsregistry.org/Part:BBa_K613007 BBa_K613007] '''T7 Promoter, LacI repressed''', 100% reported strength
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* [http://partsregistry.org/Part:BBa_K613008 BBa_K613008] '''T7 Promoter, LacI repressed''', 14% reported strength
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[http://partsregistry.org/Part:BBa_K613008 K613008]
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* [http://partsregistry.org/Part:BBa_K613009 BBa_K613009] '''T7 Promoter, LacI repressed''', 30% reported strength
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* [http://partsregistry.org/Part:BBa_K613010 BBa_K613010] '''T7 Promoter, LacI repressed''', 54% reported strength
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[http://partsregistry.org/Part:BBa_K613009 K613009]
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* [http://partsregistry.org/Part:BBa_K613011 BBa_K613011] '''T7 Promoter, LacI repressed''', 80% reported strength
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* [http://partsregistry.org/Part:BBa_K613012 BBa_K613012] '''T7 Promoter, LacI repressed''', 111% reported strength
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[http://partsregistry.org/Part:BBa_K613010 K613010]
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[http://partsregistry.org/Part:BBa_K613011 K613011]
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[http://partsregistry.org/Part:BBa_K613012 K613012]
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==== Medium-strength Plac ====
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[http://partsregistry.org/Part:BBa_K613000 K613000]
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=== Medium-strength Plac ===
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== Pre-existing parts we improved ==
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We submitted a new Plac promoter of medium strength. We characterized it with ATC inductions, using RFP as a readout.
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* [http://partsregistry.org/Part:BBa_K613000 BBa_K613000] '''pLac promoter''', medium strength
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- [http://partsregistry.org/Part:BBa_K112808:Experience Experience] - '''The Berkeley Lysis Device, BBa_K112808''': In developing our Lysis selection device, we characterised induction, and ran proof-of-principle experiments.
 
{{:Team:EPF-Lausanne/Templates/Footer}}
{{:Team:EPF-Lausanne/Templates/Footer}}

Latest revision as of 22:05, 28 October 2011

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