By Andrea Uy
Phage Hunting this semester was different from what I expected. First of all, I switched sections and was kind of nervous going back. Everyone knew each other already, and I was one of those odd new kids sitting in the corner. Then, instead of going straight back into lab, we were to spend (an additional) 5 hours a week sitting in front of our computers, a stark contrast from last semester. I didn’t really know what to expect, and with a ton of troubleshooting and technical difficulties, the idea of annotating didn’t really sound pleasant. After all, how was I suppose to know which start codon to pick or how to read coding potential? I was beyond confused. However, I quickly got to learn the process (and new people), once we were placed into groups. Soon, annotating became easy and even enjoyable. Before I knew it, we had all 103 genes of Altwerkus done, and it was time to return to the wet lab.
Upon hearing of our return to the lab, I quickly got there and took my bench. New semester, new class, but at least I got my bench. After all, it is a lovely one. I started working on some dilutions, and though I started slow, my body began to remember the notions of pipetting up and down and up and down. Everything was coming back to me – the sight of endless plates, the distinct smell of agar. It was fun annotating, but being in the lab really is a wonderful experience. I’ve been working on my independent project, and finally get to play with my phage once more. Ah my magical morphing phage, how I’ve missed you! I do hope you behave better this semester.
By Laura Hinsch
As the annotation of Phatniss came to a close and our own personal projects came to fruition, I hit the ground running. SirSheldon was a lucky little phage. He didn’t finish first at the phage Olympics, but when he shot for the moon, he landed amongst the stars and was sequenced anyway. As you might recall, SirSheldon had respectable statistics last semester. His DNA was strong and quite evidently present. Over break, he left the lab to be sequenced, his genome was sequenced using Illumina sequencing, a type that dispenses a high number of reads. When SirSheldon’s sequence returned it arrived as a large compilation of base pairs and read information. For me, this was very different and more complex than the arrival of Phatniss. Unlike Phatniss, SirSheldon’s genome had not been assembled. The responsibility of assembling his genome fell on my shoulders. An arduous task at hand, I learned how to compile a file with a smaller number of reads and assemble the genome using the Gene DeNovo assembler. When my 25k, 50k, 100k and 150k assemblies were finished, I discovered an uncanny trend in my assemblies. I would always have a primary contig with a generous number of base pairs, approximately 80k. However, I would also have several other contigs ranging from 2,000 base pairs to 100. This was unfortunately troubling and I was advised to BLAST the extra contigs. To my great distress, the results came up with a direct hit to Phatniss. I felt like a father who had raised his child for ten years, just to discover that the child was not his. Defeated, I began to pick up the pieces of my shattered project. The first step on the road to recovery was to run a PCR and discover where the Phatniss genome contamination occurred.
Today was my first day back in the lab since last semester and even though my project is still fragmented and broken, there is something truly comforting about having that micropipette in hand as you do nine-fold dilutions. I look forward to hopefully completing the assembly and annotation of SirSheldon, and I know that even with my setbacks, it’s worth it because I will have a phage to call my own.
By Marissa Totten
So probably one of the most important, non-biology, things I’ve learned this past semester is to NEVER under ANY circumstances trust that a program will actually save your work. Honestly, from now on I’m saving my documents as a new file every ten minutes. Why? Three letters and one word: DNA Master. This program made me absolutely certain that I never want to go into tech support. EVER. The problems first started with forgetting to hit post, Hunter, before moving onto another gene. Then want on to display errors that requested sums as high as $2800, Ashley. Then the best message that could pop up was the one that notified you that your previous file could not be opened. Great. Countless gene blasts lost, new ORF starts erased, and time spent waiting on HHpred function blasts (most of them turned up no known functions anyway) wasted. Word to the wise: save a new file after every couple of annotated genes. Do it. After plenty of corrupt files and the phrase “I swear I finished the annotation,” I’ve learned my lesson. I currently have 55 Phatniss files saved. After all of this, I honestly think that annotating by hand is the best way to go. It’s actually a lot faster because you don’t have to keep switching tabs and the only way you’re going to lose your data is by losing the papers yourself. Despite all of the annoyance and frustration this program presented, annotating is a really cool process! It was a great look into what genetics entail and what scientists have to go through to figure out gene functions.
