The Daily Grind
- Met with Paul, and he encouraged that I try electroporating the Arabad::GFP in pDRIVE into as many endophytes as I can, then test them out on potatoes and corn at huge concentrations of arabinose (to get maximum expression at sublethal levels) to force expression. This is a good idea and better than messing around further with vector construction. Once I have a few more, non-pathogenic endophytes tagged it Arabad:GFP, I will try the assay with both potatoes and corn. Already have 1A1, 1A8, 1B12, 1C4, 1C8, 1E9, 1G6, and Kp342. Now growing 1A5, 1B2, 1C12, 1D9, 1D12, 1E2, 1E10, 1E12, 1F1, 1F6, 1F7, 1F12, 1G4, 1G8, 1G11, 1G12, 1H2, 1H7 as well, to try them out. Lets see which are the best canditates for further work (ie. best at arabinose inducible expression while being non-pathogenic) - of these, only 1E10 and 1F7 took up the plasmid and express GFP.
- So I have 1A1, 1A8, 1B12, 1C4, 1C8, 1E9, 1E10, 1F7, 1G6, and Kp342 as pDRIVE transformable endophytes. Will characterize these again for Arabinose response at levels of 0, 500, 1000, 2000, and 5000 ug/mL of R2A media. Since I already have 0.01% Arabinose media (1 ul of 10% arabinose per mL of PNCM) I will innoculate these 10 into potatoes and see which are good or bad for plants, B) whether plasmids are stable/maintained for a month and C) in alive plants, where are the endophytes located (are they fluorescing and where are they?)