All You Need is LOV • 20

12 iGEM Bonn

All Yo u N e e d I s

LO

V •

Kevin Baßler, Katharina Düker, Tanja Funk, Rebecca Halbach, Matthias Klumpp, Nicolas Ludwig, Christine Martens, John Nguyen, Philipp Sander, Max Schelski, Erik StrickerAdvisors: Katia Schöler, Silvana Haßel, Anna Hall, Martina Bettio Instructors: Günter Mayer, Michael Famulok

University of Bonn, Life and Medical Sciences Institute (LIMES), Gerhard-Domagk-Str. 1, Bonn, Germany

TEAMBonn

Abstract Mechanism

Proof of Principle: The LOV Blues

Project Status

Application: LOV Kills

The iGEM team Bonn, established 2011, consists of a group of bachelor students in molecular biomedicine under the professional supervision of Prof. Dr. Günter Mayer and Prof. Dr. Michael Famulok, professors at the Life and Medical Sciences (LIMES) Institute, as well as PhD students in Chemical Biology.

We decided to fuse our LOV domain with mazF, a ribonuclease, as one of two application examples. Through radiation with light, the mazF will be activated, leading to cell death.

mazF is a ACA-specif-ic ribonuclease from B. subtilis and E. coli, which in nature is used as a kill device in a pro-grammed cell death pathway.

We will analyze mazF activity by monitoring bacterial growth.

The Team

Approach

Fusion protein design has always been time- and design-intensive, to say the least. We are deve-loping and characterizing a fusion construct containing a light sensitive domain, providing easy coupling and light activation of proteins, thus developing a system to modify protein activity on protein-level just by using blue light.

Using the LOV (Light, Oxygen, Voltage) domain commonly found in plants, where it enables light-directed growth, we are establishing guidelines for coupling proteins of interest to the LOV domain, offering light induced spatio-temporal control over protein activity.

Designing an activity regulation system at protein level allows quick response times (2.2 microsec-onds activation time, 85 seconds de-activation time). Furthermore, a protein fusion approach to regulation allows direct control of activity, as opposed to transcriptional regulation methods.

The LOV (Light, Oxygen, Voltage) protein consists of the LOV domain and the Jα helix. The LOV domain displays photosensitivity to blue light (420 nm - 480 nm) and undergoes a conformational change in 2.2 microseconds upon exposure. This results in unfolding of the Jα helix. In absence of blue light, the LOV domain folds back into its original state within 85 seconds, leading to reforma-tion of the Jα helix.

In its unfolded state, the rigid Jα helix forces the subsequent C-terminal effector protein in direct vicinity of the LOV domain. This leads to a steric inhibition. Upon light exposure, the steric inhibi-tion is removed, allowing the effector protein to assume activity.

We originally intended to use LovTAP (BBa_K360021) included in the distribution plate. However several restriction analyses and PCR trials with this plasmid failed. Sequencing did not show any matching part in the registry. Sequencing LovTAP samples from the TU Darmstadt’s and LMU Munich’s distribution plates did not yield a positive result either.

After two months of continued difficulties verifiying and using BBa_K360021 we asked for a LovTAP sample directly from the Sos-nick Lab. Sequencing and restriction analysis yielded positive results; however, the plasmid is not BioBrick conform as it contains to in-

ternal PstI restriction sites. In addition to introducing biobrick pre- and suffixes, we inserted a NheI restriction site for optimal coupling.

Currently, we have not fully construct-ed all planned fusion constructs, due to difficulties working with BBa_K360021.

Additionally, all of our BioBricks con-taining the LOV domain are not cur-rently RFC 10 compatible, as they still contain one or more PstI restriction sites.

We have sofar have sent in three Bio-Bricks of our own design; BBa_K820008, a lacI-inducible kill switch using ccdB, BBa_K820009, a lacI-inducible LacZα construct and BBa_K820014, which contains the mazF ribonuclease and a double terminator. We also have sent in a sample for BBa_K302033, containing mazF, which had been entered into the registry in 2010, but sofar has yet to have physical DNA submitted.

As another application example, we designed a LOV fusion construct containing ccdB, a potent cell death protein.

CcdB inhibits the gy-rase A and therefore prevents the unwind-ing of double-stran-ded DNA during rep-lication, which will be blocked as a result.

We visualize ccdB ac-tivity through a growth curve assay.

We chose LacZα as a proof of principle for the LOV fusions.

Illumination of the bacteria carrying the construct results in ac-tivation of LacZα. In bacterial deletion-mutants, which express only LacZω, activity of LacZα results in an assembly of LacZα and -ω, which together form the enzyme β-Galactosidase.

β-Galactosidase can metabolize a chromogenic substrate, X-Gal, re-sulting in blue coloration of the bac-terial colonies.

Application: LOV Cuts

Constructed The LOV Blues Vector

Constructed LOV Cuts Vector

Constructed LOV Kills Vector

Constructed Control Vectors

Designed Assays

Conducted Assays

pLac mazFPrefix

EcoRIXbaI

Suffix

SpeI PstIPstI PstI