Bioprinter Update 4

My main goal currently is to create a high titer lysate of my smegmatis phage. While there is a relatively straightforward pipeline to purifying— plating and picking a plug several times, then flooding the plates—the process has been slightly hindered by the fact that my plates are displaying very abnormal morphology. When a single phage infects a bacteria cell, it can lyse the cell, and release progeny phage to surrounding cells. Thus, a plaque, or clearing is formed in a lawn of bacteria. Typically all the plaques I’ve seen are transparent, and when the phage concentration is high enough, the entire lawn of bacteria clears.

However, this is not the case at all with my smegmatis phage. When I first started seeing the turbid plaques I assumed that I wasn’t plaque pure and that I had to keep purifying. However, after my fourth and final round of purification, I saw the same results:  turbid plaques and lawns that didn’t clear even with the highest concentration of phage possible. I am going to continue the process to obtain the lysate, but I will also try to isolate another phage from a previously picked plug. I am not quite sure what is happening, and I will talk to my PI and read more literature in an attempt to find an answer. The turbid plaques, while uncommon to this lab, are just another type of phage morphology. What is more concerning however is the lack of clearing on my 10^0 plates.

Working alongside a member of the iGEM team, I am also starting to begin the electroporation process for transforming the shuttle vectors into the smegmatis. The two plasmids have been ordered and should come within the next few days. In the meantime I have started making electrocompetent smegmatis cells.

Hopefully I will have several high titer lysates for smegmatis phage very soon, along with a kanamycin resistant smegmatis, both of which I will be able to use to examine infection dynamics using my printer.

 

image (2)Turbid plaques.