Team:EPF-Lausanne/Our Project/TetR mutants/muTetRs

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{{:Team:EPF-Lausanne/Templates/TetRtemplate|title=muTetRs}}
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{{:Team:EPF-Lausanne/Templates/TetRtemplate|title=Mutant TetRs}}
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==Why==
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=== Strategy ===
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==How==
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Having done a litterature search, we decided to focus first on mutating some key amino acids rather than going for random mutations. The most important residues seem to be the V36, E37, P39 and Y42 ones.
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==What==
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{| {{table}} style="font-size: 60%;"
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We used two distinct strategies to create these mutants:
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| align="center" style="background:#f0f0f0;"|'''TetR variants'''
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| align="center" style="background:#f0f0f0;"|'''Method'''
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* <html> <a href="https://2011.igem.org/Team:EPF-Lausanne/Protocols/Site-specific_mutagenesis"> Site-specific mutagenesis </a></html>, which consists of both a plasmid amplification step with forward and reverse primers containing the targeted mutation and a selection step (enzymatic digestion of the methylated dsDNA)
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| align="center" style="background:#f0f0f0;"|'''Promoter'''
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| align="center" style="background:#f0f0f0;"|'''C-terminal'''
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* <html> <a href="https://2011.igem.org/Team:EPF-Lausanne/Protocols/TetR_Extension_PCR"> PCR-induced mutagenesis </a></html>, which specifically amplifies two halves of the gene of interest from a linear template and introduces a mutation in one of them, followed by a stich-PCR to join the two halves and add the necessary extensions.
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| align="center" style="background:#f0f0f0;"|'''DNA form'''
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| align="center" style="background:#f0f0f0;"|'''Expression'''
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The plasmid that we used had a C-terminal eGFP fusion to the TetR, while the linear template had a C-terminal His-tag. The promoters added were SP6 in the first case and T7 in the second.
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| align="center" style="background:#f0f0f0;"|'''MITOMI vs 1-off'''
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| align="center" style="background:#f0f0f0;"|'''Biobrick'''
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| align="center" style="background:#f0f0f0;"|'''Sequenced'''
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=== Mutants obtained ===
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| align="center" style="background:#f0f0f0;"|'''Sent to registry'''
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Here is a list of all the mutants we created, their characterization statut and a link to their respective PartsRegistry page.
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{| style="font-size: 75%;"
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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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|-
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| ||||||||||||||||||
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|   V36F ||site directed||SP6||eGFP||plasmid||worked well||X||<html><a href="http://partsregistry.org/Part:BBa_K613013">BBa_K613013</a> </html>||||
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|  <html> <a href="http://partsregistry.org/Part:BBa_K613013">V36F</a> </html> ||site directed mutagenesis||SP6||eGFP||plasmid||worked well||v||BBa_K613013||||
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|  V36FW43S ||PCR-induced||T7||His-tag||linear ||(ins C-term), ITT tested||||<html> <a href="http://partsregistry.org/Part:BBa_K613014"> BBa_K613014</a> </html>||||
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|-
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| <html> <a href="http://partsregistry.org/Part:BBa_K613014"> V36FW43S</a> </html> ||PCR-induced mutagenesis||T7||His-tag||linear ||(ins C-term), ITT tested||||BBa_K613014||||
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|  E37AW43ST141A ||PCR-induced||T7||His-tag||linear ||ITT tested||X||<html> <a href="http://partsregistry.org/Part:BBa_K613015">BBa_K613015</a></html>||X||X
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| P39K ||PCR-induced||T7||His-tag||template ||ITT tested, worked||X||<html> <a href="http://partsregistry.org/Part:BBa_K613016">BBa_K613016</a> </html>||X||X
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|-
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| <html> <a href="http://partsregistry.org/Part:BBa_K613015"> E37AW43ST141A </a></html>||PCR-induced mutagenesis||T7||His-tag||linear ||ITT tested||v||BBa_K613015||v||v
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| Y42F||PCR-induced||T7||His-tag||linear||ITT tested||X||<html> <a href="http://partsregistry.org/Part:BBa_K613017">BBa_K613017</a></html>||X||X
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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>||X||X
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| P39QY42M ||site directed||SP6||eGFP||plasmid||worked well||X||<html> <a href="http://partsregistry.org/Part:BBa_K613019">BBa_K613019</a> </html>||||
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|-
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| <html> <a href="http://partsregistry.org/Part:BBa_K613016"> P39K </a> </html>||PCR-induced mutagenesis||T7||His-tag||template ||ITT tested and worked ok||v||BBa_K613016||v||v
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| P39QY42ML197S||PCR-induced||T7||His-tag||linear ||ITT tested||||<html> <a href="http://partsregistry.org/Part:BBa_K613020">BBa_K613020</a></html>||||
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| P39QY42ML52P||PCR-induced||T7||His-tag||linear ||ITT tested||||<html> <a href="http://partsregistry.org/Part:BBa_K613021">BBa_K613021</a></html>||||
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| <html> <a href="http://partsregistry.org/Part:BBa_K613017">Y42F</a></html>||PCR-induced mutagenesis||T7||His-tag||linear||ITT tested||v||BBa_K613017||v||v
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| E37AP39K||site directed||T7||His-tag||linear ||||||<html> <a href="http://partsregistry.org/Part:BBa_K613022">BBa_K613022</a></html>||X||
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| <html> <a href="http://partsregistry.org/Part:BBa_K613018">Y42FK108E</a></html>||PCR-induced mutagenesis||T7||His-tag||linear ||ITT tested||||BBa_K613018||v||v
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| E37AP39QY42F||site directed||T7||His-tag||linear ||||||<html> <a href="http://partsregistry.org/Part:BBa_K613023">BBa_K613023</a></html> ||X||
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| WT||none||T7||His&GFP||both ||||||<html> <a href="http://partsregistry.org/Part:BBa_K613023"> </a></html>|| ||
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| <html> <a href="http://partsregistry.org/Part:BBa_K613019">P39QY42M</a> </html> ||site directed mutagenesis||SP6||eGFP||plasmid||worked well||v||BBa_K613019||||
 
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| <html> <a href="http://partsregistry.org/Part:BBa_K613020">P39QY42ML197S</a></html>||PCR-induced mutagenesis||T7||His-tag||linear ||ITT tested||||BBa_K613020||||
 
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| <html> <a href="http://partsregistry.org/Part:BBa_K613021">P39QY42ML52P</a></html>||PCR-induced mutagenesis||T7||His-tag||linear ||ITT tested||||BBa_K613021||||
 
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| <html> <a href="http://partsregistry.org/Part:BBa_K613022">E37AP39K</a></html>||site directed mutagenesis||T7||His-tag||linear ||||||BBa_K613022||v||
 
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| <html> <a href="http://partsregistry.org/Part:BBa_K613023">E37AP39QY42F</a></html>||site directed mutagenesis||T7||His-tag||linear ||||||BBa_K613023 ||v||
 
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|}
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Characterization results for the most interesting mutants can be found on [https://2011.igem.org/Team:EPF-Lausanne/Our_Project/TetR_mutants/Conclusion this page]. For the detailed ''in vitro'' results, please refer to [https://2011.igem.org/Team:EPF-Lausanne/Our_Project/TetR_mutants/MITOMI_data this] page, whereas the ''in vivo'' characterizations are [https://2011.igem.org/Team:EPF-Lausanne/Our_Project/Reporter_Systems/tetR#TetR_mutant_characterization there].
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{{:Team:EPF-Lausanne/Templates/Footer}}
{{:Team:EPF-Lausanne/Templates/Footer}}

Latest revision as of 19:30, 26 October 2011

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