6/25 Project Meeting Notes by Stephanie
This week: try to boost colony count
*Potentially boost extension: perhaps increase the concentration of insert versus peptide
- As suggested by William, perhaps we should run a gel after ligation (before introducing transformation) - distinguish between circular and linear DNA
Nhe1 is actually a bad cutter - so when using it, perhaps we should use 3-4 times more than we would otherwise
One major issue of this meeting was the question of the value of "redoing" bacterial surface expression, since Bassette et al have already achieved this ... However, we should probably continue because of the following issues:
- Addition to Biobrick Library
- Different Targets
- Thought: should we ask Bassette if we can get the construct?
Pursuing the same target as their paper would resemble a positive control
Quorum was also a popular idea throughout this meeting.: Bacteria sense tumor cells, perhaps causing the downstream effect of expressing something MRI-sensitive
*Quorum is seen somewhat as a fallback plan right now, since quorum is known to work and has generally been successful so far.
Fec regulatory system
*Since Fec substrate recognition and transport domains are separate, perhaps we can change outer specificity
Signal Transduction Problems
- Ligand specificity
- Crosstalk: need normal genes: may be necessary for survival or may cross-interact
- E.coli not best organism to work with
Bottom line idea: dimerization: we may be able to hold proteins together, such that even without a ligand, it should dimerize and cause the downstream signal.
Ex: PhoR with one random ligand, which is specific for the N-term of the target protein. Another PhoR constructed with another random ligand, which would be specific for C-term of same target protein.
Another issue: many proteins are on inner membrane; perhaps we could overcome this by targeting with Lpp to outer membrane ... though this may present more problems since some necessary cofactors may not be present.
Perhaps we should look into the phosphate starvation genes: PhoR, PhoB, EnvZ. Given enough phosphate, maybe we can coopt this system.
Alain proposed that perhaps we could find a ligand through looking at the old ones and using those to guide our peptide exploration.
Another idea to approach this idea: put two known ligands @ end of artificial protein (aka strep tags)
- Strep may not be the best idea: binds biotin, cells tend to die
- maybe try His/nickel
- put 6-His at end of protein
- OR PDZ: Perry has already expressed on surface through Lpp-Omp (binds to hydrophobic, expressed on C-term)
- put 6-His at end of protein
Perhaps we should try electrocompetent cells
Note to all: look into what OmpA does
Look into signal transduction with proteins that diffuse through the outer membrane
- This has already been done a lot
- The "novelty" of this project is less than the other idea, in which we were looking toward affecting the cell surface/outer membrane protein directly.
The MIT 2005 project was brought up:
- Tried to express antigen on Fec
- Fec is nonessential
Other idea with Fec:
- Create new genes under control of sigma factor
- Put in parallel pathway (keep old genes as well)
- "First approximation": GFP
Another idea: use 2 ligands: one arbitrarily that acts as "loop" that is on iron citrate binding site; use another arbitrary ligand to release this
Also look into EnvZ as a possible pathway/mechanism?
Onward ... Plans:
- Look at Biobricks, see what might be available to us
- Create Plan (aka a single page sketch)
- Potential difficulties
- Identify prats - prep them, send them out for sequencing
- Tomorrow will act as a "decision" day
- Surface-exposed antibodies tethered on e.coli: Camel (single-stranded) antibodies
- Role of Fur gene in Fec pathway - regulator?
- MIT was able to have FecA promoter-RFP induced by iron
- Tox R (MIT 2005) - Cholera