You work with an E. coli plasmid called pPPM2, which codes for resistance to the

Question

You work with an E. coli plasmid called pPPM2, which codes for resistance to the antibiotic kanamycin and is fully self conjugative. In your studies, you have located the oriT. You clone this minimum oriT DNA sequence (25 bp in length) into the small cloning vector, pUC18 (2.8 kB), which codes only for replication functions, resistance to the antibiotic ampicillin, and production of ?-galactosidase and is not itself conjugative. You then introduce this recombinant clone, called pUC18ot into two strains of E. coli: Strain A, which contains no other plasmids Strain B, which also contains a wild-type copy of pPPM2 You then mate Strain A and Strain B independently with a plasmidless E. coli recipient, and look for recipients that have inherited pUC18ot from both crosses by selecting for recipients that are resistant to ampicillin. A. What would you expect from the mating with Donor A? What would you expect from the mating with Donor B? B. If you obtain ampicillin-resistant recipients, describe two possible mechanisms to account for them? How could you differentiate between the two mechanisms? C. Would you expect to obtain any recipients that are resistant to both ampicillin AND kanamycin? Defend your answer.

Explanation / Answer

0.3 ml donor cells contain 6.00e8 cells (0.3 X 2e9). The final volume was 0.3 + 1 + 3 ml = 4.3 ml. So the number of cells per ml is 6.00e8/4.3 which is 1.395e8 cells/ml (1.40e8 cells/ml to three significant places).

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