March 29

03/25/19 Independent Project Questions

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Rationale: 

The purpose of today’s lab was to come up with 4 testable scientific questions that could be tested for our independent projects.

Tools:

  • Canvas Module with phage genomics research topics
  • Laptop

Procedure:

  • Group met up and began discussing several project ideas and different research possibilities.
  • Asked research mentor for thoughts on different project ideas, and which would possibly offer the best research.
  • Ranked each question 1-4, 1 being the best question, 4 being the least preferred question.

Results:

Research Questions:

  • NapoleonB has both a holin and an endolysin gene, but other phages in the AM cluster do not have this (or people simply did not call it). Bioinformatic tools could be used to compare holin protein structure and the mechanism behind the interactions with the bacterial cell walls.
  • Random Reverse gene (no coding potential, was called)
    • Is gene 2 an actual gene even if it has no coding potential? If so, why?
    • Are there other random reverse genes in other phage genomes that were called and break the guiding principles of gene annotation? (maybe phylogenetic tree to suggest any relationships?)
  • Why does NapoleonB have multiple tail proteins? How does that compare to other phages?

Conclusions:

It was chosen to rank the question regarding the holin and endolysin proteins, mostly due to the fact that it was a question that posed both a level of difficulty that would challenge the group and it was different from the rest of the class. It also presented an opportunity to do a more in depth analysis of the essential functions of a lytic bacteriophage.

Next Steps:

The next steps for this project are to begin with researching the literature behind this question.

March 21

03/20/19 Independent Projects

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Rationale: 

The purpose of today’s lab was to address any last changes needed on the class poster as well as begin the process of independent projects.

Tools:

  • Laptop
  • Google Slides
  • ExPASy

Procedure:

  • Last minute concerns were addressed regarding the poster.
  • Once this was complete, independent projects were introduced as well as groups and mentors.
  • Independent projects topics were then researches to decide if anything drew up any interest in the group.

Results:

  • The group for the independent project is Me, Kathryn, and Emily, with our coach being Lathan.
  • Several topics were looked over, but none have been decided yet to research.

Conclusions:

There are several things regarding NapoleonB, but the group is still trying to find something that is unique regarding NapoleonB to research. It is possible there is something regarding NapoleonB’s relationship to other phages in its cluster as it does contain an orfam, but no clear decision has been made yet.

Next Steps:

The next steps for this are to continue with the research of these different topics so the group can begin its independent research project.

March 21

03/18/19 Class Poster Edits

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Rationale: 

The purpose of today’s lab was to split up the responsibilities of poster editing among the class to get it ready for Scholar’s Week.

Tools:

  • Laptop
  • Google Slides
  • AdobeSpark

Procedure:

  • The class looked over the edits needed on the poster that Lathan gave us.
  • Then, groups were divided tasks, our group being in charge of graphics on the poster and methods revising.
  • Methods and materials were extended to include more information regarding the in silico portion of the lab, while also creating new graphics for the added sections through AdobeSpark.
  • Figures for each heading were made as well to help make it a reoccurring theme throughout the poster.
  • The discussion was also edited and formatted in AdobeSpark, with corresponding figures as well.

Results:

  • The methods and materials were updates and corrected.
  • The final poster result with figures in each heading

Conclusions:

Though missing figures for the in silico results, the majority of the poster is complete. Centering errors, wording, and colors were altered and changed to best fit the poster, and the poster now has a unique theme and aspect with all the self-created images scattered throughout.

Next Steps:

The next steps for this are to finish the finishing touches on the class poster, begin creating a presentation to showcase at Scholar’s Week, and begin thinking about independent projects.

March 17

The Forgotten Cure Part 2

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Phage therapy has been subjected to the influences of several varying cultures and political climates in countries such as Russia, India, Poland, and the United States. Each country viewed phage therapy differently than the other, ushering in a wide array of uses for phage in some countries such as Russia and Poland, while also being completely reduced to nothing in the others such as the United States.

