Day 5: Meeting and Collaboration

2/20/17

We held another meeting as a Nubia group as planned to discuss how far everybody was on their parts of the annotation process. After the meeting, we found out that Lathan and Dr. Adair wanted us to include the Q and S values for both PHages DB and NCBI Blast, so we re-blasted the genes we worked on. After, we finished looking at the Q an S values we checked over each others work as we were almost done.

After this, we exchanged data with Navya and Christina as they were also done with their annotations. In the time left, I checked genes 32 and 34. Gene 32 was good, but their gene 34 had a different NCBI blast and conserved domain hit from what I had. I consulted with them and they realized that they forgot to change the blast results so I went ahead and corrected that.

Day 6: More Double Checking

2/22/27

I continued checking Navya and Christina’s annotations. I checked genes 38, 40, 42, and 44. My phamerator froze my computer for a while which is why I only checked 4 genes through the lab time. Out of these 4, I discovered a problem in the HHPred e-value. I consulted with Navya and Christina and we changed the value from 7.7e^-24 to 8.2e^-24. We spent the entire day checking and double checking each other’s genes.

Day 7: Meeting

2/27/17

Today we had another team meeting. We were told to split up the entire Nubia group into separate groups to do different jobs. Alex and I were tasked with creating a Master DNA file. This DNA Master file was to include the final annotations in the description of the DNA Master file which is supposed to be a part of the final submission. We worked on this the entire lab period. I added the full and complete annotations to to the odd numbered genes while Alex added them to the even numbered genes.

Day 8: AN Phage

3/1/17

Today, Dr. Adair announced that there was AN phages that we could potentially take up. I decided to take one named Saphira. Alex volunteered to take over the full burden of the final DNA Master file which I really appreciated. I started working on Saphira. I am working on this phage with Chrissy. I have genes 1-13. I had trouble figuring out gene one as I didn’t remember what to do with a gene that on DNAMaster continued on from the end to the beginning, but I consulted with Lathan and he refreshed me on the procedure. Otherwise, I worked genes 2,3, and 4 with no further complications.

Day 9: Continuation of the annotation of the AN Phage Saphira

3/13/17

I continued annotating the phage Saphira, and completed genes 5,6,7, and 8. I also helped Alex out with checking starterator and phamerator as the program was not working on her computer.

Day 10: Meeting for group project

3/15/17

Today we had a meeting for the group project. Our Group consists of me, Micheal, Alec, and Daniel. We plan to meet this Saturday after doing some homework. We each have to look over some posters, write up a possible conclusion and introduction, and search for ideas we could use in our presentation and for our topic.

Below is a picture of what we brainstormed on Saturday(3/18/17):

Day 11-Day 12: Group Project

3/20-3/22

Our group project is due on the 22nd of March so for these lab days we used these days and out of labs to prepare for the presentation and the poster. Below is a picture of our poster:

Day 13: Scholars Week Practice

3/27/17

Today we got together in our Scholars Week Presentation. Our group is me, Micheal, Alec, and Daniel. We got assigned the poster we had to present and we worked on being able to explain it properly, especially the 16/17 gene slippage and the Gepard plot that were present on the poster.

Day 14: Presenting at Scholars Day

3/29/17

Today we spent the lab time taking shifts presenting our poster at Scholars Day in the Atrium of the BSB. It was a really cool experience I thought it was really cool that people were coming to us to provide them knowledge in the subject and that we were the experts and I wasn’t on the other side of things for once. There will always be more questions, but it felt good to educate people about our poster.

Day 15: Preparing for LaTourneau

4/3/17

Today in lab, the people who were going to attend the Regional SEA-PHAGES Symposium at LaTourneau split off from the rest of the group to discuss what the gameplan was. We got split into different presentation groups again for LaTourneau.

Day 16: Preparing for LaTourneau Continued

4/5/17

Today we presented our poster to the people in the class in order to prepare to present at the Regional SEA-PHGAES Symposium. Below is a picture of our group at LaTourneau in fromt of the poster we presented.

Day 17-Now: Individual Project

4/10/17-4/26/7

After the SEA-Phages Regional Symposium, which I thought was really fun and a great learning experience, we started working on our individual projects. Below is a sheet in my notebook that we used to plan out the project.

Thomas was very interested in exploring the potential for there to be a connection between start codons and if they code for a known function or not. So going ahead with this topic, we struggled to find a paper at first that showed that our topic was possible or feasible as there hasn’t been much research on codons. Unfortunately, a program we had hoped to use to analyze these codons didn’t work and we spent two labs trying to fix the program and make it run but it didn’t. This really set us back as we had to do a bunch of number crunching by hand and by looking at the codons ourselves for each phage in each cluster so we were not able to take the project in the direction that we wanted to.

