The mysterious case of the bacterial ghost, or why I have low transformation frequency

I had a problem transforming KW20 cells with MAP7 DNA, during an experiment I started the 18th of June. I obtained 100 fold less transformation frequency (1-2e-5) from the expected (>1e-3). But the low transformation was not the only problem, number of colonies on plates were not very consistent. In sBHI+nal plates inconsistency was mild, but on sBHI+nov plates inconsistency was extreme. For example, in sBHI+nal plates, plating 20ul of transformed culture of a 1e-2 dilution gave me 4 colonies while plating 20 ul of a 1e-1 dilution gave me more than 200 colonies.

To solve this mysterious problem, I did an experiment (on the 23rd of June) transforming a frozen stock of the same competent cells from which I obtained the 1e-5 transformation frequency (TF) as well as some competent cells made by my supervisor. I tested transformants in nov and nal antibiotics, incubating transformed cells with two volumes of rich media at 37 degrees for 0, 50, 80 and 110 minutes (expression time). When using nov, our standard protocol do not use expression time so I expected to see no increase in TF as the incubation increased, while with nal plates previous lab experiments determined that optimum expression time is 80 min.

Results from this last experiment were completely different from the 1e-5 TF that I obtained before:

TF

Several results can be extracted from this figure:

1) My results are congruent with previous lab experiments showing that the optimum expression time for nal is 80 minutes.

2) I was able to obtain TF of 1e-3 but only for transformants in nal plates from my supervisor incubated 80 and 110 minutes. It is surprising (at least for me) that TF of my supervisor’s cells in nov (when time = 0) is 10 fold less (2e-4) than with nal (when time = 80).

3) I seems that a expression time of 50 minutes, increased slightly TF in nov plates.

4) There is approximately 2 fold difference in TF between my cells and my supervisor’s cells.

Since I still do not understand the reason behind the low TF that I obtained in the experiment of the 18th of June, I decided to look at all my notes carefully. The first thing I did was to think on what kind of things I did differently in both experiments:

A) In both experiments I used the same sBHI+nal plates but different sBHI+nov plates.

B) I used a new aliquot of NAD, well but …. the new aliquot was from the same batch from the old one.

C) I used a new aliquot of solution 23 (to make the minimal growth media, MIV for resuspending frozen cells).

D) For the June 23rd experiment I was very careful to use only fresh prepare reagents which include: resuspending cells in fresh MIV, and using fresh sBHI for incubating cells during the expression time.

Plates or agar did not seem to be responsible for the low TF since the same nal plates were used for both experiments. NAD was not responsible since the two aliquots used were prepared by the same person at the same time. In my opinion, from all the differences stated above, none would theoretically be responsible of a 10-100 fold decrease in TF (maybe D), but from this moment I will always use fresh sBHI and MIV. I will also code all my reagents, so I can identify which reagent aliquots were used in an experiment.

I am still puzzled about why I obtained 1-2e-4 TF with mine cells and my supervisor cells in nov plates when time = 0. All explanations so far are pure speculations, but it seems that either the cells are having problems expressing nov resistance gene or the antibiotic concentration might be too high. Since I cannot fins any logical reason why cell would have problem expressing the nov resistance gene, I can only think about the antibiotic concentration. I am sure that the antibiotic was mixed well in the melted sBHI bottle, so maybe the antibiotic stock was prepared at a higher concentration than what says on the tube.

Another results that puzzles me is the 2 fold difference that I obtained in TF using my cells compared my supervisor’s cells.  Maybe It is just part of the natural variation from cell to cell in transformation, but maybe there is a mistake in my technique when I induce competence of my KW20 cells.

The only variable that I can think is the incubation time of log KW20 cells in MIV media. Usually after cells have reached an OD of 0.2, cells are transferred to a starvation media named MIV and incubated 100 minutes.  Maybe my cells are being incubated too much time in MIV and they are starting to lose the competence state. Again but all of this explanations are pure speculations.

Finally the last possible explanation of why I had low TF is that there is a ghost in the lab that is killing some antibiotic resistant bacteria in plates. However, a human ghost would not have reasons to kill bacteria, so it is clear that only a bacterial ghost or a phage ghost could come back to life and kill my bacteria.

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