During the 1940s-50s, Russia’s state health system influenced the treatment of infectious diseases greatly as the country could not rely on the use of “superdrugs” such as antibiotics to fight off infectious diseases. The country’s healthcare was supplemented purely by the government, and due to lack of funding and resources, the Russian people had to turn to alternative treatments to prevent infectious disease. With the withdrawal of financial support from the West after the end of World War II, the Russian government turned to propaganda to turn the citizens away from the “foreign Western antibiotics” urging them to use herbal remedies that were considered more patriotic and just. This turn away from traditional Western medicine allowed alternative treatments such as phage therapy to flourish in the Soviet Union as it was cost-effective and supported by the government’s propaganda.

Despite both the Phage Therapy Center and the Eliava Institute being under the rule of the Soviet Union, both institutions had vastly different outcomes regarding their phage research. The Eliava Institute, unfortunately, gained its peak during World War II at the height of Stalin’s reign of terror. During the war, it was primarily used to further bacteriophage research so that they could control the spread of infectious diseases on the battlefield, but once the war ended so did their usefulness. The institute’s leader, George Eliava, fell victim to Stalin’s reign of terror and was executed by the regime. Many did not return to the institute and it began to fall into disarray, but the institute is now running once again trying to further bacteriophage research. Unlike the Eliava Institute, The Phage Therapy Center in Wroclaw did not share the same fate and once the USSR fell, the Polish government continued to fund the institution’s research with phages. The center focuses now more on the use of bacteriophages to treat antibiotic resistant diseases, and even has received support from the European Union.

In the early 1990s, Carl Merril, Richard Carlton, and Sankar Adhya performed a revolutionary experiment that allowed them to select for phages that could evade the liver and spleen in their host organisms. This experiment was brought about as many attempts of phage therapy in animals seemed to fail as the phages would disappear in their host way too early to fight off any infection. To perform the experiment, the team first injected a phage they called “lambda” into the stomachs of mice. They waited for 7 hours, removed blood samples from the mice, isolated phages from the blood, and reinfected the mice with the isolated phage samples. They performed this task 8 times to isolate a phage that could remain in the bloodstream for at least 18 hours. The new phage that they isolated (Argo1 and Argo2) had a survival rate thousands of times higher than their parent lambda and proved they were still capable of curing mice from lethal doses of E. coli.

As the risk of antibiotic resistant bacteria continues to rise as more and more physicians rely on treating bacterial infections with antibiotics, several companies have begun to focus on alternative solutions to superbugs. One of which, GangaGen, focuses on developing therapeutic proteins that target drug resistant bacteria. GangaGen’s leading ectolysin P128 is one of those proteins, and it focuses on antibiotic resistant strains of Staphylococcus. Its incredible effectiveness comes from its ability to only attack its specific strain and leaves the rest of the beneficial bacterial flora intact. Most of these companies are trying to eliminate the incoming threat of antibiotic resistant strains of bacteria by furthering the practice of phage therapy and are utilizing their natural ability to attack bacterial cells. Still, it is a long road ahead, and many more experiments, phages, and proteins need to be derived, isolated, and tested.

March 8

03/06/19 Class Poster Decisions/ Starterator

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Rationale:

The purpose of today’s lab was to decide as a class on which poster to present as well as checking my starterator annotations.

Materials:

  • PowerPoint
  • DNA Master
  • Starterator PhagesDB

Procedure:

  • Each group presented their poster submissions, and the class voted on which to use.
  • Starterator annotations for genes 61-64 were checked and fixed accordingly.

Results:

  • Genes 61 and 63 were changed to NI instead of SS and genes 62 and 64 were left as SS.
  • This was the poster that won the majority of the class
    •  

  • Possible additional poster elements

Conclusions:

Genes 61 and 63 were changed to NI as the starterator did not aid with any start calls as I disagreed with the starterator report. Also the poster that was chosen by the class is the poster that the class will continue to work with to present.

Next Steps:

Polish and refine the elements on the poster as well as looking to add some “meat” to the poster.

March 8

03/04/19 Final Poster Submission/ Gene Correction

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Rationale:

The purpose of today’s lab was to correct the genes that were found to have errors in them, as well as finish a group submission poster for the class to review.

Tools:

  • NapoleonB PhageNotes
  • DNA Master
  • PowerPoint

Procedure:

  • Gene 64 was found to have a coding potential error, and a gap miscalculation. In addition to this, gene 61 was found to have an RBS value error.
  • Both genes had their errors corrected, and a final presentation poster was completed and submitted as well.

