4.24.19 Finalizing Independent Projects

Rationale: Since the due date for the project is approaching, today was used to consolidate the information that had been gathered and produce a final, complete abstract.

Procedure:

• Abstract was revised using the tips that Lathan left for us
• Primary literature was used to give additional weight to our findings, and the literature found today was positive for supporting the notion that our sequence was a regulatory sequence
• PowerPoint graphics were created and consolidated
• Information and an outline were made for the PowerPoint
• Slides were created, not finished.

Results:

• Hypothesis that the sequence is a regulatory sequence is supported by primary literature, which also gave steps for continuing research to prove this.
• Abstract was successfully edited and submitted
• Powerpoint was created and distributed to group members.

Conclusions:

• Since many other logical options have been eliminated, the last remaining option that would be a regulatory sequence. This was supported by primary literature, which provides enough of a basis to present this as relevant findings.

Next Steps: Finalize PowerPoint and present to the class.

As a scientist, describe the main experiment you would like to see performed before phage therapy is approved for human use. What are the risks involved with using phage therapy?

As a scientist, two factors must be considered with each experiment that is used to prove the effectiveness/usefulness of a new treatment. Firstly, the scientist should consider whether or not the treatment is safe and ethical before beginning an experiment. A good experiment that features the testing of a new treatment should be safe and should also provide evidence to the FDA and public that the treatment is usable without substantial harm (if it is to be made public). Secondly, the experiment should be designed to determine the effectiveness of the treatment without skewing results in a direction to support or disprove a hypothesis. This increases the credibility of the work from a scientific standpoint and would also make the case presented to the FDA more convincing. If I were a scientist trying to determine if phage therapy should be approved for human use, I would like to do a direct comparison study between antibiotics and bacteriophage therapy. This study would be accomplished by first obtaining a constant bacteria strain that was antibiotic resistant. Next, a phage cocktail would be cultured (similar to those created at Phage Therapy Centers, such as those referenced by Kuchment in the text) to attack that specific strain of bacteria. This phage cocktail would be tested in conjunction with a control group with no treatment, an antibiotic treatment that contained a cocktail probable to have success against that particular resistant strain, and a general antibiotic such as penicillin. Results would include the success of the elimination of the resistant bacteria and the levels of endotoxins released by the bacteria that had died. This study would examine not only the effectiveness of phage therapy in relation to other modern treatments, but also the safety in relation to other modern treatments. If the results from this experiment returned favorably, they would play a large role in changing public perception of phage therapy (Kuchment discusses how western medicine has traditionally favored antibiotics while eastern medicine has traditionally favored phage therapy, which followed the political lines leading up to and during the Cold War) and could play a crucial role in getting the approval of the FDA.

A risk factor that would also be addressed by this experiment would be the release of endotoxins upon lysis of bacteria. This is a problem that is shared between bacteriophages and certain classes of antibiotics, so this experiment would be very useful in discerning the extent at which phages cause endotoxins to be released compared with the extent that antibiotics do. However, phages may have a unique property that could limit this risk factor. In addition to Kuchment’s description, Doss, Culbertson, Hahn, Camacho, and Barekzi describe a method that involves genetic engineering of bacteriophages to eliminate their major lysis proteins; these new phages kill bacteria by creating a hole in the inner membrane via the holin. The release of endotoxins could go from a potential problem with phage therapy to a very big advantage, as eliminating the risk of endotoxin release would reduce the worries of additional problems in the future with endotoxins causing more severe problems upon the death of bacteria. A second risk factor that would be addressed by this experiment would be the risk that the public would not have faith in the treatment. An approved treatment would remain relatively useless if the public did not trust it, so having evidence to substantiate the claims made by proponents of phage therapy before the treatment was approved could encourage the FDA to be more confident in approval of the topic, swaying public opinion in a favorable way. To summarize, this would eliminate the risk as a scientist of investing many resources into a treatment and experiments that would not be widely used. A final risk with phages is that the phage cocktails used in treatment that are designed for more breadth rather than one phage is that they can also cause bacterial resistance. Therefore, it would be important to use phages carefully and in a focused manner to ensure that bacterial resistance would be minimized.

Doss, J., Culbertson, K., Hahn, D., Camacho, J., & Barekzi, N. (2017). A Review of Phage Therapy against Bacterial Pathogens of Aquatic and Terrestrial Organisms. Viruses, 9(3), 50. doi:10.3390/v9030050

4.17.19 Abstract

Rationale: As an additional way to check progress, an abstract was made to begin to consolidate the research that has been done over the last weeks. This also signifies progress to the ‘coaches’ overseeing the experiment.

Procedure: In lab, we used various tools (Phire, PhiSite,etc.) to investigate the possibility of the sequence being a regulatory region. We also spent time summarizing our research into an abstract that was submitted at the end of the lab.

Results: Promoter sequences are present near our repeat, but more investigation needs to be done before conclusions can be drawn. The cursory abstract was successfully completed, but more revisions will need to take place before a final product is submitted.

Conclusions: The abstract highlighted some holes in the research that exist, and we now have a better plan on how to finalize our experiment and get results that could be considered more concrete.

