Interesting Mistake

Right now I am working on a protocol procedure which extracts periplasmic DNA from gram negative bacteria. My objective is to assess if I can extract sheared genomic DNA of Haemophilus influenzae from the periplasm without any contamination of either chromosomal DNA or DNA fragments that were not taken up. But first I did a control experiment assessing if Map7 sheared genomic fragments can be transform and what is their transformation frequency.

For this experiment I transformed 0.25kb average sheared Map7 DNA , 6kb average sheared Map7 DNA, and not-sheared Map7 DNA into kw20 cells that were previously made competent (5 months ago).

For my control with non-sheared Map7 DNA I obtained a transformation frequency per ml of 3.6e-4. This number was very surprising since The competent cells that I froze 117 days ago had a transformation frequency of 2.3e-3 (6-7 fold less). The cells didn’t seem to dying in the freezer because the number of CFU/ml in both cases was >1e+9 (which is expected). Then I remembered that our lab technician (Yvonne) and our visitor professor (Lauri) had a similar phenomenon happening to their experiments. So, I asked them for their results to compare with mine:

 

Days since fresh Trans/ml CFU/ml Researcher
0 2.1e-3 1.29e+9 Yvonne
1 2.3e-3 1.1e+9 Marcelo
1 1.4e-3 8.1e+8 Yvonne
55 7.5e-4 1.38e+9 Yvonne
117 3.6e-4 1.26e+9 Marcelo

Yvonne and Lauri had a 3 fold decrease in competence when cells were on the freezer 55 days, while I had a decrease of 6 -7 fold when cells were 117 days in the freezer. Even though this results might sound very simple they are important to consider when doing experiments because researcher might check competence only once after making the cells but no after a certain time in the freezer. This results suggest that a bias from 2 to 10 fold could be introduce when using old cells or even worse when comparing experiments with cells of variable age (since frozen).

Implications for my experiments mean that I will have to make new stocks of all my competent cells, except maybe pilA mutants with should not be transformable anyway.

I am really glad I made this preliminar experiment : )

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