Results:

  • It was decided that gene 64 did not cover all of the coding potential marked by genemark and that it has an overlap of 11 instead of 9.
  • Also, the Z-value for gene 61 was inputted incorrectly and was corrected to the proper 3.113 instead of 3.118.
  • Final poster submission:

Conclusions:

It was decided to change the coding potential originally annotated for gene 64 as the atypical coding potential extended past where the gene was called. The gene could have been pulled back to a longer ORF, however that would have violated the overlap rule that guided phage genome annotations. The overlap miscalculation was an error on my part as I did not add, I subtracted. Also, the z-value was an input error.

Next Steps:

The next steps for this experiment are to decide on a poster to represent as a class and check my starterator hits.

March 1

02/27/19 Poster Design

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02/27/19 Poster Design

Rationale: 

The purpose of today’s lab was to create a poster design on PowerPoint for Scholars Week based off of the rough draft created.

Tools:

  • Microsoft Powerpoint
  • DNA Master Results
  • Microsoft Excel

Procedure:

  • Created a poster through Microsoft Powerpoint by setting the dimensions to 48 x 48 inches, selecting a color scheme, and inserting all the necessary charts, graphs, and figures.

Results:

  • The poster was not yet completed, but significant progress was made.

Conclusion:

The poster making process was incredibly difficult to complete within the allotted time period as there were several learning curves to figuring out how to adjust for the extreme size of the powerpoint.

Next Steps:

The next steps for this poster are to continue tweaking little details until a fully polished and presentable poster is ready.

March 1

02/25/19 Poster Rough Draft

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Rationale: The purpose of today’s lab was to look at past poster designs and create a rough outline of a possible poster deign for scholars week.

Procedure:

  • Previous years poster designs were examined as the class determined what they liked and what they didn’t
  • Poster design groups were then assigned
  • Sheets of paper were taken out and poster design outlines were drawn out and drafted for submission.

Results:

  • The resulting poster was drafted with some possible figure ideas and layout.

Conclusions:

Although a very rough draft was made, there are still several elements and figures that have yet to be added to the poster.

Next Steps:

The next steps for this experiment are to take the draft that we have created and apply it to creating a poster for Scholars Week.

February 18

Forgotten Cure Post Chapters 1-4

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The Forgotten Cure, by Anna Kuchment, offers us insight into phage biology and therapy well beyond the textbooks, as well as offering us the story behind scientific discoveries such as phage biology. After reading the first four chapters of the book, here are my thoughts about what I have discovered about the process of scientific discovery:

The father of bacteriophages, and a pioneer for one of the most incredible advances in modern science, was self taught. How incredible is that? There are so many things that culminated into the discovery of the world of microbes, and more specifically, phages. All building off the knowledge and discoveries made by those prior. D’Herelle, of course, discovered bacteriophages purely by chance while plating bacterial cultures during his work with sick locust. The war brought forth new evidence for the plaques D’Herelle discovered on his agar plates as patients recovering from dysentery yielding plaques in the bacterial colonies taken from their stools, as he tested the introduction of phages to bacterial solutions. Of course, a discovery wouldn’t be complete without opposition right? The concept of bacteriophages contradicted many long-held beliefs in the world of microbes, but that did not stop D’Herelle from holding strong to his beliefs. He was able to successfully treat diseases such as the bubonic plague, a disease that had troubled mankind since the dark ages by using phage therapy 15 years before the discovery of penicillin. Also, to me it is incredible how fast D’Herelle was able to achieve legitimate results by jumping straight to human trials as there were no regulatory agencies in his way. I know that those agencies are put in place to ensure proper ethical and legitimate research is being conducted, but in the case of phage therapy, nothing screams legitimate results like human phage treatment.

It surprised me just how much human conflict drives scientific discovery, especially in the case of phage therapy. In times of war, countries will put forth their efforts into the sciences, in the hopes that something will turn up that will give them the edge on their competitors. That was the case of the USSR in the early to mid 30s, following World War I and leading up to the front of World War II. I was completely unaware of the “war on disease” Stalin had called upon the USSR, establishing institutions dedicated to acquiring knowledge of bacteriophages.

