Although this semester of Phage Lab we had to deviate from the exciting and hands-on aspect of phage hunting, we still learned so much about another aspect of phage research. The purpose of the first half of this semester was to annotate Manatee’s (my phage that won the Phage Olympics last semester) genome. It took a little while for us to receive it, but a few weeks into the semester we finally received Manatee’s genome! For me it was such a rewarding and gratifying experience to actually see my phage’s genome mapped out in front of me after spending last semester isolating, purifying, getting plagued with unwarranted bacterial contaminations, and physically struggling to filter and isolate my phage’s DNA- sounds weird, I know, but trust me, it took some serious elbow grease to squeeze a liquid through a tiny little DNA-clogged filter using just a flimsy syringe.
At the beginning of the semester we had downloaded several programs to aid us in completing the annotation of Manatee’s DNA. My favorite program that we used was Phamerator. There are so many fun hidden features to this program, but the best I’ve seen yet Dr. Fisher and Dr. Schildbach showed us. With the click of a button all of these rainbow-colored bubbles magically appeared and floated across the screen landing on different genes (shown as colored blocks on the page) of the phages’ DNA that we were comparing. Specific colored bubbles represented individual “phamilies” of genes.
In addition to Phamerator, we annotated Manatee’s entire genome sequenced by the Howard Hughes Medical Institute using another program called DNAMaster. While this program didn’t quite have all the frivolous features of Phamerator, it excelled in practicality. Within a matter of weeks (it went by much quicker than I thought it would!) collectively as a class we annotated Manatee’s DNA in its entirety, all 51040 base pairs. Along the way we eliminated a few genes based on their lengths and many other qualifications including coding potential, Shine-Dalgarno sites, HHPred hits, and nucleotide blasts. HHPred allowed us to submit a specific gene sequence to be compared against all other phage DNA’s to find similar genes. Through this program we found out functions for a few of the genes like for example, a protein coding for capsids.
Although all of this genome annotating is fascinating I’m switching topics to explain my individual project. With a month and a half left in the semester our professors set us free to explore a phage-related topic of our choice. My partner-in-crime, Eleni, and I decided to vary the environmental conditions in which our phages typically grow in and observe and changes in growth. Both Manatee and Phido, Eve’s phage, came from the same soil sample on the Beach and I wanted to see if they reacted similarly of differently to the growth conditions I imposed on them. The results would hopefully provide me with a better understanding of the two phages and whether they are closely related.
While I am still in the midst of experimenting, I have already gotten back results that I am really excited about! To start of the process I re-titered both Manatee and Phido so that I could recalculate their titers and make sure the high titer lysates had not degraded in the past months while we were doing the annotation work. Both titers were fairly similar to their original ones: success! Moving on, the first growth condition modification I made was temperature. I plated the dilution 10-7 3 times for both Manatee and Phido because in their titers, the 10-7 plate produced a countable number of plaques and then I placed one plate of each phage in an incubator set at 42 degrees Celsius, another at 37 degrees Celsius (the normal incubation temperature), and one plate each sitting out at room temperature. To my surprise, plaques grew on each plate! The main difference between the growth at the different temperatures was the carrying plaque sizes. At the lower temperature the plaques were smaller than the normal size and at 42 degrees Celsius the plaques were approximately 1-2 mm larger in diameter than normal. I repeated the same experiment and followed the same growing temperatures as before and plaque growth on the plate came back the same for both phages. Unfortunately, I do not know if the plaques are just physically larger or if there are more plaque forming units in the 42 degree Celsius plaques. To figure this out I must titer each of the plates that I tested in the temperature trials. Although this is tedious this will give me a better idea if the concentration of phage on the plates at the different temperature varied or if just the physical plaque size changed.
Another growth condition I am testing is calcium dependence. Usually we add a small amount of calcium to top agar before plating the phage-infected M. smegmatis. We’ve been told the calcium aids in phage growth, but I want to know if this is essential to the phage’s growth or if it’s superfluous. I plated the same 10-7 dilution with the usual amount of M. smegmatis but when adding the TA and plating it out, I used calcium enriched TA as a positive control and then used plain TA without any calcium to plate the other two plates for Manatee and Phido. The results came back and Manatee grew without any calcium! There were not any plates on either of Phido’s plates, even the positive control, so I must have done something wrong with the dilution. Today in class I repeated this experiment to try to get consistent results for Manatee and to get decent results for Phido.
The final environmental test I plan to do involves drying out a sample of the 10-7 dilution of each phage then reconstituting it in phage buffer and finally plating it with the normal amount of bacteria. We are still working out the kinks of this experiment, but hopefully we will get interesting results! I have been very surprised with the results we have gotten thus far, and I cannot wait to pull together all of the observations and make conclusions about Manatee and Phido’s similarities. At the same time I am incredibly sad that a wonderful year in Phage Hunting is coming to an end. This class has truly been an amazing experience and I feel that all of the students, TA’s and professors have become a phamily: persevering through empty plates and frustrating contaminations, while also reveling in the achievement of isolating a plaque and discovering new things about our phages. Cheers to a phantastic year of phage hunting!