Pranav Kapoor

4/25/17

Prompt 1: Describe the differences between Intralytics 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?

The last section of The Forgotten Cure is mainly about the four companies, and their struggle to become approved by the Food and Drug Administration (FDA). The author, Anna Kuchment primarily focuses on two companies who take different approaches to try to be approved by the FDA.

Intralytics is focused on developing phages for food safety(fruits, vegetables, eggs, and meat) and human diseases. The leader of Intralytics, Sulakvelidze worked on phage cocktails to hopefully solve these problems. He realizes that there is no consensus on phages, which is why he is working so hard to show people that this is a viable solution. The other pathogen he was very interested in was Stapholococcus aureus. Poultry companies liked the idea of bacteriophages because of its inexpensivity. It was in 2002 that Intralytics fisrt began petitioning the FDA for permission to launch clinical trials. In 2006, the FDA changed their regulation on phages and declared them safe. Intralytix was able to use their phage cocktail on luncheon meats to fight listeria. Their phage cocktail was mainly used to clean the processing plant, rather than the fruits. After a couple of setbacks, Intralytics received a $1.5 million contract with the Army to create a cocktail to fight against E. Coli in infected soldiers.

The second of the main bacteriophage companies, GangaGen, was started by Dr. Ramachandran. Their company was based in India. They primarily focused on phage therapy. Initially he focused on Stapholococcus aureus  but after having a meeting with his scientsits and investors, he realized he should be focusing on E. Coli. Unfortunately, due to insufficient funding, he could not conduct clinical trial sand get past the FDA. As a result, many investors and board members jumped ship. However, GangaGen was able to isolate a phage tail and ultimately show that phages can kill E. coli in a cow’s intestines.

Currently, Intralytics is still working on food safety, and have expanded on safety for pets as well as humans. Their main product is a product targeting Shingella which was just recognized by the FDA as Genreally Safe. GangaGen still targets bacteria pathogenic to humans, particularly antibiotic-resistant strains. They want to pursue a more humanistic application. GangaGen’s lead product is a recombinant protein, P128, that binds to and kills Staphylococcus, including methicillin-resistant strains of S.aureus (MRSA).

Pranav Kapoor

Chapter 5 starts with how in the early stages of phage therapy, the USSR was actually ahead of the rest of the world when it came to phage therapy. Because of politics, the USSR couldn’t properly communicate and share ideas so western countries would send representatives to them to share their research, products, or ideas. Unfortunately, after Western influence and funding was removed after World War II, they couldn’t  continue to function at the level they previously were. Then when the USSR was disbanded, the phage program deeply sunk; They went from producing 200L per day to 200-300L per year. The resources were so scarce that scientists would store samples at home in their own refrigerators. It was even harder to generate funding because of the fact that USSR was a government/State funded health system. This led to a more dependence and development on herbalistic and naturalistic methods in Russia because of the scarce availability of other medicines.

Similarly, Hirzfeld had the same problems when it came to funding. He had many obstacles to overcome. He had to convert religions from Judaism to Catholicism to avoid anti-semitism but this greatly backfired as he was still put in the Warsaw Ghetto where he was also disliked by fellow Jews because he converted. He lost his daughter because of the Ghetto. He was devastated. I feel so bad for him that must have been a huge blow. The Eliava Institute and the Phage therapy Center are similar, however the Eliava Institute focuses more on research while the Phage Therapy Center is focused on treatment.

The United States didn’t really get into Phage Therapy until Carl Merril. He was interested early on in his career where he took a class during Medical School, but he didn’t start experimenting until he read an article which sparked his interest again. He came up with an experiment to isolate phages that didn’t get removed by the liver or the spleen. He injecting the phage in  mice, took blood samples from the mice, then re-injecting the isolated phage into the mice. After eight rounds of this procedure, Merill was able to isolate Argo 1 and Argo 2, which were two phages that were could not be destroyed by either the spleen or the liver. He then went on to show that Phage Therapy, in tandem with traditional medicine, is highly effective and more effective than traditional methods alone. I believe that one of the main reasons doctors in the United States don’t trust phage therapy is that they are not exposed o]to it during medical school. That coupled with the fact that the U.S. government an its programs are also hesitant, provide a bad combination for phage therapy to grow. We need to accept it as this is an important and useful tool that w have at our disposal that we don’t use. It is up to private companies to innovate as the government isn’t helping.

Pranav Kapoor

2/15/17

The goal for today is to finish our part of the annotation. We have Genes 13-17 left. Alex already started on 13, so I took Gene 14 and 15. We ended up not having to annotate Gene 17 as the second group who was doing Genes 17-32 (Chrissy and Caroline) found that they had a frameshift at Gene 17 so they decided that it would be easier for them to work on Gene 16 also.

