Friday, July 8, 2005
- Got the sequences
- In the process of modifying the scFv and ToxR, and optimizing ctx sequences
- Have inserts for scFv
- Have specified: PhoA, MalE, ctx promoter WT, ToxR WT, ToxR', reporter ctx-GFP
- Would like help with "in framing" the scFv sequence with ToxR
- specify scFv, make composite parts, get florescein primers
- run by advisors --> order from BlueHeron
- set up experiments
- Heard back from UCSF --> going to ask more about
- Looking into topology and binding mechanism of antibody and scFv
- Need help with topology
- Read up on binding mechanism of antibody and scFv
- Find sequences
- Specify them
- Two conflicting mechanism on how it works
- Creative ideas
Monday, June 27, 2005
- We want an input/output system that is modular
- Summer: finish research for pathways, antibodies, and antigens, and successfully build a gene fusion
- We should name a few applications just so people see the significance of our project
- OMP-R (Outer Membrane Protein)
- Mating factor -> G protein
- Cell Wall Integrity -> does not dimerize
- 2-component (Che, NTR)
- TGFB (mammalian)/Cytokine/RTK
- E. coli: preferred but we haven't figured out how to deal with the cell wall
- Yeast: (same as above)
- Many pathways are in mammals so if we can't figure out how to work with yeast/mammalian cells
Friday, June 24, 2005
General Issues to Consider
- What will team define as a successful summer?
- will fusing antibody to protein and testing to see that you got expression be "enough"
- is planning out and presenting a theoretical project "enough"
Antibody signaling group
- Distance between epitopes that would cause transmembrane domains to dimerize?
- Epitopes on the antigen--same or different?
- Can you dimerize the two fusions @ all….are the primary antibodies too big?
- Look @ light receptor from Texas project…Omp system from E. coli?
- Single chain variable regions instead of entire antibody
- Polyclonal antibody instead of monoclonal for the “primary”
- Where to look for sequences of antibodies?
- Antibodies to latex, starch, glycogen, other biological polymers that are unstructured
- Is the metagoal to have a biological sensor?
- Surface expressed/membrane associated antibodies: how do they react to antigen binding? Can we copy that mechanism? Might that allow team to get away from dimerization of the antibody-protein fusions?
- Could we use a yeast pathway that signals in similar ways to a mammalian pathway (like Epo) to get around the issue of moving a pathway from mammalian cells into yeast cells?
- Are their receptor tyrosine kinase pathways in yeast? (Epo signals that way)
- What about bacterial signaling pathways?
- What pathways might be exploited for sensing in yeast?
- mating pathway
- HOG pathway (osmoregulation?)
MAJOR ISSUE: the problem of the yeast cell wall--can't get the antigen or primary antibodies across it!!
Antibody fusion group
- explanation of epitope tags and how they were used in yeast surface display system (Wittrup)
- What is the probability of having an antibody that actually binds to your target of interest?
- Creating and screening a library would take YEARS
- Might you use antibody from the Wittrup lab as a starting point?
- How to fuse the antibody and other protein: how are enzyme linked secondary antibodies made? Are they made as fusion proteins, or by in vitro crosslinking the enzymes and proteins themselves? If fusion protein, then then company should have the sequence encoding the antibodies.
Advisors would like to see by Monday's briefing
- Decision on what system the team will use (yeast, bacteria, etc.) and WHY.
- Decision on what protein will you fuse to for signaling/what signaling pathway will be exploited?
- Clear description of application/vision, for both v1.0 and v10.4 of your system. How will you build on this prototype, which can only do XYZ, to meet the goal you think is really neat which is ABC....
Monday, June 20, 2005
- We need to plan to plan.
Friday, June 17, 2005
- Goals and Objectives
- Immediate goal: glucose induces bacterial cells to make proinsulin (picture demonstration of system)
- Further goals:
- standardizing the system to allow for all types of inputs and outputs (diagram)
- proinsulin processing and insulin exporting out of the cell
- quantification of signal
- Steps We Have Taken
- Biobrick Diagram (show and explain)
- Found a list of "available and working" parts in registry to test in parallel (have a backup list of "available parts")
- Found insulin available on e. coli plasmid from invitrogen (and obtained insulin sequence), and other "available" parts to test in parallel
- What We Need to Do
- Find and test parts chosen
- Plan out how to make our own biobrick pieces if necessary
- Order registry parts, insulin plasmids, BlueHeron parts
- Pour/buy plates and lab materials (by Monday)
- Culture cell strains
- Find and test parts chosen
- Can someone else sign so we can get lab materials so that we don't have to wait for Isadora for Monday?
Probably...I don't do ordering for 7.02, so I'm not much help. We can use 7.02/BE109 reagents for now until our order comes in. Shouldn't be a big deal to wait until Monday, really. -KBS
- Putting parts together??
- CAP/CRP help polymerase bind alpha subunit bind.
- Book on Lac operon - Beckwith(sp?) --Drew has one!
- Muller-Hill book --TK has it. Description of state of knowledge of system, historical version.
- Lac Operon is not completely in the library (truncated)
- Start thinking about linking two insulin bonds
- Signal peptide sequences
- LSP gene
- LSP A; protein secretion pathways
- Derman - worked with disulfied bonds.
- Think of some tests we want to use for quantification.
- beta Gal
- System = Vector NTI. DNA editing tool for annotation
- This system is very doable!! Yay!
- NEED TO DO
- We need to plan a time to be in lab. Rough sketch for Kate.
- Design experiment to test the system
- Synthesis of necessary parts (BlueHeron) and get insulin (also check the DNA seq. of cI of Or2 and Or3 against seq. in registry)
- Get pre-existing parts --> Jemma's group
- Get stamp and get cells for streaking
- Culture sample cells for FACS certification
- Gather books and reference materials