I am characterising a plasmid found in a Salmonella species. from a foodborne outbreak of disease. The plasmid carries a tetracycline resistance gene and I would like to perform a mating experiment to determine if the plasmid is conjugative. The Salmonella species donor strain is sensitive to ampicillin so I have chosen a recipient strain that carries an ampicillin resistance gene. What antibiotic(s) do I need to include on my agar plate to select for transconjugants?

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The scenario involves a donor Salmonella strain that is tetracycline-resistant but ampicillin-sensitive, and a recipient strain that carries ampicillin resistance. The goal is to identify transconjugants—cells that have received the plasmid by conjugation.

Option 1: 'Any two antibiotics as long as one is not ampicillin' — This is incorrect. Transconjugants must be selected for both traits that indicate transfer: the recipient’s ampicillin resistance and the donor’s tetracycline resistance. Using two arbitrary antibiotics could allow growth of bacteria that did not acquire the plasmid, or fail to distinguish donor from recipient, leading to false positives or negatives.

Option 2: 'Ampicillin only' — This would select for ampicillin resistance, but would not differentiate transconjugants that simply retain the original recipient or donor background. It would fail to require tetracycline resistance, so plasmid transfer might not be detected if the transconjugants do not carry the tetracycline resistance gene.

Option 3: 'Tetracycline only' — Similar problem as Option 2, but reversed. It would select for tetracycline resistance, which the donor carries, but would not require ampicillin resistance from the recipient. This could allow parental donor bacteria or contaminants to grow rather than true transconjugants.

Option 4: 'Any antibiotic as long as it is not tetracycline' — This is overly broad and nonspecific. Without forcing co-resistance (both markers), you cannot confirm that the bacteria acquired the plasmid via conjugation. It also risks selecting organisms that do not have both resistance traits.

Option 5: 'Ampicillin and tetracycline' — This is the correct approach. By using dual selective pressure for ampicillin (recipient’s resistance) and tetracycline (donor’s resistance), you enrich for cells that received the plasmid through conjugation and thus express both resistance traits, distinguishing true transconjugants from parents or contaminants.

In summary, to reliably identify transconjugants carrying the tetracycline resistance plasmid while retaining ampicillin resistance from the recipient, the appropriate selection is a medium containing both ampicillin and tetracycline. The other options fail to enforce this dual selection and thus are not suitable for discerning successful conjugation events.

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