Out of all the options presented, I think that option 3 appeared as the most appealing. It seemed to be the most obtainable given the circumstances that are presented to us today. A lot of companies are already looking for solutions and making technological advances towards combatting climate change. It also seemed to be the path that was responsible for the least amount of disruption to others. Options 1 and 2 would undoubtedly cause the lives of many to change, and would, therefore, face some opposition.

A majority of the solutions presented in option 2 were solutions that I had not considered previously. Moving and relocating individuals in areas that were susceptible to climate change was a solution that I had not thought about before. I was able to learn from others who live in susceptible areas that that change is currently happening, which was surprising to hear. Our group also discussed possibly creating incentives for energy efficient products to encourage consumers to choose that option.

I thoroughly enjoyed the climate change deliberation forum as it was a good way for me to hear the opinions of my peers. I was exposed to new ideas and heard evidence to claims that I had not known to be true. I would assume that communities would feel the same way. I think it would be interesting to facilitate a public deliberation forum, but I do not think that I possess the skills to do so.

Rationale

Today we will finish data collection and complete our final abstract for the independent research project.

Procedure

• Each AM specific gene in NapoleonB’s genome was BLASTed on NCBI
• Results were recorded if it hit was of low %GC content
• Our abstract was edited and revised to account for the comments made previously. The results presented in the abstract were updated to match the data collection conducted today.
• Slides for the final presentation were created.

Results

Abstract: Arthrobacter is a gram-positive soil bacteria that has a guanine and cytosine nucleotide composition (%GC) of 63.41% in its genome. NapoleonB and other Arthrobacter AM phages have a comparatively lower %GC of 45.3% and 45.2%, respectively. This is interesting because phages tend to have similar %GC compositions relative to their host bacteria. This project tests the hypothesis that lateral gene transfer is responsible for this discrepancy in %GC. %GC3 analyzes the GC content of the third codon, which is significant in the analysis of codon usage. In order to test this hypothesis, phams were used to separate NapoleonB’s genes into AM specific genes and genes shared with phages outside of the AM cluster. The percent GC and GC3 were analyzed for each gene and compared using Student’s T-test. AM specific genes were compared using NCBI BLAST, to search for matches from host or viruses with low %GC. The %GC of AM-specific genes was significantly lower than non-AM specific genes (P= 0.0029). However, no significant difference was seen between the GC3 values. 51 AM specific genes were checked for BLAST hits with low %GC species, and 12 genes had matches. The evidence indicates that lateral gene transfer from low percent GC bacteria is a potential cause of low %GC content in AM cluster phages, compared to their host. Future analysis of other sequenced genomes could help to strengthen this hypothesis.

An example of results for a single AM-specific gene is shown below:

• Gene 6
  • Clostridium
    • Hypothetical Protein
    • E value: 3 x 10^-7
    • Identity: 34.34
  • Candidatus Moranbacteria bacterium
    • Hypothetical Protein
    • E value 3 x 10^-14
    • Identity: 42.70

Conclusion/Next Steps

It was concluded that lateral gene transfer could be a probable cause for the exceptionally low %GC content in AM cluster phages and genes. Next, we will continue working on our final presentation.

Question: Describe the differences between Intralytix and GangaGen. Can you locate their “best selling products”? What are the main struggles these companies have to deal with? Look at their current web page. What changes have taken place since the writing of The Forgotten Cure?

Alexander Sulakvelidze was the co-founder of Intralytix, a company that initially focused on the use of phages to combat human diseases and bacteria found in produce and poultry. Vancomycin-Resistant Enterococci (VRE) was Intralytix’s main target during their starting days in the world of bacteriophage therapy. Intralytix faced some expected challenges that surrounded bacteriophage therapy in human diseases. The Food and Drug Administration was an obstacle that was proven tough to get around when it came to initiating clinical trials. Intralytix’s bacteriophage concoction to combat VRE was rejected by the FDA, and as a result, the company switched gears to focusing on bacteriophage therapy of meat and poultry.

Dr. Janakiraman Ramachandrian was the primary founder of GangaGen and “leader” of a Scientific Advisory Board that was composed of many notable figures. GangaGen appeared to have not have battled the same amount of financial troubles that Intralytix had in their time, due to donors and investors in the company. GangaGen centered their focus around E. coli 0,157, with the ultimate goal of passing clinical trials and placing itself on the market. After a large investment from Otsuka, a Japanese pharmaceutical company, GangaGen was highly encouraged by Otsuka to concentrate on human diseases in comparison to Intralyix.

Perhaps the largest struggle that Intralytix and GangaGen encountered was the FDA and gaining approval to move forward to clinical trials. There was a large amount of skepticism surrounding bacteriophage therapy. Companies were also hesitant with investing and utilizing bacteriophage therapy. Nevertheless, Intralytix was able to secure a distributor, manufacturer, and producer of their products, all without the product receiving approval from the FDA. However, in 2006, strides were made from the FDA approving the use of phages in prepared foods.

