Did any of the Options appeal to you more than the others?

Option 3 appealed the most to me since it seemed the most realistic; however, this option still is not ideal. In option 1, forcing individuals to only drive electric cars would cost a lot of money and create waste since all the cars being replaced would need to be discarded. In option 2, relocating people would cause problems by invading property rights and forcing people to move and adapt to areas they did not want to live. Option 3 suggests giving incentives to companies to encourage research for more innovative products and methods which is a very positive step. I disagree with the easing regulatory processes example since this could potentially expose the market to products that have not been proven safe over time.

Did you hear or think of any new way of addressing the issues associated with the warming of the climate?

When reading through option 1 which addressed reducing carbon emission and option 2 which addressed protecting communities, I was surprised that there was no point made about cows. Raising cows requires many acres of grass, and the animals can be cited as major contributors to methane gas emissions. Ranchers also have difficulty discarding the manure in an environmental-friendly way. However, cows are excellent sources of protein and dairy. As the human population continues to increase and as more communities can afford to purchase meat, the need for more sources for protein has increased. In a Ted Talk, entrepreneur and conservationist Mike Velings gives the frightening statistic that the human population will be approximately “9.7 billion by 2050” and that “70 percent more protein” will be required (1). One alternative, suggested by the Ted Talk, addressed these problems by suggesting aquaculture. In aquaculture, fish are grown for food. This would significantly reduce both cow methane and mature problems while also providing a source of protein. It would also allow previous dairy farms and ranchland to be use for homes or for growing other crops for human consummation. However, two problems come with this solution. First, by raising fish in small spaces, they become more prone to spread disease, and studies have shown the farmed fish are not as healthy as the wild fish (2). If the farmed fish manage to escape the chamber and mate with wild fish, they can spread contamination and disease. The second problem would be convincing individuals to change their diet.

1. https://www.ted.com/talks/mike_velings_the_case_for_fish_farming?language=en#t-57905
2. https://science.sciencemag.org/content/303/5655/226

What are your thoughts on the use of Public Deliberation in the classroom or the community? Is this something you would like to facilitate?

I think the best way to inform individuals about these issues would be through social media. If a public deliberation is held, it should be livestream. As an individual, I sometimes feel like lifestyle does not have a significance impact on climate change. If a public deliberation could focus more on things individuals can do, show how the little things do matter, and convince others to motivate their friends and family to make these subtle changes, then the public it could create an impact on the community. I might be interested in facilitating a public deliberation, but I have a heavy course load next semester so I am not sure.

Rationale:

Practice presenting presentation for CURES in BIO symposium.

Procedure:

1. Presented presentation in front of peers.
2. Answered questions and received critiques from peers.
3. Listened and critiqued other peers’ presentations.

Results:

The following list is a of suggestions and critiques my presentation received.

• start strong by explaining the significance behind the whole project at the beginning
• be clearer that the methods are the not just one slide
• mix up speakers, instead of having person A talk then person B talk then person C talk

The following list are critiques directly for me.

• pronunciation and remembering the different names of the structures
• explain all the aspects of the video first before showing it
• loop and slow down the video

Conclusion:

Practicing in front of peers is a great tool because it allows for feedback which allows for improvement before the actual presentation.

Future Work:

Present individual project at CURES in BIO symposium.

Rationale:

Performed a final check on the presentation for CURES in BIO Symposium.

Procedures:

1. Corrected abstract for CURES in BIO.
2. Calculated approximately the number of holins required to oligomerization.
3. Finished presentation.

Results:

Final abstract submitted for CURES in BIO is shown below.

The following images shown were used as measurements when calculating the number of holins required to oligomerization.

The following calculations were made to find the number of holins required for oligomerization.

The following image shows the slides of the CURES in BIO presentation.

The following is the video in the CURES in BIO presentation.

Conclusion:

Depending on the orientation the endolysin exits the cell, it would take approximately 11 holins (if endolysin was oriented vertically) or 17 holins (if endolysin was oriented horizontally). This is an estimate. The estimated number would have more accuracy if calculations used calculus and/or physics-based algorithms and if factors such as spacing between holins were taken into account. Due to limited time, research was not conducted into these variables and methods.

Future Work:

Practice giving presentation in front of class.

Rationale:

Rewrite abstract and work on PowerPoint for CURES Symposium.

