Team:Evry/Biology/CellCharacterization/Antibiotics

After having succeeded to make our bacteria grow, we tested their resistance to different antibiotics. Knowing their resistance to antibiotics is very important because it will allow us to finalize protocols of selection after transformation.
We chose to test the most commonly used antibiotics. We included the three antibiotics used in iGEM (kanamycin, chloramphenicol and ampicilin), plus the erythromycin and the tetracyclin. We chose the erythromycin to test a conjugation protocol which required this antibiotic for E.coli, and we chose to test tetracyclin because it is quite often used for inducible systems.


We tested several concentrations of each antibiotics (cf: Table2), and we added a supplementary concentration for erythromycin because it is known to be not very effective on GRAM- bacteria.

The sensitivity tests were performed in four different conditions (cf: Figure3). We tested on Marine Broth (MB) 1X plates, MB 0.5X plates (used for conjugation) and M9-CASA 1X (+3% NaCl). We also tested in liquid M9-CASA 1X (+3% NaCl) to have a test in liquid culture, but unfortunately we cannot test on liquid MB cultures because this media is no usable for the spectrometer, so we cannot have a good information about the cells' growth.
The cells' sensitivity was measured on our strain Pseudovibrio denitrificans, E. coli, and different clones of E. coli transformed with plasmids and carrying an acquired resistance AmpR, KanR, CamR ou ErmR.

We let all the media incubating at 30°C (with shaking for liquid cultures), and they have been checked at day 1, day 2 and day 3. In the following figures, find the results obtained after three days of cultures on plates and one day of liquid cultures.
NB: The purcentage of growth of cells on plates were calculated by taking the controls without antibiotics as reference.
Unfortunately, results for tetracyclin on MB media are not useful. However, this year, biobricks have to be cloned in PSB1C3, and we have very good results for chloramphenicol. According to these results, we know that we can select transformed Pseudovibrio denitrificans on a concentration of chloramphenicol adjusted to the ratio 1/1000 (cf: Table3).
To use other drugs, best ratio (Growth on antibiotic/Growth on medium only) allowed to determine optimal concentrations to use on our different media (cf: Table3).