Recombination Assay Sample Clauses

Recombination Assay. In order to characterize the dynamics of recombination mediated by transformation in S. pneumoniae we inoculated chemostats with a mix culture of two otherwise isogenic R6 strains carrying two different antibiotic resistance markers, rifampicin (R6-Rif) and streptomycin (R6-Str). We estimated the number of recombinants by counting assessing the number of doubly marked strains resistant to both rifampicin and streptomycin. The The culture conditions were as described previously for single strains chemostats. Before inoculation, the flow of the chemostats was stabilized and frozen aliquots of R6- Str and R6-Rif were grown in THY to OD600 ~ 0.3 nm and adding 100 μl. To follow the densities of the parental and recombinants strains, we plated samples at appropriate dilutions on THY (5 % blood) agar supplemented with streptomycin (40 μg/ml) to follow streptomycin resistant strains (R6-Str), on agar supplemented with rifampicin (25 μg/ml) to follow rifampicin resistant strains (R6-Rif); and agar supplemented with both antibiotics at the same concentrations mentioned before to follow recombinants carrying both markers. Sampling was performed at hourly intervals for 80 hours. Control chemostats were followed to assure that the double resistant strains did not arise by mutation. For this individual chemostats carrying R6-Str and R6-Rif strains respectively were inoculated in a similar way, and in parallel to, the mixed culture chemostats. This chemostats were sampled hourly for 80 hours in THY (5% blood) agar to estimate total density of the culture and with THY (5% blood) agar supplemented with streptomycin (40 μg/ml) and rifampicin (25 μg/ml) to follow the rise of double resistant mutants. In addition to the previously mentioned controls, we performed fluctuation tests on R6- Str and R6-Rif in order to estimate the mutation rate for the gain of rifampicin resistant in R6-Str backgrounds and streptomycin resistance in R6-Rif backgrounds. For this, 24 parallel cultures of R6-Str and 24 parallel cultures of R6-Rif were grown overnight in 2 ml of THY yeast (media as described earlier). 5 cultures were sample and plated at appropriate dilutions to estimate the average population size of the cultures and the totality of the cultures were centrifuged at 14,000 rpm for 10 minutes, re-suspended in 200 μl of saline solution and plated in the corresponding selective agar. Plates were incubated at 35oC for 48 hours, and the number of colony forming units in each culture was a...
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