35. Two auxotrophic strains of E. coli: A (met^– bio^– thr⁺ leu⁺ thi⁺) and B (met⁺ bio⁺ thr^– leu^– thi^–) were incubated together for 18 hours in liquid medium and then ~10⁸ cells were plated on minimal medium. Prototrophs were observed at frequency of 1 X 10^-7 cells. This may have happened by process of genetic recombination between two strains or by mutation of strains. Which of the following control experiment would help rule out the

possibility of mutation?
(1) Plating strains A and B directly on minimal medium
(2) Growing the mixture of strain A and B for 18 hours and then plating on complete medium
(3) Growing strains A and B individually in a liquid complete medium for 18 hours and then plating on a minimal medium
(4) Growing the obtained prototroph in a liquid complete medium for 18 hours and then plating them on minimal medium

Auxotrophic E. coli strains A (met⁻ bio⁻ thr⁺ leu⁺ thi⁺) and B (met⁺ bio⁺ thr⁻ leu⁻ thi⁻) produce prototrophs at 1 × 10⁻⁷ frequency after 18-hour incubation, indicating genetic recombination (conjugation) rather than mutation, as spontaneous mutation rates are far lower (~10⁻⁹ to 10⁻¹⁰). The correct control to rule out mutation is option (3): growing strains A and B individually for 18 hours in complete medium, then plating ~10⁸ cells each on minimal medium—if no or negligible prototrophs appear, mutations cannot explain the mixed-culture result.​

Experiment Background

In the classic Lederberg-Tatum setup, complementary auxotrophs (unable to grow on minimal medium due to specific biosynthetic defects) are mixed to detect gene transfer via conjugation, yielding rare prototrophs (met⁺ bio⁺ thr⁺ leu⁺ thi⁺) that grow on minimal medium. The 10⁻⁷ frequency matches bacterial recombination rates, not back-mutation, which requires multiple independent reversions (probability ~10⁻³⁰). Controls distinguish these mechanisms by isolating strain interactions.​

Option Analysis

• Option 1: Plating A and B directly on minimal medium
  Both strains are auxotrophic, so no growth occurs, confirming initial phenotypes but not addressing mutations during 18-hour incubation.​

• Option 2: Growing mixture for 18 hours, plating on complete medium
  All cells grow on complete medium regardless of genotype; no selection for prototrophs, failing to test minimal-medium growth or mutation/recombination.​

• Option 3: Growing A and B individually for 18 hours in complete medium, then plating on minimal medium (Correct)
  Mimics experiment duration/numbers without mixing; zero prototrophs prove no revertants arose via mutation in either strain alone, confirming recombination needs both strains.​

• Option 4: Growing prototrophs for 18 hours in complete medium, then plating on minimal medium
  Tests prototroph stability but assumes they exist; cannot distinguish original mutation vs recombination origins.​