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.
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in The Biology Files
Not your typical science blog, but an 'open science' research blog. Watch me fumbling my way towards understanding how and why bacteria take up DNA, and getting distracted by other cool questions.
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