I want to test whether their ability to bind DNA helps competent cells form or join biofilms.
The standard way to test for biofilm formation is to incubate cells in a polystyrene microtiter-well plate, remove the unbound cells, and stain the remaining bacteria with crystal violet. But I don't know anything about whether DNA sticks to polystyrene (I can only find bapers about chemical methods of attachment, so I don't think it spontaneously sticks), and I think it's better to do the test under conditions where biofilms don't form easily.
As a preliminary test, I first want to coat the inside of glass culture tubes with a film of DNA. I'll then add dilute H. influenzae cells, either wildtype or constitutively competent, and incubate overnight (with or without constant mixing? probably I'll try both). The on-line sources say that DNA binds 'avidly' to glass under conditions of high salt and low pH (below 7.5).
So I'll prepare a high-salt low-pH stock of H. influenzae chromosomal DNA (100 µg/ml DNA, 1 M NaCl, 50 mM Tris pH 7.0). I have an old high-concentration DNA stock somewhere. I'll add 2 ml of this to new (sterile)glass culture tubes and let them sit for 1 hr at 37 °C. Then I'll pour out the DNA (saving it for next time), and leave the tubes to dry at 37 °C overnight. In the morning I'll fill the tubes with 1M NaCl 50 mM Tris pH 7.0, leave them for 30 min, vortex them and discard the wash solution. Then I'll add 5 ml of cell culture in sBHI, at a density of ~ 10^7 cfu/ml, and incubate the tubes at 37 °C for 5 hr or overnight. Then I'll remove the cells, and add crystal violet (0.05%) to stain the biofilm. After 10 min I'll wash the tubes with water twice, and then dissolve the remaining crystal violet in 95% ethanol and measure its absorbance at 570 nm.
What cells will I use? Wildtype cells, hypercompetent mutants (sxy-1, murE749), noncompetent mutants (sxy-, cya-, pilA-). I'll have control tubes with no DNA, and tubes with added DNase I.
9 hours ago in Variety of Life