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Team:Evry/Biology/CellCharacterization/Antibiotics
From 2014.igem.org
Sensitivity to 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.
Table 1: Concentrations tested for each antibiotic
We tested several concentrations of each antibiotics (cf: Table 1), 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: Figure 1). We tested on Marine Broth (MB) 1X plates, MB 0.5X plates (used for conjugation) and M9 1X (+3% NaCl). We also tested in liquid M9 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.
Figure 1: Protocol of antibiotics' tests on Pseudovibrio denitrificans.
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.
Figure 2: Results of antibiotics' tests on MB plates.
On the left: Results on MB 1X plates.
On the right: Results MB 0.5X.
Figure 3: Results of antibiotics' tests for M9 media.
On the left: Results on M9 1X plates.
On the right: Results in liquid M9 1X.
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: Table 2).
Table 2: Concentrations which can be used for selection of Pseudovibrio denitrificans