The Intralytix website displays its list of products, all of which food safety, pet food safety, or harvest of animals. None of the featured products on the Intralytix website directly combats human diseases. This indicates that after Intralytix switched over to meat and poultry bacteriophage therapy as seen in The Forgotten Cure, they did not switch their focus back to human diseases. Notable products include SalmoFresh and EcoShield. The GangaGen website puts emphasis on Ecto-Lysin (P128), which presents strong evidence that it is the “signature GangaGen gene” that was inserted in the place of the lysin gene as described in The Forgotten Cure. The GangaGen website cites research that seems to focus on the Ecto-Lysin as their main recombinant protein. MRSA was also on the radar of GangaGen during The Forgotten Cure. The GangaGen website indicates that MRSA is still a focus of the company.

Rationale

Today we will look more into our independent research project and hopefully discover a reason for why the %GC in AM cluster phages are significantly lower than that of Arthrobacter.

Procedure

• Each group member read a large portion of review articles to see if there was a reason why some bacteriophage %GC content was significantly lower than that of their host.
• We began to see if the trends were present in Arthrobacter using NCBI Blast, PhagesDB, and Phamerator

Results

We found from article reading that horizontal gene transfer is a large reason for why the %GC can differ between host bacteria and their bacteriophage. Lucy found that Staphylococcus had a reportedly low %GC and after using NCBI Blast, it was found that gene 97 in NapoleonB had a CDD hit for Staphylococcus. Gene 98 in Mudcat also had a hit.

Conclusions/Next Steps

Next, we will research more into horizontal gene transfer and if it played a role in the low %GC of other genes.

Rationale

Today we aimed to analyze more %GC and %GC3 content of AM phages and dive more into our independent research project question.

Procedure

• Reasons for why there was a lower %GC content in AM phages were sought after, as the percent difference is approximately 20% between %GC of Arthrobacter sp ATCC 21022
• It was decided that the difference between the %GC content in AM Arthrobacter phages and Arthrobacter would be researched as we proceeded for further questioning.
• An abstract was devised.

Results

There were no notable results from today’s research, as the course of the project changed.

Conclusion/Next Steps

Next, we will research more into our question and investigate the probable differences.

Rationale

Today we will continue working on our independent research projects and analyze the data that we have collected.

Procedure

• Two-sample t-tests were performed on the data previously collected.
• Pham numbers were changed since data on PhagesDB was updated. We spent the remainder of lab time double-checking to ensure that the same genes there were declared as AM specific before remained AM specific.
• Our groups were altered and our results were changed.

Results

The computed averages are shown below.

Conclusions/Next Steps

Next, we aim to collect more data on AM phages and non-AM cluster phages.

Rationale

Today we will continue working on our independent research projects. We aim to gather more data on NapoleonB and other AM cluster phages.

Procedure

• The annotations created by the class were uploaded onto DNA Master.
• The %GC and %GC3 were calculated for each gene in Napoleon and the results were recorded into an excel sheet.
• The genes and their %GC and %GC3 values were sorted into two categories of whether the gene was AM specific or non-AM specific.
• The average %GC and %GC3 values were calculated for each group created.
• The process was repeated for a non-AM cluster phage.
• Average %GC was also collected for each specific type of Arthrobacter phage.

Results

Shown below are the recorded average %GC for each cluster of Arthrobacter phage.

Conclusion/Next Steps

Next, we will perform a t-test with our results and move onto data collection for other Arthrobacter phages.

Rationale

Today we will finalize our independent research questions and begin looking more into the topics at hand.

Procedure

• Topics were reviewed to determine if the would be fit for an independent research project.
• A question was determined for use in the project
• Scientific literature was investigated to find articles that included topics in the realm of our project.

Results

The question that we created stated: What is the relationship between the percent GC and GC3 in AM-specific genes and genes present in AM and non-AM phages?

We utilized the following links establish a background on our topic:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3923770/

https://www.omicsonline.org/open-access/the-gc-content-of-bacterial-genomes-2329-9002-2-e108.php?aid=26236

https://www.pnas.org/content/111/39/E4096

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4576692/

Conclusions/Next Steps

Next, we will begin collecting data regarding our scientific question and dive deeper into our independent research project.

Rationale

Today we will practice presenting our posters for URSA Scholars Day.

Procedure

• Each pair that signed up to present for Scholar’s day presented in front of the class.
• Questions and critiques were made to each group that presented.
• Helpful tips for presentations were given.

Results

Each group was able to create an outline for their presentation the following days. We all gained a better understanding of what a poster presentation consisted of.

Conclusions/Next Steps

Next, we will present our poster at URSA Scholar’s Day and then begin to work on our independent research projects.

Rationale

Today we will finalize our independent research question and explore the tools needed to answer the question.

Procedure

• A final question was created using a mixture of the questions proposed on 3/25/19.
• DNA Master and a variety of external tools were used to explore the %GC in NapoleonB.
• DNA Master was also used to look for repeated sequences in NapoleonB.

Results

The new research question devised explores the characterization of the coding regions versus the non-coding regions of AM cluster phages by comparing the %GC and the number of repeated sequences present.

Shown below is a picture of DNA Master and the %GC data for gene 1.

Conclusion/Next Steps

Next, we will determine the %GC of the non-coding regions and the coding regions of NapoleonB. We will also look for repeated sequences in the genome. After analyzing NapoleonB, we will expand our research to more AM cluster phages.