Next Steps: Find results to concretely determine what our sequence really is.

4.15.19 Work on Independent Projects

Rationale: More work needed to be done to correctly determine the identity and function of the sequence of interest. Therefore, time today was spent using online tools to determine a potential function for the sequence.

Procedure:

• Found a tool that was able to identify transposable elements (IS Finder)
• Read primary literature to obtain any other ideas surrounding sequences similar to the one found and being investigated
• Looked for other tools that could be used to help predict the function of the sequence

Results:

• The transposable elements finder showed no direct hits to any organism in the database
• Primary literature search did not reveal any new information, but pointed to new tools that needed to be tested out – tools did not have a very good success rate

Conclusions

• Since the sequence is officially not a transposable element, the new struggle is finding something that the sequence could be. We have disproved the notion that the sequence is other likely components, and therefore we need to seek a new function or path that can hopefully contextualize the sequence and give direction to a potential function.

Next Steps: Finding a new function that could be applied to the sequence of interest

4.10.2019 Investigating the Possible Transposon

Rationale: Since we hypothesized that a transposon may be the cause of our highlighted repeat, it was beneficial to take a closer look to examine the evidence to see what it supported.

Procedure: The same tools (DNA Master, Phamerator, NCBI BLAST, and HHPred) were used to process data about this repeat and to begin to draw conclusions about it.

Results: Today’s results were less than encouraging. NCBI BLAST did not match the repeat to anything other than bacteriophages, which makes the notion that the possible transposon was from a different organism improbable. HHPred did not match to anything significant, and DNA Master offered no further results for the sequence other than the other members shared the same TAAA inverted repeat on either side of the sequence.

Conclusions: Since the other evidence supporting a transposon or a transposable element is not present, the initial thought that the repeat is a transposon may be incorrect. This would be difficult, as it would leave many more questions than answers about what kind of function this noncoding region could have.

Next Steps: Despite the negative results, the theory that the sequence is the result of a transposon is not completely debunked. More tests and analyses need to be run before a definitive conclusion can be made.

4.8.2019 Furthering Investigation of Repeats

Rationale: At the end of the previous lab, we focused on one particular repeat of interest. Today, it would be beneficial to gain more knowledge and information about that repeat to determine whether or not there are interesting findings to be had.

Procedure: Today, DNA Master and NCBI BLAST were used to analyze this particular repeat along with Phamerator. We examined sequences in DNA Master and used Phamerator and NCBI BLAST to contextualize our results.

Results: The repeat in NapoleonB along with the other members of the AM bacteriophage cluster had an inverted repeat of TAAA and AAAT, leading us to the hypothesis that this sequence may be a transposon.

Conclusions: Since there was an inverted repeat that was present in a conserved sequence, we hypothesized that this could be a transposon, as it has all of the basic qualities of that phenomenon. However, more investigation to confirm this idea is needed before any direct and influential data can be compiled and had conclusions drawn from it.

Next Steps: On Wednesday, we will take a more detailed look at trying to determine the classification of this sequence.

4.1.19 Practicing Presentations

Rationale: It was found to be necessary to pause the independent research projects to practice our URSA presentations as a class. Therefore, the time was dedicated to reviewing important information about our poster and highlighting key parts of the presentations that should and should not be emphasized.

Tools/Procedure: Each duo of presenters went up to either give a full or abbreviated presentation, with feedback and live situations simulated by the entire class.

Results: As the presentations were practiced and refined, they were found to get better and be more cohesive. The feedback from our presentation guided me in my practicing for my time slot during URSA week.

Conclusions: Group practicing of presentations was very helpful, as some interesting and potentially problematic situations were encountered and worked through. As I was hearing others present the sections I was going to present, I was able to learn different phrases and techniques for presenting the topics that were more cohesive or easier to comprehend for someone without full experience in bacteriophages. Therefore, this additional time to practice and prepare as a class was helpful and I believe that it will drastically improve the quality of presentations given by those at the research poster at a given moment.

Next Steps: Each pair will go to their assigned 15 minute time slot and present the rehearsed and polished information!

3.27.19 Preliminary Research

Rationale: Since Leo approved our preferred research question about repeats in NapoleonB, he encouraged us to begin finding information to answer our question. Therefore, we began looking for repeats in the genome of NapoleonB

Tools/Procedure:

Gepard dot plots and DNA Master.

I used the Gepard dot plot software updated with NapoleonB’s fasta file to begin to search for repeats within the genome. When a possible repeat was found, I used DNA Master’s scan feature to determine the quantity and location of the repeats in the genome.

Results:

Over the course of the lab hour, a few repeats were found to have relatively minor significance. There was one repeat of approximately 50 base pairs that showed two repeats, and other shorter repeats that were repeated a maximum of five times.

Conclusions:

Repeats are more difficult to find upon initial examination. A bigger challenge that was not as anticipated was a lack of immediate success with repeats. While I understood immediate success was unlikely, I had anticipated the process would involve weeding through many more significant repeats to find the most significant ones rather than weeding through many insignificant repeats to rare significant ones. With more time and effort, I think that we will eventually have more success – resilience!

Next Steps:

More time will be spent looking for and analyzing repeats.