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

Wouldn‘t it be nice … • direct, peptide-level protein control • photo-sensitivity via a simple cloning strategy • modularized photo-control toolkit

Let‘s talk about LOV „ L ight O xygen and V oltage “ • protein commonly found in plants • light-directed growth • Wu et al. 2009 • LOV-Rac1 fusion • Strickland et al. 2009 • improved LOV

Mechanism LOV LOV J α Effector

Molecular Structure of LOV LOV FMN J α Source: Wu et al., Nature. 2009 Sep 3;461(7260)

Previous Constructs vs. Our Idea LOV mRNA Repressor Effector

Previous Constructs vs. Our Idea LOV Repressor

Concept • fuse effector proteins with LOV • establish a fusion system for simple protein photo-activatability LOV Effector

Fusion Constructs The LOV Blues LOV Kills LOV Cuts LOV LOV LOV LacZ α ccdB mazF RNA

Approach LOVTAP LOV TAP BBa_K360021 PCR LOV LOV Restriction Ligation LOV Effector fusion constructs

Approach – LOVTAP ( I ) PCR Restriction Analysis GTC ACT GCC CGC TTT CCA Sequencing GTC GGG AAA CCT G

Approach – LOVTAP ( II ) LOV LOV LOV TAP TAP TAP iGEM Team Bonn LMU Munich TU Darmstadt

Approach – LOVTAP ( II ) LOV LOV LOV TAP TAP TAP iGEM Team Bonn LMU Munich TU Darmstadt

Approach – LOVTAP ( III ) LOV TAP Sosnick lab Prefix LOV Suffix EcoRI XbaI NheI SpeI PstI PstI PstI Additional step: • removal of two PstI restriction sites

Approach – Coupling Sequence J α LOV Effector Ligation J α J α Effector LOV Fusion Sequence -Glu-Leu-Asp- Regular Sequence -Glu-Ile-Asp-

The LOV Blues - Concept • LOV-LacZ α Fusion LOV • proof of concept LacZ α • LacZ α • split version of LacZ

The LOV Blues - Assay LOV LOV J α LacZ α LacZ ω LacZ ω + LacZ α + LOV LacZ α LacZ ω

The LOV Blues - Results LacZ α LOV Prefix pLac Suffix EcoRI XbaI SpeI PstI C 1 C 2 C 4 C 5 C 6 C 7 C 8 C 9 C 10 C 3 1000 bp 500 bp

LOV Kills - Concept LOV LOV J α ccdB ccdB Gyrase

LOV Kills – Results LOV Prefix pLac ccdB Suffix EcoRI XbaI SpeI PstI C 2 C 1 C 3 C 4 C 5 1000 bp 500 bp

LOV Cuts - Concept LOV LOV mazF J α mazF RNA RNA bacterial êndonuclease

Fusion System Status LOV Prefix Suffix EcoRI XbaI PstI PstI NheI SpeI PstI • added Prefix and Suffix • added NheI restriction site • removed one PstI restriction site

Project Status • constructed LOV Blues vector • constructed LOV Cuts vector • constructed LOV Kills vector • constructed Control vectors • designed assays • conducted assays

LOV in the future LOV • prove functionality of fusion constructs • prove modularity of fusion system

Human Practice • poster at two conferences • national SynBio Day • video translation http://www.youtube.com/watch?v=bykAeGMMosI

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:

Thank You For Listening! Any Questions?