1. I tested cell adhesion to the coverslips the RA had cleaned for me - it wasn't dramatically better or more consistent than either uncleaned or acid-washed coverslips (after all had been coated with poly-L-lysine using his spreading method. Overall plenty of cells are adhering, given that I'll only need a small number of cells adhered for each chamber. I think part of the trick was inverting the slides for 10 minutes before washing away the nonadherent cells, so the cells settle on the coverslip. I'm also giving the slides a few brisk taps during the washing, so that loose cells detach and wash away.
2. Incubating the chambers with concentrated BSA protein didn't significantly reduce the number of H. influenzae or B. subtilis cells that stick to the poly-L-lysine coated coverslips. Nor did it prevent the polystyrene beads from sticking to the coverslips. A Google search for treatments that might block/inhibit the poly-L-lysine surface revealed only that people are coupling poly-L-lysine and polyethylene glycol or other materials into 'block' copolymers, and testing the ability of poly-L-lysine to 'inhibit' cell growth and various enzymatic reactions. I didn't test BHI directly, but cells growing in BHI did adhere to the coverslips so I don't think it's much of an inhibitor either. I don't know how big a problem the unwanted sticking is going to be, but I'd welcome suggestions for other possible blocking agents to test. Just for fun I'm going to test milk. Not reconstituted powdered skim milk, but whatever's in the food fridge (2% milk, I think).
3. I made my big batch of competent cells and froze 34 tubes (0.5 ml), as well as 14 tubes of log-phase cells. But I haven't yet tested how competent they are.
4. Several tests showed that the H. influenzae cells that are stuck to the coverslips are otherwise healthy. In the first test I filled the chambers with sBHI plus low-melt agarose and incubated them overnight. I didn't seal the ends of the chambers but put the slides in a humidified box, but the chambers dried out completely overnight. But I could see that there had been lots of cell doublings before the medium dried up. So next I tested sealing the chamber ends with nail polish. The tweezers lab people use wax from a candle, because they worry that the nail polish might be toxic, but I didn't have a candle. The nail polish is indeed toxic (probably because acetone is quite water-soluble); the cells close to the ends of the chamber didn't divide at all. But the cells in the middle part of the chamber grew well, producing nice tight microcolonies by the end of the day. I brought in a candle, and today I'll see if I can improve my skill at applying melted wax to chamber ends.
5. I think today I'll also test whether the cells on the coverslips can be transformed, by washing in MAP7 DNA, and then DNaseI and then sBHI agarose with added novobiocin. Provided I have thousands of cells stuck to the coverslip, I should be able to find rare transformant colonies. I'll try this with B. subtilis too, selecting for Trp+.
I'd suggest trying the PEG to block, as it is more hydrophobic than poly-lysine, which is likely positively charge under your experimental conditions.
ReplyDeleteNancy, thanks for the suggestion. Might you be able to point me to a protocol, or to a PEG type (there are different types, right?) and a concentration?
ReplyDeleteRosie:
ReplyDeleteYes there are many "types" of PEG, mostly based on molecular weight. But there are also many modifications that might be useful as well. Have a look at:
Hyperbranched Fluoropolymer and Linear Poly(ethylene
glycol) Based Amphiphilic Crosslinked Networks as Efficient
Antifouling Coatings: An Insight into the Surface
Compositions, Topographies, and Morphologies
GUDIPATI et al. Journal of Polymer Science: Part A: Polymer Chemistry, Vol. 42, 6193–6208 (2004)
(use GOOGLE Scholar and a .pdf will be found)
Am not sure if this is exactly what you need, but it should help.
While I'm here I wanted to follow up on a question posed around the 20th concerning EcoRI cutting inside the resistance gene and if so - then how do resistant cells come about?
ReplyDeleteThanks Nancy, I'll take a look at that paper.
ReplyDeleteClem, EcoRI cutting in the gene doesn't completely destroy the mutation's ability to recombine with its chromosomal allele. It just greatly increases the likelihood that exonucleases will chew up that part of the DNA before it gets a chance to recombine.