Alec Ingros

4/26/17

Goals: Insert the RaptorX results and cross cluster analysis, present to the lab

Methods:

• RaptorX returned with a predicted structure after folding the amino acid sequences
  • Figure 1 is CapnMurica
  • Figure 2 is Gordon
  • Figure 3 is Glenn
  • Figure 4 is DrRobert
  • Figure 5 is Laroye
  • Figure 6 is Salgado
• The gepard plots for cross cluster analysis were also inserted before presenting
  • Figure 7 is AU vs AK
  • Figure 8 is AU vs AL
  • Figure 9 is AK vs AL
• Presented to our peers

Figure 1: This is the hypothetically folded endolysin for CapnMurica

Figure 2: This is the hypothetically folded endolysin for Gordon

Figure 3: This is the hypothetically folded endolysin for Glenn

Figure 4: This is the hypothetical endolysin for DrRobert

Figure 5: This is the a hypothetically folded endolysin for Laroye

Figure 6: This is the hypothetical endolysin used by Salgado

Figure 7: This represents the similarities of endolysin in the AU and AK clusters. AU is on the Y axis and AK is on the X axis

Figure 8: This dot plot is showing the lack of similarity between the endolysin proteins of the AU and AL clusters. Cluster AU is on the Y axis, while cluster AL is on the X axis

Figure 9: This graph shows the slight significance in relation between the AL and AK clusters. The AL cluster is on the Y axis, and the AK cluster is on the X axis

Conclusions and Next Steps:

• We received great feedback on our presentation, which allowed us to make changes and practice before presenting on Friday April 28

Alec Ingros

4/24/17

Goals: Recenter our project, finish collecting results from software, and finish the presentation

Methods:

• We began by refocusing our project. We did this by discussing everything we have read up until this point, and making sure that everyone was on the same page
• Lathan introduced us to RaptorX, a service that will fold a hypothetical protein that is more accurate than a GDP prediction, a software we were looking at before this.
• I finished the gepard plots comparing Glenn to DrRobert (Figure 1), CapnMurica to Gordon (Figure 2), and Salgado to Laroye (Figure 3).
  • To run this, I found the sequence for the endolysin in a fasta file by running the genome on DNA master
  • I compiled the fasta files to create dot plots that would compare similarities of the two sequences that code for endolysin
• We also submitted the amino acid sequences of Glenn, DrRobert, CapnMurica, Gordon, Salgado, and Laroye to RaptorX to be folded

Figure 1: Glenn is on the Y axis, and DrRobert is on the X axis. The solid lines that form indicate similarity between the two sequences.

Figure 2: CapnMurica is on the Y axis, and Glenn is on the X axis. There is little relation between the endolysin of these two phages, as there is very little hits for similarity

Figure 3: Salgado is on the Y axis, and Laroye is on the X axis. These two phages had the exact same sequence, supported by the full solid line.

Conclusions and Next Step:

• We were on the same page after this lab, and just needed our results from RaptorX so we could enter them in the presentation
• I ran gepard plots comparing the AK cluster vs AL cluster, the AK cluster vs AU cluster, and the AU cluster vs AL cluster

Alec Ingros

4/19/17

Goal: Continue reading primary literature on our topic, and begin to interpret our results

Methods:

• Roshni took our data from our previous excel sheet and compiled it into a graph showing each cluster’s presence or lack of either endolysin or holin (Figure 1)
• Micheal blasted the endolysin genes for the phages Salgado DrRobert and Glenn who were from the AL AK and AK clusters respectively then made Chimera structures. Figure 2 is Salgado’s endolysin, Figure 3 is DrRobert’s endolysin, and Figure 4 is Glenn’s endolysin.
• I continued to read primary literature, fixed a section of the excel sheet before Rosh made the graph, continued to practice gepard, and looked for more software to incorporate in our presentation

Figure 1: Rosh compiled all of the excel data into this graph

Figure 2: Endolysin Gene product of phage Salgado, blue represents the hydrophilic region and red represents the hydrophobic regions

Figure 3: Endolysin Gene Product for DrRobert, blue represents the hydrophilic region and red represents the hydrophobic regions

Figure 4: Endolysin Gene Product for Glenn, blue represents the hydrophilic region and red represents the hydrophobic regions

Conclusion and Next Steps:

• The proteins show large amounts of similarities in structure, which may indicate that the phages and clusters are heavily related when it comes to endolysin
• Make the gepard plots comparing the endolysins interclusterally and intraclusterally

Alec Ingros

4/12/17

Goal: Finish collecting data to begin analyzing and developing results

Methods:

