Preparation of plate lysates of P1 virus grown on two E. coli strains

 Phage Lysate: A Brief Rundown of the Procedure

October 29th, 2021

Introduction

In the laboratory, it is absolutely essential to plan for experiments ahead because they take time to perform. For instance, it is much more practical if growth media and/or agar plates were already prepared before a growth experiment, as this both saves time and effort for research students. Similarly, a transduction procedure requires a bacteriophage, and if the stock of that is low or if the transduction procedure is planned to be performed again in the near future, it is logical to prepare a plate lysate of the bacteriophage from the products of previously infected bacterial cells. This week's objective is to produce a plate lysate solution from two E. coli strains, JW5055 and JW0391, that were infected with the bacteriophage P1. This procedure involves extracting the P1 viruses from intact E. coli cells grown in top agar for it to be used in future transduction procedures.

Strategy

Since the concentration of the P1 virus used to infect JW5055 and JW0391 was high (with a dilution of 1/100), many P1 viruses are still alive inside of E. coli cells that have not been lysed by the virus. This is helpful because a plate lysate solution with a good concentration can still be retrieved from the growth plates. There are four growth plates to begin with, two for each of  JW5055 and JW0391. Using a spatula, the top agar of the two JW5055 growth plates is scraped off and transferred to a 50 ml centrifuge tube. Then, 2 ml of LB broth is poured on top of the remaining top agar in the two plates to be collected with a transfer pipette. The content is dispensed into the 50 ml centrifuge tube. This method is then used again to collect the top agar from the two JW0391 growth plates into a separate 50 ml centrifuge tube. 

After both tubes have the collected content, 2-3 drops of chloroform are added to each of the centrifuge tubes followed by vigorous vortexing for 60 seconds. This action efficiently lyses the E. coli cells to release the P1 viruses. Afterwards, the tubes are set on the lab bench for 10 minutes to ensure all remaining bacterial cells have been lysed and a good amount of viruses has been collected. Following, the two 50 ml tubes are centrifuged at 9,500 x g for 10 minutes to obtain a pellet of dead bacterial cells on the bottom and a supernatant containing P1 viruses. Finally, the supernatant in each tube is decanted into a separate 15 ml conical centrifuge tube, and both conical tubes are then refrigerated for future use. 

Conclusion

The newly collected plate lysates of the two E. coli strains JW5055 and JW0391 are ready to be used in a transduction procedure, and they should only contain P1 viruses since all bacterial cells were eliminated by the chloroform. This method makes it extremely efficient to maintain stock of the P1 bacteriophage because lysate solutions, if stored at 4 degrees Celsius, can be functional for years. It is especially helpful to maintain a good amount of a highly concentrated phage solution because it can be used numerous times if diluted. After all, bacteriophages never fail to amaze scientists with their countless applications (given they can enter both the lytic and lysogenic life cycles), durability, and remarkably long shelf life. 

References

Deutch, C.E. (October 2021) Preparation of plate lysates of P1 grown on E. coli JW5055 and JW0391. Put Project at GCC.