Bacteriophage Lab

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.

Titer of T4 rII stock 29 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 12 1.2 x 10 9 10 6 B 10 6 0.1 mL 280 2.8 x 10 9 10 5 B 10 5 0.1 mL <<300 (280/0.1) (10 6 ) = 2.8 x 10 9 T4/mL Table 3: Titer of T4 rII wild-type reversion stock 29 E. coli K Plate Fold Dilution Volume Plated (mL) Number of plaques Titer (a/b*c) T4/mL 10 3 K 10 3 0.1 mL N/A N/A 10 2 K 10 2 0.1 mL 37 3.7 x 10 4 10 1 K 10 1 0.1 mL 111 1.11 x 10 4 Titer of Mutant Stock= (37/0.1) (10 2 ) = 3.7 x 10 4 Phage particles/ml (111/0.1) (10 1 ) = 1.11 x 10 4 Reversion Frequency: wild-type titer/ mutant stock titer : (p. 80) (3.7 x 10 4 )/ (2.8 x 10 9 ) = 1.32 x 10 -5 Table 4: Titer of T4 rII wild-type reversion stock 31 E. coli K Plate Fold Dilution Volume Plated (mL) Number of plaques Titer (a/b*c) T4/mL 10 3 K 10 3 0.1 mL <1 <10 -5 10 2 K 10 2 0.1 mL <1 <10 -6 10 1 K 10 1 0.1 mL <1 <10 -7 Titer of Mutant Stock= (<1) / 0.1 mL (10 1 ) = 10 2 Phage particles/ml Reversion Frequency: wild-type titer/ mutant stock titer: ( (<10 2 phage/mL)/10 9 phage/mL)=<10 -7 Table 5:

Total Phage Progeny after infection of E. coli B (A series) Plate Dilution Factor Volume Plated (mL) Number of plaques Titer (T4/mL) Average 0.1 mL 10 4 10 4 0.1 mL 96 9.6 x 10 6 9.6 x 10 6 0.2 mL 10 4 10 4 0.2 mL 135 6.75 x 10 6 0.1 mL 10 5 10 5 0.1 mL 3 3.0 x 10 6 0.2 mL 10 5 10 5 0.2 mL 15 7.5 x 10 6 0.1 mL 10 6 10 6 0.1 mL 10 1.0 x 10 8 0.2 mL 10 6 10 6 0.2 mL 25 1.25 x 10 8 Titer (T4/mL): (96/0.1) (10 4 ) = 9.6 x 10 6 The plate of 0.1 mL 10 4 is the most reliable plaque count because it follows the 30 to 300 rule. Table 6: Unabsorbed rII Mutants (U series with E. coli B) Plate Dilution Factor Volume Plated (mL) Number of plaques Titer (T4/mL) Average 0.1 mL 10 3 10 3 0.1 mL 72 7.2 x 10 5 0.2 mL 10 3 10 3 0.2 mL 120 6.0 x 10 5 6.0 x 10 5 0.1 mL 10 4 10 4 0.1 mL 15 1.5 x 10 6 0.2 mL 10 4 10 4 0.2 mL 26 1.3 x 10 6 Titer (T4/mL): (72/0.1) (10 3 ) = 7.2 x 10 5 (120/0.2) (10 3 ) = 6.0 x 10 5 Table 7: Total Wild Type Phage Progeny after infection of E. coli K Plate Dilution Factor Volume Plated (mL) Number of plaques Titer (T4/mL) Average 0.1 mL E 1 0.1 mL >>>300 >3.0 x 10 3 0.2 mL E 1 0.2 mL >>>300 >1.5 x 10 3 5.2 x 10 3 T4/mL 0.1 mL 10 1 10 1 0.1 mL 52 5.2 x 10 3 0.2 mL 10 1 10 1 0.2 mL 21 1.05 x 10 3 0.1 mL 10 2 10 2 0.1 mL 22 2.2 x 10 4

need to be accounted for. Yet, the recombination frequency and map distance are not always reliable because when multiple crossovers take place they can go undetected. On the other hand, the reversion frequency showed the rate at which the mutants T4 rII 29 and T4 rII 31 changed back to the wild-type gene. In the case of this experiment, the reversion rate was found by dividing the wild-type titer by the mutant titer. The mutants T4 rII 29 and T4 rII 31 infected the permissive E. coli B and restrictive E. coli K. Once infected, it was seen how the wild-type phage could grow on the E. coli K, but both mutants were unable to due to E. coli K’s restrictive capabilities. Yet, the mutants T4 rII 29 and T4 rII 31 could easily grow on the permissive E. coli B. So, the wild-type T4 phages could only be observed on the E. coli K cultures. Works Cited: Hartwell, Hood, Fischer, Goldberg. Genetics: From Genes to Genomes. 7th ed. New York: McGraw Hill, 2021. PDF. Biology Dept. Staff. 2023. Biology 2296 Lab Manual . Temple University.