Bacteriophage Lab

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Feb 20, 2024

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Madison Karpinski Biology 2296, section 11 Dr. Bichenkov April 5, 2023 Bacteriophage Reversion & Recombination Objectives: Measure the reversion rate of the bacteriophage mutants T4 rII 29 and T4 r II 31 to wild-type recombination. Determine the recombination frequency of the mutants and use that data to find the map distance for the mutants present on the rII region on the phage’s chromosome. Methods: Permissive E. coli B and restrictive E. coli K was infected with the mutant T4 rII 29 and T4 rII 31 and closely watched as the mutants reverted into wild type (70). The permissive E. coli B allows the T4 rII mutants to lyse and reproduce in the culture. On the other hand, the T4 rII mutants cannot lyse and reproduce in the E. coli K culture, only T4 wild type are able to lyse the E. coli strain K. A serial of dilutions was created using varying 10-fold additions of phage stock and phage buffer. Each dilution was made up of both mutants T4 rII 29 and T4 rII 31 mixed together, three containing the E. coli B and three containing E. coli K. The dilutions are plated on the

agar plates and incubated for 24 at 37 o C and 6 days at 4 o C. From infecting each permissive and restrictive E. coli strain, a reversion frequency could be calculated. This rate was calculated by dividing the wild-type titer by the mutant stock titer. As mentioned before, the restrictive host E. coli K was only able to be lysed by the wild- type phage 29. Log phase E. coli B and the mutant phage mixture (T4 rII 29 and T4 rII 31) had a 1:20 ratio and was incubated at 37 o C for 9 minutes without shaking to allow the bacteriophage to absorb to the bacteria (p. 76). The infected mixture was then added to the growth broth and incubated in a shaking water bath at 37 o C. This growth flask was then used for plating. Within 10 minutes after the incubation the titer of the unabsorbed phage is started by the addition of chloroform. The chloroform kills and lyses the E. coli cells. Thus, the chloroform mixture was only free phage (not attached to bacterial cell wall) and were able to reproduce to form new plaques. This aids in finding the total concentration of unabsorbed phage. With chloroform added later, infected cells will lyse and release a progeny phage. 4 U dilutions are made to titer the unabsorbed phage. The unabsorbed phage is mixed with E. coli B and then given time to grow a lawn on the agar plate. The lawn of plaques was resulted from the infection of an E. coli cell in the assay by a free phage particle. The plaques formed were scored.

Titering the progeny phage, began with the incubation growth flask for 60 minutes to allow for bacterial growth. After initial incubation, chloroform was added before another 15 minutes of incubation at room temperature. The chloroform was added before the titer to allow for the intracellular phages to form during the 60- minute incubation period and allow for bacterial growth. Adding the chloroform at any other time would have skewed the recombination frequency of the progeny. A series of A dilutions were made to titer the progeny phage capable of infecting E. coli B and capable of infecting E. coli K. The total progeny phage was measured by infecting E. coli B. The wild-type phage was measured using E. coli K and the diluted lysis series. The E. coli B and E. coli K were incubated at 37 o C for 1 day and 4 o C for 6 days. Following incubation, recombination frequency was found by multiplying the titer on E. coli K by 2 and dividing the titer on E. coli B minus the titer on U. Results: Table 1: Titer of T4 rII stock 31 E. coli B Plate Fold Dilution Volume Plated (mL) Number of plaques Titer (a/b*c) T4/mL 10 7 B 10 7 0.1 mL 46 4.6 x 10 9 10 6 B 10 6 0.1 mL 220 2.2 x 10 9 10 5 B 10 5 0.1 mL <<300 Titer of Mutant Stock= (46/0.1) (10 7 ) = 4.6 x 10 9 Phage particles/ml (220/0.1) (10 6 ) = 2.2 x 10 9 T4/mL Table 2:

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