Procedure:

1. Made corrections to the abstract.
2. Worked on PowerPoint presentation.
3. Created structures using Jmol.

Results:

The following abstract was submitted for the CURES Symposium.

The following image is of the Gly-Gly structure created.

Conclusion:

The Phage Symposium PowerPoint and the modified abstract was used to create a rough draft for the PowerPoint presentation for the CURES in BIO Symposium. Jmol can be used to create 3D molecules.

Future Work:

Finish CURES in BIO Symposium PowerPoint and endolysin-holin interaction film.

Phage therapy needs to be beneficial for the patient. Potential side effects, such as: phages causing bacterial toxins to spread during lysis, phages mutating and then start harming good bacteria, and phages transforming bacteria to become more anti-resistant, should be addressed before phage therapy receives approval for human use. Along with these risks, other factors that should be considered when using phage therapy include which phage components are manipulated, the pathway of administration, and best practices for ensuring patient recovery. One main experiment that should be performed before phage therapy is approved for human use would investigate ways to make phage therapy the most beneficial and safe for use. With this in mind, I propose an experiment that specifically examines what would be administered to patients and would answer the question of what phage therapy design would ensure patients with the best recovery with the lowest amount of risk.

In Kuchment’s The Forgotten Cure, many phage therapy companies have designed different therapies to be administered. These companies therapies can be divided into the categories of whole-phage focused or “single biological entity” focused (98). Intralytix uses the whole-phage focused approach when they created a multi-lytic phage cocktail. The FDA opposed their work since they had no reliable method of ensuring only lytic phages were present in the cocktail. Also, the VRE cocktail was only tested on mice which are not immunocompromised to VRE, and company findings did not report the speed and the effects of mutations. Shifting towards component-focused therapies, GangaGen addressed the problem of determining whether the phage is virulent or temperate by removing the its endolysin, a protein responsible for cleaving the bacterial host’s peptidoglycan wall. Ramachandran, the founder of GangaGen, hypothesized that “without a ‘burst,’ no toxins or phages would be released into the patient’s body. The fewer the number of phages, the lower the chance that a virus would swap genes with a bacterial cell” (94). In 2001, an experiment was performed using phages without endolysins which resulted in inactive E. coli and no plaques. Another protein GangaGen looked into was tail proteins responsible for the initial hole allowing for DNA to be transferred into the bacterial cell. Their idea was to have the phages create the initial hole but not allow them to seal it back up. It was hypothesized since there was “no DNA going in to repair the hole, so it remains open, and eventually the cell dies because the membrane collapses” (110). Later, this phage tail protein was combined with lysostaphin, a protein that binds and kills bacterial cells, to create the drug StaphTame. Fischetti’s private company decided to abandon using phages for their enzymes.

With the creation of many different phage therapies, an experiment should look into comparing the effectiveness of therapies to each other. Each group would receive one of the following phage therapies: singled lytic phage cocktail, Intralytix’s multi-phage cocktail, GangaGen’s phage tail protein, StaphTame, Ramachandran’s endolysin-less phages, Fischetti’s phage enzymes, or no treatment, which would serve as a control. Also, the experiment should consider having an endolysin cocktail group to determine its effectiveness when compared to the edited phage tail proteins. Another set of groups should receive an antibiotic paired with one of the other therapies. Multiple trials would be performed on different organisms. Many control variables, such as the bacterial host used for infection and the way and rate of administration, would remain consistent among all groups to provide reliable results. This experiment would record the amount of infection and any side effects over the course of the experiment. The results from this experiment would reveal which therapy out of the ones mentioned in The Forgotten Cure would be the most beneficial and safest to use.

Rationale:

Compose an abstract about the individual research project.

Procedure:

1. Utilized outline created previously to write.
2. Read over abstract draft for mistakes.

Results:

The following image shows the abstract written.

Conclusion:

Abstract was written.

Future Work:

More research will be performed using Jmol to find catalytic regions of other Arthrobacter lysins.

Rationale:

Outline an abstract about the individual research project and use bioinformatic tools to analyze different lysins that infect Arthrobacter.

Procedure:

1. Outlined an abstract about individual research project.
2. Used tools such as PhagesDB, NCBIp BLAST, and TMHMM to analyze AL, AM, and AN cluster phages’s lysins and holins.

Results:

The following image shows the outline created.

The following images show the chart created to organize data collected from search.