The biggest thing that frustrated me regarding the story of phages was the overwhelming amount of ignorance that impeded the process of scientific discovery. Despite physician experimentation occurring in the 1930s in the United States, they were incapable of producing legitimate results simply due to their inability to perform consistent experiments. In addition to this, many held fast to their beliefs that there existed no organism smaller than a bacterium, and those who held the correct viewpoint on phages were in the overwhelmingly small minority. Due to these failures in research, phage therapy results were few and underwhelming, leading bacteriophage results to be overshadowed by new drug advances in the form of sulfa pills and of course, penicillin. These two untimely discoveries helped solidify phage therapy’s fate in the early 1940s, as the people in the scientific community simply lost interest and moved on to the next thing that caught their eye. Despite experimentation continuing with scientist like Delbruck and Luria discovering that DNA was the genetic material and not protein through the use of bacteriophages, not much was done in terms of their use in medicine. Technology did not help push phage researcg forward as it allowed humans to work with larger animal cells, completely leaving bacteriophages behind in the 70s. If only American medicine had continued to keep phage therapy in their sights instead of sweeping it aside, the possible discoveries we could have made by now in 2019 after almost 80 years of research would be incredible.

In conclusion, the road to scientific discoveries is a long one, and this holds true in the case of phages. Initially deemed as agents of “immunity”, bacteriophages seemed to offer humanity the cure to bacterial infection, only to be forgotten after new discoveries pushed it away. Now, over 100 years after their initial discovery by D’Herelle, they have made their way back under the sights of scientists’ microscopes as they once again offer us a solution to the issue of super-bacterial infections, beginning the road to scientific discoveries for phages once again.

 

 

 

February 15

02/13/19 Finishing NapoleonB Genes 63-64

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Rationale: 

The purpose of today’s lab was to finish annotating the final 2 genes assigned in NapoleonB

Tools:

  • DNA Master
  • NCBI BLASTp
  • HHPred
  • Phamerator
  • Starterator
  • PhagesDB BLASTp
  • Genemark (pdf)

Procedure:

  • Lab began immediately with finishing up final annotations of assigned genes from the previous lab.
  • Ran genes 63 and 64 through a series of BLASTp searches in both NCBI and PhagesDB, searched for protein folding structure in HHpred, analyzed the starterator analysis through PhagesDB, and checked for any supporting information of synteny by examining similar phages in the AM cluster with NapoleonB.

Results:

  • Final Annotation for Gene 63:
    • SSC:41221 – 41427, CP:Yes, SCS:BothGM, ST:SS, BLAST-Start:Aligns with Arthrobacter Phage Arcadia gp62 NCBI BLAST q1:s1 0.95 2E-32, Aligns with Nason gp62 PhagesDB BLAST q1:s1 0.96 1E-28, Gap:4bp overlap, LO:NA, RBS:Kibbler7 and Karlin Medium 1.104 -6.698 Yes, F:NKF, SIF-BLAST:NKF, SIF-HHPred:NKF supported by gp , SIF-Syn:NKF
  • NCBI BLASTp Results:
  • PhagesDB BLASTp Results:
  • HHPred Results:
  • Phamerator Results:
  • Final Annotation for Gene 64:
    • SSC:41417 – 41782, CP:Yes, SCS:Both, ST:SS, BLAST-Start:Aligns with Arhtrobacter Phage Correa gp60 NCBI BLAST q1:s1 0.64 1E-46, Aligns with Arthrobacter Phage Tribby gp65 PhagesDB BLAST q1:s1 0.76 1E-38, Gap:9bp overlap, LO:NA, RBS:Kibbler7 and Karlin Medium 3.16 -2.354 No, F:NKF, SIF-BLAST:NKF, SIF-HHPred:NKF supported by gp , SIF-Syn:NKF
  • NCBI BLASTp Results:
  • PhagesDB Results:
  • HHPred Results:
  • Phamerator Results:

Conclusions:

For both genes, the protein call was decided to be NKF as there was not enough supporting information to call a protein function with confidence. Despite there being a potential hit on HHPred, the probability was not enough to be sufficient for a call. Also the RBS for gene 63 despite having a low z-score was chosen as other suggested RBS shortened too much of the gene.

Next Steps:

The next steps for this lab are to edit and finalize any errors in the gene annotations made.