Tools:

Phamerator, Starterator, NCBI Blast, phagesDB Blast, HHpred, DNA Master, GeneMark

Results:

Gene 14

Start: 10412bp Stop: 10822bp FWD GAP: 1bp Gap SD Final Value: SD Score: -2.942 (Best score) Z-Value: 2.786 CP: The gene is covered SCS: Agrees with Glimmer, Agrees with GeneMark NCBI BLAST: hypothetical protein SEA_OXYNFRIUS_14 E-Value: 4e-92 CDD: No good hit PhagesDB BLAST: Oxynfrius_14, function unknown E-Value: 1e-72 HHPred: 5a21_G Tail-TO-head joining pr E-Value: 3E-17 LO: Yes ST: Agrees with Starterator F: NKF FS: NCBI, Phages DB Notes:

Gene 15 

Start: 10838bp Stop: 11746bp FWD GAP: 15bp Gap SD Final Value: SD Score: -3.442 (Best score) Z-Value: 2.679 CP: The gene is covered SCS: Agrees with Glimmer, Agrees with GeneMark NCBI BLAST: hypothetical protein SEA_OXYNFRIUS_15 E-Value: 0.0 CDD: No good hit PhagesDB BLAST: Oxynfrius_15, function unknown E-Value: e-171 HHPred: No good hit LO: Yes ST: Agrees with Starterator F: NKF FS: NCBI, PhagesDB, HHPred Notes:

Next Steps:

The next step is to check our own work, then meet up with a different pair and have them check our work also.

Pranav Kapoor

2/13/17

Today I annotated Gene 6, then after that I Starterated all the previous Genes and the Genes that Alex and I annotated today(1-12).

Tools:

DNA Master, GeneMark, Starterator, NCBI Blast, PhagesDB Blast, HHpred

Results:

Gene 6

Start: 4652bp Stop: 6670bp FWD GAP: 18bp Gap SD Final Value: SD Score: -3.512 (Best score) Z-Value: 2.647 CP: The gene is covered SCS: Agrees with Glimmer, Disagrees with GeneMark GeneMark calls the gene shorter, but that leads to a higher SD score and Z value. NCBI BLAST: hypothetical protein JOANN_1 [Arthrobacter phage Joann] E-Value: 0.0 CDD: Nucleoporin_FG2 E-Value: 4.75e-05 PhagesDB BLAST: Joann_1, function unknown E-Value: 0.0 HHPred: No good hit LO: Yes ST: Agrees with Starterator F: FS: Notes:

Gene 9

NCBI Blast:

PhagesDB Blast:

Starterator:

Start: 8722bp Stop: 8925bp FWD GAP: 1bp Gap SD Final Value: SD Score: -5.78 (2nd best score) The reading frame for that gene would only be 9 bp. I wouldn’t cover the entire coding potential. Z-Value: 1.453 CP: The gene is covered SCS: Agrees with Glimmer, Agrees with GeneMark NCBI BLAST: capsid decoration protein [Arthrobacter phage RcigaStruga] E-Value: 1e-37 CDD: No good hit PhagesDB BLAST: RcigaStruga_9, Capsid decoration protein E-Value: 9e-30 HHPred: No good hit LO: Yes ST: Agrees with Starterator F: capsid decoration protein FS: NCBI, PhagesDB Notes:

Gene 10

NCBI Blast:

PhagesDB Blast:

Starterator:

Start: 8997bp Stop: 9191bp FWD GAP: 71bp Gap SD Final Value: SD Score: -2.589 (Best score) Z-Value: 2.943 CP: The gene is covered SCS: Agrees with Glimmer, Agrees with GeneMark NCBI BLAST: hypothetical protein JOANN_10 [Arthrobacter phage Joann] E-Value: 2e-37 CDD: No good hit PhagesDB BLAST: Oxynfrius_10, function unknown, E-Value: 2e-30 HHPred: No good hit LO: Yes ST: Agrees with Starterator F: NKF FS: NCBI, PhagesDB Notes:

Gene 12

NCBI Blast:

PhagesDB Blast:

HHPred:

Start: 9782bp Stop: 10120bp FWD GAP: 0bp Gap SD Final Value: SD Score: -5.6 (3rd best score) This allows for the best combination of Sd score and Z value while completely covering the coding potential Z-Value: -1.545 CP: The gene is covered SCS: Disagrees with Glimmer, Agrees with GeneMark GeneMarks call is better as it coveres the entire coding potential. NCBI BLAST: hypothetical protein SEA_OXYNFRIUS_12 [Arthrobacter phage Oxynfrius] E-Value: 8e-79 CDD: No good hit PhagesDB BLAST: Oxynfrius_12, function unknown E-Value: 3e-62 HHPred: 1 3f3b_A Phage-like element PBSX E-Value: 9.5E-17 LO: Yes ST: The extended gene coveres the entire coding frame and has better SD and Z values. F: FS: Notes:

Gene 4 Starterator

Gene 5 Starterator:

Gene 6 Starterator

Gene 7 Starterator

Gene 8 Starterator

Gene 11 Starterator

Conclusions and Next Steps:

I realized that I didn’t take screenshots for Gene 6. I did not have too much trouble annotating these genes however I did have to extend the start of Gene 2 from 9914 to 9782 at the 3′ end. The next step is to finish annotating our genes by next lab.

