Team:Gothenburg-Sweden/Project

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Chalmers University of Technology

The project

Synthetic readout of cellular stress

Project Description

The conceptual idea is to use the conformational change in the SNF1 protein complex to establish a FRET (Förster Resonance Energy Transfer) system for cellular stress detection. There are two chromophores tagged at appropriate locations and upon undergoing the conformational change, they come in close proximity such that the emission energy from one can excite the other, thus resulting in different emission energy. There are two possibilities to be tested:

1. To fuse the chromophores to the subunits of the Snf1 complex to build an active readout of the conformational change
2. To fuse the chromophores to a short peptide (SAMS peptide) that can be phosphorylated by Snf1 and is commonly used as a read-out of Snf1 activity.

• Working package 1: Find positions for fluorescent markers in SNF1
For WP1 all present information about the protein complex is utilized, so that the fluorescent tags do not interfere with active sites or evolutionary conserved regions. Two alternatives should be investigated and presented with regard to present information and distance between fluorophores. The working package is ended with a 3D modeling of the tagging suggestions to further investigate functionality.

• Working Package 2: Decide what fluorescent protein to use
To apply FRET a compatible fluorphore pair is necessary. Also, efficiency and duration in respect to e.g. photonic bleaching should be taken into consideration.

• Working package 3: Find DNA sequence for SAMS-peptide
Beside the two tagging alternative from WP1 a SAMS-peptide will be used. Both ends will be tagged with fluorophores and conformational change should be indicated with FRET-signal during conformational change at phophorylation by active SNF1.

• Working package 4: Decide on plasmid backbone
The plasmid containing fusion protein should be able to amplify in E.coli and also contain a strong promoter for protein expression in yeast. Use VectorNTI to find good alternatives and locate RE sites.

• Working package 5: Design primers for fusion PCR
When WP 1-4 is finished the primer design can be initiated. Primers should be designed so that they have a high melting temperature, preferably around 60°C, with at least 20 bp homology with each side that should be fused. Primers at utter end of the fusion proteins should include restriction enzyme sites with GGCC repetition at the end. After rigorous proofreading the primers should be ordered together with a primer representing the SAMS peptide.

• Working package 6: Acquire plasmid backbone for transfection with fusion protein
Look up how the plasmid backbone from WP4 can be acquired. If it is a common backbone it should be avaible in the plasmid bank at the laboratory, otherwise it should be ordered.

• Working package 7: Perform fusion PCR
When the primers from WP5 has arrived the fusion PCR can be initiated. As a template for the protein subunits in SNF1 genomic DNA from yeast should be used, this should be avaible at the laboratory, and as template for fluorescent proteins dried DNA provided by iGEM will be used. Create a PCR mastermix that includes all ingredients except the DNA template. Each fusion is run in five steps according to the manual and each step is analyzed on agar gel so that the correct fragments are produced.

• Working package 8: Ligate insert into plasmids
After WP7 is successful the fusion protein should be inserted into the plasmid backbone. Use restriction enzymes as decided in WP6 and follow ligation protocol. Afterwards the plasmids should be purified with miniprep, they should be cut again and put on a gel to verify that the insert was successful.

• Working package 9: Amplify transfection plasmids
After WP8 the plasmid need to be amplified before being tranfected into yeast. Tranfect E.coli and amplify over night. Purify the amplified plasmids with a miniprep kit.

• Working package 10: Sporulate diploid yeast strains to acquire Snf1 deletion mutants
Grow diploid yeast strains with Snf1 deletions on sporulation plats to acquire haploid deletion mutants. Use protocol to clear the population from diploids and unwanted haploids.

• Working package 11: Transfect yeast with tagged fusion protein
After WP9-10 the hapoloid yeast deletion strain should be tranfected with the fusion protein plasmid. When the yeast has recovered from transfection the strain should be grown on sucrose overnight to show transfection result.

• Working package 12: Apply FRET and analyze results
By using a microscopy with FRET set up the success of previous WP can be concluded from the presence or absence of a FRET signal. Analyze results and make conclusions together with Marcus Wilhelmsson.

• Working package 13:Write report
WP 13 is running alongside with the other WPs during the whole project. Initially aims and theory is the major work load and as the project progress results and analysis claims the major part of this WP.

• Working package 14: Apply for funding
Also running alongside the other WPs. Funding is initially applied at science funding committees and later, as there are results to be presented, at major companies interested in the possibilities presented by this project. Funding should cover laboratory expenses, travel to Boston and team t-shirts.

safety questions

to be continued

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