Katie Entry #4: Transduction

My fifth week in the lab was very busy and exciting. At the beginning of the week,  I was told that I would present all of my work from the past few weeks to my lab during lab meeting on my last day so I began working on my presentation.  I also finished my growth curves from the previous week and streaked and grew cultures of the wild type and two mutant strains of S. aureus  that I would be using later in the week for transduction.
Over the next few days, I made two phage lysates that would each carry a mutated region from a mutant strain that exhibited cold sensitivity in the primary and secondary screen.  I did this by mixing a phage lysate that was previously grown on wild type cells, each mutant strain, TSA and TSB. Then, we plated the mixtures on hard agar and incubated overnight.  The next day, we scraped off the soft agar and sonicated and filtered it, resulting in the two phage lysates.  We then repeated this procedure using the mutant phage lysates and the wild type cells and plated the mixture on TSA with erythromycin.  Since the transposon insertions in the NARSA mutant library strains contain a plasmid encoding macrolide resistance, only the wild type bacteria that were successfully transduced and carry the mutated region would be able to grow on the plate containing erythromycin.

These are the plates of the wild type cells transduced with the mutant phage lyates.

This is what happens at the cellular level during transduction.  We used a phage lysate grown on wild type cells to infect mutant bacteria cells, incorporate part of the bacterial genome containing the mutation into their own DNA, and infect and pass on this mutation to wild type cells.  The resulting wild type cells will then be tested for cold sensitivity by measuring the growth rates through ODs like in the secondary screen.

Unfortunately, the transduction did not work very well and only one plate of the transduced wild type cells had a few colonies growing.  This may have been because we did not use enough bacteria or we did not incubate the plates for long enough. So I grew more cultures of wild type and mutant strains for transduction the following week.

Also, Mrs. Terhaar visited the lab midweek, which was really fun.  I got to show her my work and explain what I have been doing so far, which was also great practice for my presentation.