052608 notes


Nora's presentation (secretion systems):

 . 1 vs 2-step
 . Dependency on size of protein/folding ability
 . Type I
         o Subsets for exporting large/small proteins
 . Do we need different secretion pathways for small/large (Mgfp vs.
   tyrosinase)
 . Links to reviews will be posted online
 . Type II - two-step = sucky
 . Type III - Usually involved in virulence factor secretion (would have to
   be inserted in E. coli exogenously)
 . Flagellar secretion systems?
 . Type IV - Virulence factor-associated; not well characterized - unclear 1
   vs. 2 steps.
 . Type V - self-secreting proteins; not regulated by any cellular mechanism
         o Is there a sequence that you can put on a protein to make it
           secrete itself?
 . Registry of parts
         o Salmonella type III secretion pathway under development (Voigt
           lab) - very touchy with regard to protein stability ("Voigt
           secretion pathway")

Big question: What system should we use?
 . E. coli

Laura's presentation (mechanism of tyrosinase/mussel glue)
 . Tyrosine residues are oxidized to dopa and then further oxidized to a
   quinone (either by tyrosinase or spontaneously by O2 at basic pH)
 . The quinone can react with the amine groups of lysines, thereby
   crosslinking the Mgfp proteins
 . Nora thinks that she's read that metal ions in salt water catalyze
   oxidation
         o Could heavy-metal contaminants catalyze this?  Would this be
           practically useful?
 . Metals in rocks can get coordinated by hydroxyl groups on dopa; this
   allows the glue to stick to rocks underwater


Damon (mechanism of tyrosinase/mussel glue)
 . Trouble with expressing Mgfp due to difficulty in cloning repeat sequence
 . Mcfp-6 acts as a crosslinker; forms thiol adduct with dopa residues;
   increases stickiness, but not absolutely essential
 . Current product: Cell-Tak; they purify it from gazillions of mussels

Adhesion proteins
 . 6 different proteins in californicus
 . Mefp-5 has the highest dopa content - probably so sticky that it can't
   even be purified.

How do we want to characterize the protein that is produced - do we want to
purify it or just characterize the really sticky bacteria?

Laura's idea: build in a quorum-sensing mechanism that requires the cells
to reach a certain density before they can start secreting adhesive.  Maybe
build an AND gate such that adhesive genes are only activated IF (signal)
AND (quorum).

Daniel's presentation (bioengineering theory)
 . Need to develop a theoretical basis for our work.
         o May be able to adapt principles from electrical engineering.
 . Need standardized logical theory of biological networks
 . Goals of theory:
         o Develop a formal language of gene circuits


For next week:
 . Laura
         o Do more advanced reading into tyrosinase system
         o Has gene been cloned and have people tried to express it?
         o Work on funding
 . Damon
         o Talk to Sean Crosson - culture conditions, etc.
         o Review papers about Caulobacter
         o Meet with Chris Schoenbaum (Wednesday)
 . Thomas
         o Look up more about B. subtilis - look for availability
         o Look up Pseudomonas
 . Nora
         o More research on exogenous Type III systems for E. coli
         o Look up pre-existing secretion systems in the registry
 . Rob
         o Quorum sensing mechanism (check out work of Ron Weiss)
 . Daniel/Ioana
         o Also look into quorum sensing mechanism
         o Read some stuff about reaction-diffusion mechanisms
 . Jata
         o Look up other secretion systems
         o Look up other things at will