Field of Science

DNA and biofilm formation, planning

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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