To find pathogens capable of conjugation, we screened Carb-resistant isolates for the ability to transfer β-lactamase into Carb-susceptible recipients. Pathogen cultures were grown overnight in 1 ml of LB media with Carb (100 μg/ml) in 96–deep well plates covered in a gas-permeable membrane. Recipients were grown overnight in 3 ml of LB media with either Kan (50 μg/ml) or Cm (70 μg/ml). Pathogen isolates were then diluted 10× in LB media and regrown under similar conditions for 2 hours to enter exponential phase. Exponential-phase pathogen isolates and recipient were diluted 100× into 200 μl of LB media and mixed 1:1 in a 96-well plate with dual antibiotic selection for transconjugants. Mixed cultures were then covered with 50 μl of mineral oil and grown in a plate reader for 24 hours.

After 24 hours of exposure to transconjugant selection, mixed pathogen and recipient cultures that displayed growth were diluted 1000× into fresh LB media with antibiotic selection for transconjugants. These cultures were covered with 50 μl of mineral oil and regrown in a plate reader for 24 hours. Cultures that repeated growth under high dilution and strong transconjugant selection were plated on agar also selecting for transconjugants. Individual colonies were isolated and grown overnight in 3 ml of LB media with transconjugant selection. These clonal transconjugant cultures were glycerol stocked for later analysis.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.