Pranav Kapoor

2/8/17

Today I finished up Gene 1 and completed Genes 2 and 4. I did not take screenshots today as I was not aware but I will take screenshots for future labs.

Tools:

Phamerator, Staterator, HHpred, NCBI, phagesdb, DNA Master, and GeneMark.

Results:

Gene 1

Start: 77bp Stop: 1696bp FWD GAP: bp Gap SD Final Value: SD Score: -4.387 (Best score) Z-Value: 2.103 CP: The gene is covered SCS: Agrees with Glimmer, Disagrees with GeneMark NCBI BLAST: hypothetical protein JOANN_1 [Arthrobacter phage Joann] E-Value: 0.0 CDD: Nucleoporin_FG2 E-Value: 4.75e-05 PhagesDB BLAST: Joann_1, function unknown E-Value: 0.0 HHPred: No good hit LO: ST: Agrees with Starterator

Gene 2

Start: 1706bp Stop: 2521bp FWD GAP: 9bp Gap SD Final Value: SD Score: -4.325 (2nd best score) THis SD score allows for the whole coding potential to be covered. Z-Value: 2.103 CP: The gene is covered SCS: Agrees with Glimmer, Disagrees with GeneMark NCBI BLAST: hypothetical protein SEA_OXYNFRIUS_4 E-Value: 6e-99 CDD: No good hit PhagesDB BLAST: Oxynfrius_4, function unknown E-Value: 9E-76 HHPred: No good hit LO: Yes ST: Agrees with Starterator F: FS: Notes:

Gene 4

Start: 2689bp Stop: 3135bp FWD GAP: 20bp Overlap SD Final Value: SD Score: -4.811 (Best score) Z-Value: 2.123 CP: The gene is covered SCS: Disagrees with Glimmer, Disagrees with GeneMark We extended the gene to cover the coding potential. This also led to a lower SD score and Z value. NCBI BLAST: hypothetical protein SEA_OXYNFRIUS_4 E-Value: 6e-99 CDD: No good hit PhagesDB BLAST: Oxynfrius_4, function unknown E-Value: 9e-76 HHPred: No good hit LO: ST: We extended the gene based on SD score and Z value and because the new range now covers the entire reading frame. F: FS: Notes:

Conclusions and Next Steps:

Today, we were getting a handle on the speed and the process of how we should annotate which is why I only got three Genes done today. One thing to note is that I extended Gene 4 in order to cover the coding potential. However I had to move to a lower SD score and Z value.

Pranav Kapoor

2/6/17

Rationale:

After downloading the FASTA file for Nubia and letting DNA Master Auto-annotate it, we met as a group. There are 8 people in our group, and 63 genes in Nubia. We split one another into 4 groups of 2. Our group is Alex and I. We then divided the genes equally among us and got to work annotating them. Alex and I got genes 1-16. We decided to work on a gene individually, and check on each other’s work as we went.

Tools:

PhagesDB

Procedure:

Download the FASTA file from PhagesDB. The meeting and the downloading took so long that we couldn’t start working on our genes.

Next Step:

Start annotating the gene.

Before reading this book, I didn’t realize the difficulty people went through to discover bacteriophages. They were discovered partly because of the bloodies war in history, World War One. Originally calling them “taches vierges”, Felix D’Herelle discovered bacteriophages on accident while studying locusts. Clear spots had developed on the cloudy base of the typical bacterial culture in a dish, which  was unusual at the time. With the war bearing down, D’Herelle focused on creating a cure for big diseases that were problems. In his case, dysentery. He discovered this in a time where people were frantically searching for cures an pandemics were spreading throughout the world. D’Herelle traveled the world trying to spread his findings. Unfortunately, there was also severe political polarization. The popular opinion at the time was that those clear spots were due to enzymes produced, and not bacteriophages. As time went on, bacteriophages started to die down especially after the discovery of Sulfanilamide by the Beyer group. However, in the mid-1900’s, two scientists, Luria and Delbruck, founded The Phage Group. Scientists in this informal group focused on bacteriophages and believed that they were essentially the biological equivalent of hydrogen atoms. Delbruck, along with Hershey and Luria even won a Nobel Prize in 1969 for their discovery of the replication mechanism and genetic structure of bacteriophages. This was however short lived as The Phage Group began to study human cells. As a result, interest in bacteriophages started to die.