• We finished the collection of data from PhagesDB on all the Arthrobacteriophage clusters
• We gave our topic proposal to the class
  • We did this so people were not doing the same project, and so that we could determine if our project was appropriate
• Christina Gaw, Caroline Addison, and Chrissy Sessa had a similar project to ours, so we met with them to determine the directions we would take as not overlap in projects.
  • We worked it out, and our groups went our separate directions
• We finished this day by researching into softwares that will help us highlight the differences between the Holin and Endolysin, as well as the cross cluster analysis
  • We found that Gepard, Chimera, NCBI, and SplitsTree would be most useful in identifying discrepancies

Conclusions and Next Steps:

• Ultimately, creating a phylogenetic tree with SplitsTree was not valued, so we decided to drop this software
• We wanted to focus why some clusters have both Holin and Endolysin, while others may have one or the other
• A final step moving forward was to find a new software source that would help analyze and emphasize the deviation between the two proteins

Alec Ingros

4/10/17

Goal: Make a group, and begin the narrowing of ideas for our project.

Methods:

• I chose to work with Micheal Munson and Roshni Jaffery
• Previously, Micheal and I were discussing the functions and purpose of Holin and Endolysin in the Arthrobacteriophages
  • We wanted to explore the relationship between these two proteins when it came to lysing the bacteria. This came from the fact that Caterpillar, Shrooms, and Nubia all presented different combinations of the proteins.
• From here, we wanted to expand the project to include all Arthrobacteriophage clusters: AK, AL, AM, AN, AO, AP, AQ, AR, AS, AT, AU, and AV. We began collecting information from each of these clusters, only including published phages and put all the information into an excel sheet that was shared by the group.

Conclusions and Next Steps:

• This was a good start to the project, we have a direction to follow as well as a group!

• Finish collecting data for all the clusters

2/20/17

Josh and I reviewed annotations for Caterpillar genes 1 through 22, making sure that the annotations were called properly, and had the best scores. If our call was questionable, we made a pro and con list for our call to help our decision making process.

2/22/17

Today the Caterpillar group held a meeting to compile the FASTA file after everyone had completed and reviewed their individual annotations. In addition, we split into smaller groups to complete other tasks like finishing the annotations for AN phages, creating the cover sheet, and compiling the final annotation file for Caterpillar.

2/27/17

Josh and I were assigned the cover sheet. We meet with each pair of Caterpillar members and asked if they made any decisions that were questionable. After compiling a list of these genes, we began making the cover sheet.

3/1/17

Today we (Josh and I) had to edit the cover sheet due to some unnecessary genes that were not needed. After, we reviewed the sheet with Ashely and then submitted it alongside the other files in a box folder.

3/13/17

Today we picked groups to make a poster for Scholars Week. My group consisted of Micheal Munson, Daniel Zeter, and Pranov Kapoor. After getting in our group, we determined that we would bring ideas to Wednesday to begin making the poster.

3/15/17

Today we (Micheal, Daniel, Pranov, and I) worked to develop the design and layout of our poster for Scholars Week. In addition to this, we scheduled to meet on Saturday, March 18 to finish our poster.

3/18/17

Our group did a majority of the poster today, so on Monday we could quickly finish the poster.

3/20/17

Today we finished the poster, and began to think about how to split up the presentation for Wednesday.

3/22/17

Today the whole class presented each poster they designed. After hearing all of the presentations, we voted on the best two posters we wanted to present at Scholars Week.

3/27/17

Today the class split into two groups, and each was assigned a poster. In our groups, we practiced presenting for the Scholars Week presentation. Finally, we signed up for presentation times for Scholars Week.

3/29/17

Today our individual groups meet at Scholars Week. We each presented the two chosen posters to the professors and Baylor students.

4/3/17

Today the class met to finalize the posters for the regional SEA-PHAGE conference with Lathan and Dr. Adair.

4/5/17

Today some of the class presented these new posters to each other as a final way to prepare for the conference.

Alec Ingros

Prompt 2: One of the biggest problems in phage therapy has been in the approval process.   Describe the trouble surrounding FDA approval and recommend some suggestions to improve the process of phage therapy approval.

The FDA constantly makes very difficult decisions. They need to be certain about a product with out a doubt will be safe to all human uses. This being the case, sometimes the organization is too strict in some aspect. The way in which the FDA felt with Intralytix serves to support some of this groups shortcomings.

