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]
 
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== New parts ==
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== Overview ==
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we must have 3 favourite, then the others come in a "other biobrick" section
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=== Summary ===
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Variants without additional lac operator (constitutive):
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[[File:EPFL-Summary_drawing.png|right|thumb|250px]]
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[http://partsregistry.org/Part:BBa_K613001 K613001]
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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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[http://partsregistry.org/Part:BBa_K613002 K613002]
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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.
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[http://partsregistry.org/Part:BBa_K613003 K613003]
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=== Systems ===
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[http://partsregistry.org/Part:BBa_K613004 K613004]
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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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[http://partsregistry.org/Part:BBa_K613005 K613005]
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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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[http://partsregistry.org/Part:BBa_K613006 K613006]
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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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== Favourite parts ==
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Variants with additional lac operator (LacI repressed):
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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_K613007 K613007]
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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_K613008 K613008]
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== Pre-existing parts characterised ==
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[http://partsregistry.org/Part:BBa_K613009 K613009]
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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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[http://partsregistry.org/Part:BBa_K613010 K613010]
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== All other parts submitted ==
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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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blablabla
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=== TetR Mutants ===
=== TetR Mutants ===
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{| {{table}} style="font-size: 75%;"
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In addition to our three favourite tetR mutants, we created these four:
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!| TetR variants
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!| Mutagenesis
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!| Promoter
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!| Cterminal
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!| DNA form
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!| Expression
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!| MITOMI vs 1-off
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!| Biobrick
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!| Sequenced
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!| Sent to registry
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|  E37AW43ST141A||PCR-induced||T7||His-tag||linear ||ITT tested||v||<html> <a href="http://partsregistry.org/Part:BBa_K613015"> BBa_K613015 </a></html>||v||v
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| P39K ||PCR-induced||T7||His-tag||template ||ITT tested, worked||v||<html> <a href="http://partsregistry.org/Part:BBa_K613016"> BBa_K613016 </a></html>||v||v
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| Y42F||PCR-induced||T7||His-tag||linear||ITT tested||v||<html> <a href="http://partsregistry.org/Part:BBa_K613017">BBa_K613017</a></html>||v||v
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| Y42FK108E||PCR-induced||T7||His-tag||linear ||ITT tested||||<html> <a href="http://partsregistry.org/Part:BBa_K613018">BBa_K613018</a></html>||v||v
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|}
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=== T7 promoter variants ===
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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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=== T7 Promoter variants ===
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== Pre-existing parts ==
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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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add something about the lysis cassette, and add also on its page on the partsregistry!
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* [http://partsregistry.org/Part:BBa_K613001 K613001] '''T7 Promoter''', 100% reported strength
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== In vivo characterization - Readout systems ==
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In addition, we submitted the following mutants:
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We created two different readout systems to characterize TetR mutants ''in vivo''. This is the last step of our whole method, after having selected the interesting TetR-Ptet mutant pairs with the lysis device and having characterized them ''in vitro''.
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Variants without additional lac operator (constitutive):
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=== TetR - RFP ===
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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_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_K613006 BBa_K613006] '''T7 Promoter''', 111% reported strength
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The first readout system is composed of TetR with a constitutive promoter, followed by RFP with a Ptet promoter. If TetR '''binds''' to Ptet, then RFP is ''' repressed'''. For more details about them and the experimental results, please refer to the [https://2011.igem.org/Team:EPF-Lausanne/Our_Project/Assembly#TetR_-_RFP_system Reporter systems] page.
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Variants with additional lac operator (LacI repressed):
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[[File:EPFL_2reporters_ss.JPG|400px]]
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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_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_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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=== TetR - LacI - RFP ===
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=== Medium-strength Plac ===
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This second readout system is composed of 3 genes: TetR under Pconst control, LacI under Ptet control (playing the role of an inverter) and finally RFP under pLac control. Here, RFP is '''induced''' when TetR '''binds''' to pTet. The experimental results are available on the [https://2011.igem.org/Team:EPF-Lausanne/Our_Project/Assembly#TetR_-_LacI_-_RFP_system Reporter systems] page.
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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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[[File:EPFL_3reporter_ss.JPG|400px]]
 
{{:Team:EPF-Lausanne/Templates/Footer}}
{{:Team:EPF-Lausanne/Templates/Footer}}

Latest revision as of 22:05, 28 October 2011

Retrieved from "http://2011.igem.org/Team:EPF-Lausanne/Our_Project/Data"