(Clusters AL (Shrooms) –– AN (Muttlie)-21ktwwj)

Conclusion:

After looking through phages from Cluster AL (Shrooms) to Cluster AN (Muttlie) it was found that generally AL cluster phages had a lysin A with a PGRP superfamily CDD with a holin. AM clusters generally had an endolysin around gene 5 with a Peptidase_M23 superfamily CDD with holin. AN cluster phages generally either had two lysin A with one lysin A with a Peptidase_M23 superfamily CDD at gene 16 and the other lysin A with a PGRP superfamily CDD and a holin around gene 18. The other commonly found pattern followed in AN cluster phages was an endolysin and a holin around gene 18. Despite having two different trends all AN cluster lysin genes had the same Pham numbers. Only AM cluster phages had the “HLH” catalytic region.

Future Work:

Continue analyzing different phages lysins and holins.

Rationale:

Analyze endolysins and holins to collect more data to create a presentation to present at SEA-PHAGES symposium.

Procedure:

1. Printed out and analyzed RaptorX structures created previously.
2. Outlined SEA-PHAGES symposium presentation.
3. Used Blender to model holin’s hole formation.

Results:

The following images shows the endolysins and holins structure predicted with RaptorX.

The following image shows the unfinished holin creating holes in the plasma membrane model.

Conclusion:

Compared with Multiple Sequence Alignment and phylogenetic tree results, the predicted protein structure visualized with RaptorX revealed that there are although sequences were quite similar, the structures varied.

Future Work:

Finish creating presentation and present poster and presentation at SEA-PHAGES symposium.

Rationale:

Start analyzing endolysins and holins in the AM cluster.

Procedure:

1. Performed Multiple Sequence Alignment with AM cluster endolysins along with FF cluster phage Elesar’s and FE cluster phage Corgi’s endolysins.
2. Performed Multiple Sequence Alignment with AM cluster holins along with BI cluster phages Madamoto’s and Bing’s holins.
3. Folded protein structures with RaptorX.
4. Explored and discovered NapoleonB’s endolysin’s catalytic site with Jmol.

Results:

The following image is the results from Multiple Sequence Alignment with AM cluster endolysins along with FF cluster phage Elesar’s and FE cluster phage Corgi’s endolysins from Clustal Omega.

The following image is a phylogenetic tree from endolysin Multiple Sequence Alignment.

The following image is the results from Multiple Sequence Alignment with AM cluster holins along with BI cluster phages Madamoto’s and Bing’s holins from Clustal Omega.

The following image is a phylogenetic tree from holin Multiple Sequence Alignment.

The following image shows NapoleonB’s endolysin’s catalytic site.

Conclusion:

The Multiple Sequence Alignment and phylogenetic tree show high similarly between phages. NapoleonB’s endolysin’s catalytic site, which has the responsibility to cleave the bacterial host’s peptidoglycan membrane, appears to be located in beta-sheets.

Future Work:

Predicted endolysin and holin structures will be analyze more.

Rationale:

By reading through published articles, more information on holin and Peptidase_M23 endolysin proteins’ chemical mechanisms will be discovered to increase my team’s background on the subject.

Procedure:

1. Performed PubMed searches with various topics relating to holin and Peptidase_M23 endolysin proteins.
2. Looked at various hydropathy graphs for the holin protein.

Results:

The following modified question was determined to be the research question.

Question: Through using bioinformatic tools, are phage holin and endolysin structures and functions conserved across different Arthrobacterphages, and what key characteristics in each protein determine the mechanisms?

The following articles were of interest.

https://www.sciencedirect.com/science/article/pii/S0005273614002107?via%3Dihub

https://www.nature.com/articles/srep14833

https://www.sciencedirect.com/science/article/pii/S0065352718300563?via%3Dihub

https://www.microbiologyresearch.org/docserver/fulltext/micro/161/12/2269_mic000190.pdf?expires=1554401307&id=id&accname=sgid026869&checksum=07E768FFC4737D052907C768EF754256

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6354061/

The following hydropathy graphs were created using NapoleonB’s holin protein sequence.

(The graph above was created from DTU Bioinformatics TMHMM Server.)

(The graph above was created from HMMTOP.)

(The graph above was created from Transporter Classification Database.)

Conclusion:

A key characteristic in Dp-1 phage holin is its loop. As shown in the hydropathy graphs the loop was predicted to be found outside the lipid bilayer membrane.

Future Work:

Finish literature research and specifically research more into holin’s structure.