I've been reading the classics - papers on Haemophilus influenzae competence and transformation published in the 1960s. They contain lots of information that, if true, is important for our work. But we know it's all too easy to 'discover' things that turn out to not be true, so we'll need to proceed cautiously.
Here are some factoids that contradict my current assumptions:
The media additive hemin loses potency after for a few weeks in the fridge . (We've been keeping it for years.)
Washing competent cells removes one or more factors needed for DNA uptake.
Cells have a nucleoside phosphorylase that is active in the periplasm - this means it acts on nucleosides that haven't been taken up by the cell yet. This may be important for understanding why purine nucleotides and nucleosides inhibit competence but purine bases don't.
Cells bind about five times more DNA than they take up.
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Any references, particularly for the last one?
ReplyDeleteBarnhart and Herriott 1963 Biochem Biophys Acta 76:25-39.
ReplyDeleteI made new hemin a few months back and compared uptake with this and old hemin, and could not see any startling difference. Maybe in early experiments, people didn't pasturise it and so it got contaminated over time?
ReplyDeleteI have not noticed differences in Hemin either. When I first arrived you and I also did some transformation experiments where we varied the Hemin stocks used and also saw no difference (if I recall correctly).
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