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All you need is LOV
University of Bonn Europe Jamboree, Amsterdam October 6th, 2012
~450 nm
Source: http://www.bioinformatik.uni-leipzig.de/Leere/PRAKTIKUM/Protokolle/SS04/1/Bilder/protein_einf.jpg
All you need is LOV University of Bonn Europe Jamboree, Amsterdam - - PowerPoint PPT Presentation
All you need is LOV University of Bonn Europe Jamboree, Amsterdam - - PowerPoint PPT Presentation
All you need is LOV University of Bonn Europe Jamboree, Amsterdam October 6 th , 2012 ~450 nm Source: http://www.bioinformatik.uni-leipzig.de/Leere/PRAKTIKUM/Protokolle/SS04/1/Bilder/protein_einf.jpg Wouldnt it be nice direct,
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- direct, peptide-level protein control
- photo-sensitivity via a simple cloning strategy
- modularized photo-control toolkit
Wouldn‘t it be nice…
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„Light Oxygen and Voltage“
- protein commonly found in plants
- light-directed growth
- Wu et al. 2009
- LOV-Rac1 fusion
- Strickland et al. 2009
- improved LOV
Let‘s talk about LOV
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LOV Jα LOV Effector
Mechanism
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Molecular Structure of LOV
LOV Jα FMN
Source: Wu et al., Nature. 2009 Sep 3;461(7260)
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LOV
Repressor
Effector
Previous Constructs vs. Our Idea
mRNA
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Previous Constructs vs. Our Idea
LOV
Repressor
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Concept
- fuse effector proteins with LOV
- establish a fusion system for simple protein photo-activatability
LOV Effector
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The LOV Blues LOV Kills
LacZα ccdB
LOV Cuts
mazF
Fusion Constructs
RNA
LOV LOV LOV
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Approach
LOVTAP LOV fusion constructs
LOV TAP LOV LOV Effector
PCR Restriction Ligation
BBa_K360021
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Approach – LOVTAP (I)
PCR Restriction Analysis Sequencing
GTC ACT GCC CGC TTT CCA GTC GGG AAA CCT G
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LOV TAP iGEM Team Bonn LOV TAP LMU Munich LOV TAP TU Darmstadt
Approach – LOVTAP (II)
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LOV TAP iGEM Team Bonn LOV TAP LMU Munich LOV TAP TU Darmstadt
Approach – LOVTAP (II)
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LOV TAP
Sosnick lab
Prefix LOV Suffix
EcoRI XbaI PstI PstI NheI SpeI PstI
Approach – LOVTAP (III)
Additional step:
- removal of two PstI restriction sites
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Jα Effector
Regular Sequence
LOV
Ligation
- Glu-Leu-Asp-
- Glu-Ile-Asp-
Jα
Fusion Sequence
Effector
Approach – Coupling Sequence
LOV Jα
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The LOV Blues - Concept
- LOV-LacZα Fusion
- proof of concept
- LacZα
- split version of LacZ
LacZα
LOV
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The LOV Blues - Assay
LacZα LacZω + LacZα
LOV LOV
Jα LacZα
LOV
+ LacZω LacZω
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The LOV Blues - Results
LOV pLac LacZα Prefix
EcoRI XbaI
Suffix
SpeI PstI
C 1 C 2 C 3 C 4 C 5 C 6 C 7 C 8 C 9 C 10 1000 bp 500 bp
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LOV Kills - Concept
Gyrase
ccdB
LOV
Jα ccdB
LOV
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LOV Kills – Results
LOV pLac ccdB Prefix Suffix
C 1 C 2 C 3 C 4 C 5 500 bp 1000 bp
EcoRI XbaI SpeI PstI
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LOV Cuts - Concept
mazF
LOV
Jα mazF
LOV
RNA RNA bacterial êndonuclease
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Fusion System Status
LOV
PstI NheI
Prefix
EcoRI XbaI
Suffix
SpeI PstI
- added Prefix and Suffix
- added NheI restriction site
- removed one PstI restriction site
PstI
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Project Status
- constructed LOV Blues vector
- constructed LOV Cuts vector
- constructed LOV Kills vector
- constructed Control vectors
- designed assays
- conducted assays
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LOV in the future
LOV
- prove functionality of fusion constructs
- prove modularity of fusion system
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Human Practice
- poster at two conferences
- national SynBio Day
- video translation
http://www.youtube.com/watch?v=bykAeGMMosI
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Give some love to
- very special thanks to
- Prof. Dr. Michael Famulok and Prof. Dr. Günter Mayer
- University of Bonn and LIMES Institute
- our collaborators:
- LMU Munich, TU Darmstadt
- special thanks to
- Vincent Noireaux, University of Minnesota (for the mazF plasmid)
- Sosnick Lab (for LOVTAP)
- our sponsors:
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