Team:UCSF/Andrej Ondracka Notebook

Week2 (6/30/08 – 7/6/08):

-PCRed alternative DBDs from plasmid sources as AB and BD parts, as well as with XhoI and BamHI sites. Topocloned, sequenced.

-digested vectors with GFP reporters (for insertion of operator sequences)

 

Week3 (7/7/08 – 7/13/08):

-sticky end PCRed or annealed operator sequences. Ligated into vector with GFP reporter. Miniprepped, sequenced. Obtained 2 different orientation of operators.

-Repeated bad topoclones. AarI digested DBDs, Sir2 AB/BD, and acceptor vectors with 3 different promoters. Ligated, transformed, miniprepped

 

Week4 (7/14/08 – 7/20/08):

-Repeated bad ligations.

-transformed reporter plasmids into Sf992 yeast. Selected 5 colonies of each, re-streaked them to select for integrants.

-Prepared the chemicals for the growth essay with strains with mammalian GPCRs. Did the growth essay. It failed, decided not to work on that anymore.

 

Week5 (7/21/08 – 7/27/08):

-Repeated bad ligations again (?)

-Ran FACS on Sf992 with reporter plasmids. Selected for appropriate level of expression of GFP. Lac operator plasmid didn't work well – low expression of GFP in all of the clones. Others worked well.

Transformed DBD-Sir2 fusions driven by Gal promoter into selected strains.

-Wrote the first version of our model in Matlab

 

Week6 (7/28/08 – 8/3/08):

-Re-streaked transformants onto new selective plate and onto +/-gal plates. Ran FACS on them. All C-terminal fusions to Sir2 did not work. As for N-terminal fusions, TetR showed a little silencing. Decided to increase the number of operator repeats.

-Miniprepped catalytic domain of Sir2 as BD part. Digested with AarI, ligated with DBDs and vectors with 3 different promoters, transformed. Miniprepped.

-Worked on implementing second version of model in Matlab.

 

Week7 (8/4/08 – 8/10/08):

-Increased the number of operators by digesting the plasmid and re-ligating with operators. Used various insert:vector ratio to obtain a range of number of insert repeats. Transformed, miniprepped, sequenced. Transformed into Sf992 yeast.

-Transformed DBD-cat domain of Sir2 constructs into reporter strains. Re-streaked onto new selective plate, and on +/-gal plates.

 

Week8 (8/11/08 – 8/17/08) :

-Ran FACS on new reporter strains with more operator repeats. Screened for colonies with appropriate levels of GFP expression. Transformed silencing construct into them with various promoters.

-Ran FACS on Sir2 cat domain constructs. Did not work – seemed that catalytic domain is not enough to silence.

-Ordered new oligos for Lac operators, as well as oligos for smaller number of LexA operator repeats to try to create a range of numbers of operator repeats. Annealed them, ligated into plasmid with GFP. Miniprepped, sequenced.

 

Week9 (8/18/08-8/24/08):

-Ran FACS with TetR silencing system. It worked even with much weaker promoters than LexA (pCyc).

-Repeated some of the unsuccessful operator ligations.

-Transformed successful ones into Sf992 yeast, as well as yeast strains that have the silencing construct in them. Re-streaked onto new selective plates. Ran FACS.

-Worked on nucleation - polymerization version of the model.

-Started cloning DNA-methylation constructs.

-Started designing biobrick shuttle vector.

 

Week10 (8/25/08-8/31/08):

-Re-run FACS with TetR silencing system with addition of aTc to see if it can disrupt silencing. Results showed with that it can, but there was some fluorescence probably caused by aTc itself.

-Designed and ordered oligos for biobrick shuttle vector. Obtained pSB1AC3 vector from registry of parts. Checked by diagnostic digest that the vector is OK.

-Started preparing for the presentation

 

Week11 (9/1/08-9/7/08):

-Re-run FACS with TetR silencing system with trying washing the cells before running (did not work – the fluorescence in control was still high), and trying another tetracyclin analog, Dox (worked to some extent but not completely).

-Digested the biobrick vector and ligated with the oligos with Aar1 sites. Transformed, miniprepped, sequenced.

-Preparing for the presentation