Bioprinter Update 3

I’ve taken a break from the printer for the past week to focus on the wetlab portion of the project. Now that my current goal is precise spatial control of phage infection on a biofilm, I need to make sure I have the bacteriophage to support this endeavor. A few weeks ago a high school student interned at my lab and brought in soil samples that contained a Mycobacterium smegmatis phage. I have been working on isolating and purifying that phage so I can infect my biofilms.

At the same time I’ve been working on growing my biofilms. I was able to use M63 media from the iGEM team to grow my cells in smaller petri dishes. After four days I rinsed the non-adherent cells with sterile water and stained the remaining cells with crystal violet. Thus, only the cells that had attached themselves to the surface of the plate in a film would remain. The crystal violet binds to the peptidoglycan layer of the cell wall, and is useful in imaging the presence of biofilms.

Biofilms grew on my first set of plates using my m.smeg culture. However I quickly realized after trying to use the same culture for my phage purifications that the culture was contaminated, and I had to halt that portion of wetlab while I grew new cultures of m.smeg. The culture was also very old, and should probably not have been used in the first place.

image (1)Result of crystal violet stain on my biofilm.

My next steps are to continue my phage purification process to obtain a high titer lysate that I can use to infect my bacteria. I also am starting to look at using a shuttle vector to confer kanamycin resistance to my bacteria. While E.coli can be transformed merely by using a heat shock, M.smeg needs to be electroporated, which is not something that I have done before. However, I am very excited to try!