IGEM:MIT/2005/Wednesday 7/20 Experiment Meeting "Minutes"
Experimental Planning Meeting:
PURPOSE: Do run through of experimental meeting What do you have What do you need
- Permeablizing membrane -- like making cells competent
- Attach FFK peptide to antisense agents (RNA) -> lets antisense agent enter cell -> proteins to nucleic acid -> Drew knows things -- proteins that splice out ? -- catch splicing process -- make a tadpole -- protein head and oligo tail
- mutant cells: actinomycin (anti biotic) -- strain that takes big things, or just actinomycein?
- DNA transport: what exists: conjugation? comEC system (B. subtlis)
- Tests this morning didn't go so well
- Grew Mc4100 in M9 (doesn't fluoresce), LB
- no pellet in M9 solution
- LB cells resuspended in flur. diluted in M9 media
- Severe contamination -- Will != sterile
- Do the whole set of tests again (can do MC4100 when I do overnights)
- Kate says: this expt. was planned at the end of the day, and that was no good at all.
- Would like to start dealing with OligoFlurs. --> need assembly
- scFv's moving forward
- MalE and PhoA -- PhoA had multiple PCR products: TK says raise annealing temperature; Kate: Blast primers against e. coli genome. Though we can just excise band and use that, it makes sense to go forward and do it again
- Took protein sequences from cholera toxR -- vibrio species have similar genes, v. fischeri -- also serratia marcescens (don't really believe it is there) -- (sprayed over San Fran)
- ATCC -- cholera dna is BL1
- go ahead and get it -- use this as an exercise
- Annotation of translation start on cholera toxR was initially wrong -- there is ambiguity -- ack!! We need to check what we ordered!!
- track down fur- strain -- hoping to receive -- how do we get it? RAY = Point of contact
- jen and annie talking with samantha to work out recombineering
- where do we fuse our scFv? 3 domains: 7 fusions for one domain alone -- working out details -- having a team meeting about this at 4.30 -- should be a team decision -- can we do it in parallel?
- FecA in process, FecA prom -- know by end of day tom. if need new primers
- FecR and FecI starting pcr
- Testing parts, RNA based regulation from IAP 2003
- waiting on promoters, going to assemble with gfp when those are ready
Side note: lets talk about risk and danger! Side note: are we overextending ourselves? -gathering materials != doing expt -- might as well gather materials
TO DO: Look into B. Subtleis