Team:Bonn/Project

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Revision as of 22:05, 26 September 2012

Project Summary

Drafted crystal structuce of the LOV-domain. The Jα-helix is shown in blue, the flavin mononucleotide and the functional cysteine residue are depicted as balls and sticks.

Control of protein activity at the peptide level offers spatial-temporal control and quick reaction times, but so far has always involved target-specific tools, such as specific chemical inhibitors or proteases. We are developing and characterizing a fusion construct containing a light sensitive domain that provides quick, universal peptide-level light control of proteins of interest within the framework of easy biobrick-conform coupling.

We are engineering the LOV (Light, Oxygen, Voltage) domain – commonly found in plants where it enables light-directed growth – to control protein activity through blue light. Upon exposure, the LOV domain undergoes a conformational change and shifts away from the protein of interest, uncaging the coupled protein and allowing it to resume activity. In our project, we coupled a small part of the beta-galactosidase (which offers a simple assay) to the LOV domain as a proof-of-principle. We also built a cell death device using ccdB, a gyrase inhibitor. Furthermore we will design a MazF construct, as an example for a Nuclease.

Potential applications of our LOV fusion system include bioreactor regulation or site-specific drug activation.

Project Details

LOV-Blues / Lov-LacZalpha

In our proof-of-principle, we are coupling LacZalpha to the LOV domain. LacZalpha is one of two parts of a split-version beta-galactosidase, which upon exposure to light will resume galactosidase activity in mutants containing LacZomega, the complimentary second part of beta-galactosidase. Using a chromophore substrate for our beta-galactosidase gives us a simple blue-to-white assay.

LOV Kills / LOV-Ccdb

To show one of the many, wide-ranging possible applications, we will fuse a cell death protein, ccdB, to our LOV construct. Upon light exposure, the cell will go into apoptosis.

CcdB is the toxic part of a toxin/antitoxin system on the Fplasmide of E.coli. It is coexpressed with its labile antitoxin CcdA, which inhibits the toxic activity by forming a CcdA:CcdB complex. It serves mainly as a plasmid maintenance system: If the plasmid is lost, CcdB is freed due to the rapid degradation of the labile CcdA. It will now attack gyrase, a topoisomerase II, either in its free from or bound to DNA to form a covalent gyrase:DNA adduct, which leads to breakage of double-stranded DNA and plasmid and blockade of polymerases. It eventually results in cell death. But CcdA is also able to remove CcdB from its gyrase again as the CcdA:CcdB bond is stronger and therefore rejuvenate the cell.

LOV Cuts / LOV-MazF

MazF is a ACA-specific ribonuclease from B. subtilis which in nature is used for toxin/antitoxin addiction systems. It leads to programmed cell death in bacteria upon high-scale expression. As a consequence we want to use the LOV-MazF fusion construct as a cell death device. The functionality can be verified through a simple assay.

Fusion System

We want to develop a simple Fusion System, which allows anyone a simple coupling of any potential effector to LOV. This system contains a Biobrick compliant LOV domain with the additional restriction site NheI at the C-terminal end for coupling the effector to it. NheI will easily fit with the Biobrick standard, since it is a isocaudomere of XbaI and SpeI. Furthermore the resulting Scar imitates the regular sequence of the Lov Domain leaving no additional Amino-Acids in the fusion construct.

Our Approach

LOV-Blues

In order to prove the funtionality of our LOV-LacZalpha construct qualitatively.

LOV Kills

LOV Cuts

For detailed information about the experiments we did in the lab, visit our Notebook.