But now we’ve started the best part of the course, of course: web lab experiments! The hands-on work in this class is the best. I’ll admit my experiment isn’t the most interesting, I wrote it up the day before it was due while I was at a soccer tournament, but I’m still super excited to be working with my phage Drac109 again (despite the fact that I have to count and measure 150-300 plaques every class). I plan on switching the top agar with agarose because agarose doesn’t contain sulfates and other molecules that are suppose to inhibit virus propagation. Proven by the entire lab last semester, many phage aren’t bother by these molecules in the top agar in the first place, so it’s possible that the agarose will actually allow plaque size to increase. Agarose doesn’t have the same nutritional content as top agar though, so I may have to throw a couple of things in there so I end up only testing one variable.
And maybe, hopefully, some things will go right on the sequencing side and I’ll be able to annotate my phage’s genome…
By Alyssa Khan
“This semester we’re going to be annotating a genome.”
Sounds pretty cool, right? I don’t know exactly what my expectations were, but it wasn’t to be spending hours on my laptop, trying to make sense of diagrams I had never seen before. We spent the first few weeks attempting to get the necessary software to work on our laptops. Then we spent the following weeks learning how to do the annotations and doing them in groups to present. My group could only meet on Sunday morning (the best time to get work done!) and working on the annotations took hours. As the semester progressed though, it became much easier to understand how to interpret all the information we had available to us.
However overall, annotating a genome turned out to be one of those things that sounds a lot cooler than it actually is.
Last week, we returned to “wet lab” to work on individual projects. I am doing an immunity assay on my phage from last semester, FuzzBall (yes, I named my phage “FuzzBall” – if you’re planning on taking this class be sure to start thinking about your phage name early so you don’t end up choosing one that you’re slightly ashamed to tell others). When I walked into lab, I started to smile uncontrollably. I didn’t even realize how much I missed seeing my all-too-familiar ‘bullseye’ plaques on agar plates. So far, this part of the semester promises to be a lot more exciting. I’m really looking forward to seeing if the lysogens of my phage are immune to infection by other phages!
By Sean Melucci
When you think of biology lab work, the last thing that would come to your mind is working on a computer. However, surprisingly enough, computer software tools are an integral part to biology lab work and research. In this, the second half of the phage-hunting course, the work on the computer was indeed shown to be invaluable. What good is isolating a phage and having it sequenced, if the sequencing data cannot be interpreted and shared with the world? It was our job this semester to interpret the data and annotate the genome of the winning phage “Phatniss”.
Just as in the lab, the annotation of the genome did come with its own trials and tribulations, but in the end they helped us learn and exercise certain computer skills that are very valuable to research involving bacteria or beyond that. It would frustrate me greatly getting the Virtual Machine to install correctly, or DNA Master to start up without immediately shutting down due to failure, or even to BLAST all the genes, but in the end I pushed through it and finally got everything to work so I could begin annotating. Yes I learned important computer skills through this process, but I also learned how to think through problems and continue to persevere, just as I had done in the lab portion of this course. This course, both in the lab and outside of it, will challenge you and make you think outside of the box, all in training you to think like a researcher.
Now, as we enter the last stage of the course being individual projects, I am more excited than ever. I feel like this course taught me a lot about the workings of a lab as well interpreting the data collected, and I feel like this final project is our chance to show the knowledge we have learned as well as learn something new about our phages. This class really has come full circle and truly prepared me to become apart of the amazing research that is being done at this great university.
