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Team:Bonn/Project
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=== Fusion System === | === Fusion System === | ||
| - | We | + | We want to develop a simple Fusion System, which allows anyone a simple coupling of any potential effector. This system contains a Biobrick compliant LOV domain with the additional restriction site NheI 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, so leaving no additional Amino-Acids in the fusion construct. |
== Our Approach == | == Our Approach == | ||
For detailed information about the experiments we did in the lab visit our [[Team:Bonn/Notebook|Notebook]]. | For detailed information about the experiments we did in the lab visit our [[Team:Bonn/Notebook|Notebook]]. | ||
Revision as of 21:43, 26 September 2012
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Project Summary
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.
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. This system contains a Biobrick compliant LOV domain with the additional restriction site NheI 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, so leaving no additional Amino-Acids in the fusion construct.
Our Approach
For detailed information about the experiments we did in the lab visit our Notebook.