By Jessica Bauer
I can honestly say that this semester of Phage lab was not what I expected. Coming back from winter break, I was excited to start looking at the genome and doing whatever “annotating” entailed. I was not sure exactly what we would be doing, but I assumed it would be just as interesting and fun as being in lab was last semester. I may have been slightly disappointed. Don’t get me wrong, I liked debating start codons and staring at coding potentials just as much as everyone else, but it got boring after the 5oth or so gene. Finding SD scores, ORFs, and blast hits got really repetitive and did not have the same excitement that being in a lab and waiting for a surprising turn of events to happen gave me. Thankfully, all this annotating is mostly done though and we can finally get back to lab! Yay! I will say though that one thing I liked about annotating was how smart it made me feel to explain to someone else not in the class what I was doing. Just throwing around words like Shine Delgarno scores, ORFs, coding potential, and BLAST hits left so many of my friends with blank stares on their faces and made me feel like some mad scientific genius who does cool things like annotate a genome. Luckily they didn’t figure out how little I actually knew about what I was talking about.
Annotating is done, though, and finally we can all return to our lab benches, put on our favorite white coats, and start doing whatever we want to our phages. One thing about this semester that I am starting to really enjoy is all the different projects people are doing. We are no longer all streaking and plating and diluting, we have a wide array of topics and experiments to do. I personally have decided to do experiments testing how my phage responds to changes in pH. I figured being from Ohio, my phage is used to changing environments since weather in my town can move from spring to fall to winter to summer all in the same week. Going into lab last week I was a little worried since I was not sure on all the details of my project, but I just dove right into it and so far it has been successful. My little phage even grew in it’s titer count. This was a big surprise seeing as I was a little disappointed in him/her (it feels too impersonal to call my phage “it”) for not making it to phage olympics because of too little DNA. This growth in titer is a big accomplishment. The latest thing that I have done for my experiment up to today was make different phage buffers with an array of pH values. Word to the wise, although the name of the stuff is “phage buffer” it does not have a great buffer itself. I added just a little bit of concentrated hydrochloric acid to it and it’s pH dropped from 7.6 to almost 1. A lot of diluting had to be done to fix that accident seeing as although I have faith that my phage will be able to survive changing pH, I don’t thing he/she would survive in that acidic of an environment. I’m excited to see where my experiment will take me in the next few days. Although surprises can be bad during an experiment, I’m kind of hoping for maybe a few harmless ones just to make things more interesting.
Although Phage Lab this semester did not start off how I thought it would, it has definitely made a turn for the better. It will be interesting to hear about the results of everyone’s experiments and what they deduced from them. I’m not too thrilled about having to go back to finishing the annotation, but I know that it is something that we have to do so I’m fine with it. I’m also not too thrilled about it though because it will mean that Phage Lab is actually over which is a very depressing idea, probably for everyone.
By Jessica Kahan
Unlike the majority of the other students in my class, I am new to Phage Hunting this semester. At first, when we were annotating genomes, everyone was new to the material, and we all learned together. However, now that we are in the lab I am totally out of my league–it’s a little intimidating! Luckily, everyone around me has already isolated a phage before and is available to answer my millions of questions. Since this is only my second week in wet lab, I’m optimistic that as the semester progresses I will get more comfortable. After all, nothing drastic has happened..yet.
Last week, I went outside to collect my soil sample. I didn’t really know where to look, so I picked a spot near a newly placed statue that looked promising. After checking out my plates today, it looks like I made the right choice! My phage was so potent that it killed all the bacteria on my first three dilutions, and I only had visible/countable plaques on my lowest dilution plate. Needless to say, I am very excited to continue isolating my phage, and I hope that my phage has yet to be discovered by another person so that I can claim it officially as my own.
Although I was nervous at first about the prospect of wet lab, I’ve found that it is highly engaging and fun. The hours fly by, and I always feel satisfied after accomplishing the day’s work. Phage Hunting is definitely the highlight of my week, and I can tell that each day I am learning more about phages and lab research in general. I’m looking forward to completing the semester!