Intralytix, a biotechnology group focusing on phage use, was in the final processes of moving to clinical trial for using phage therapy as treatment against Vancomycin Resistant Enterococci. After being able to restore infected mice consistently to full health, the company, being led by Marissa Miller and Torrey Brown, applied for permits to begin clinical trials in 2001. Ultimately, the FDA were too expensive to continue onto the clinical trials. The trials came to a halt, quickly lost funding, and the company stopped further research.

Each aspect of the experimental process needs to be examined, which leads to an approval process that is too long and expensive for most companies. To satisfy the concern over each step of the new process, the FDA aquires specialists from the National Institutes of Health. The average cost is around $800 million from start to finish for an experimental procedure to gain FDA approval. In addition to this, it is about a 10 year process. In this lengthy period, the drug may no longer be as important, which could lead to companies losing investors leading to the abandonment of the project. These qualities of the FDA approval process make the production of new treatments in a timely manner a dream. This stunts entrepreneurship, continuing to  keep drug production in control of major companies.

I would recommend that this process is shortened. If shortened, the promising smaller companies may still have investors to help pay for the production. This would open new possibilities for businesses. The expenses seem that they are unavoidable, so if the drug or procedure shows promise then the smaller companies may be able to gather enough investors to complete the FDA approval process.

Alec Ingros

3/17/17

These few chapter were extremely interesting especially when it came to making comparisons and contrasting the development of phage therapy India, Poland, Russia, and the United States. Furthermore I found it intriguing how the views of phage therapy changed during World War II and post World War II. On top of this, some nations, Russia in particular, made foolish decisions based on predisposed mindsets rather than evaluating pros and cons of a choice.

The development of phage therapy was of utmost concern medically during World War II. It was used as a treatment for various lethal infections that were plaguing the Axis as well as the Allies. To me, it is surprising how after World War II the United States decided not to pursue the possibility of phage therapy, despite it becoming the common practice on the battlefield. In addition to this, Russia’s refusal to develop antibiotics during the 40’s and 50’s because they were Western was also rather unusual. Would it not make sense to pursue medical advancements that will lead to saving the most amount of lives?

A reoccurring theme through the history of phage therapy has been the negative mindset, and skepticism from the scientific community. Despite producing many positive results in both Russia and Poland, the United States scientific community were very close minded toward accepting phage therapy as an appropriate treatment option. This made Merril’s attempts to develop phages as an alternative to antibiotics extremely difficult in the face of great criticism. This reinvigorated the development of phages, and began the creation of start-up companies that are invested in further testing of phages. Before phage therapy becomes a common practice and a primary method of treatment on resistant bacteria, these companies need to figure out how to get more consistent results from the phages.

Alec Ingros

February 6, 2017

Rationale: Preparation for annotating the genome for the phage Caterpillar

Methods:

1. Download fasta file of “Caterpillar” from phagesdb
2. Export fasta file to DNA master and auto-annotate
3. On Genemark, upload Fasta file and select: Protein sequence, Gene nucleotide sequence, phage, LST, and PDF
4. Open starterator on virtual box and select “whole-phamerated phage”

Conclusions and Next Steps:

• We prepared for annotating the genome of the phage Caterpillar. We divided the 88 genes between four groups of two. My partner, Joshua Baker, and I will be responsible for genes 1-22. We will officially begin annotating next lab (2/8/17).

Alec Ingros

February 15, 2017

Rational of today’s work: Annotating genes: 4, 7, 8, 9, 20, and 11

Tools used and/or Methods:

• HHpred
• GeneMark
• Starterator
• PhagesDB
• Phamerator
• NCBI blast

Results:

Gene 4

Start: 1620bp Stop: 2729bp FWD GAP: 20bp Gap SD Final Value: SD Score: -2.077 (Best score) Z-Value: 3.188 CP: The gene is not covered The gene can’t be extended to cover all – approximently 20 bp left uncovered. SCS: Agrees with Glimmer, Agrees with GeneMark NCBI BLAST: endolysin [Arthrobacter phage Gordon] Q:1 S:1 E-Value: 0.0 CDD: PGRP and Cpl-7 domain-containing protein E-Value: 2.2e-13 PhagesDB BLAST: Gordon_4, endolysin Q:1 S:1 E-Value: 1e-164 HHPred: Endolysin, putative lys E-Value: 2.3e-23 LO: Yes ST: Agrees with Starterator F: FS: HHpred, NCBI, PhagesDB Notes: Endolysin; PGRP and Cpl-7 domain-containing protein

Gene 4 GeneMark Results

Gene 4 PhagesDB Best Results

Gene 4 NCBI Blast Best Results

Gene 4 NCBI Blast Conserved Domain Results

Gene 4 HHpred Best Results

Gene 4 Starterator Results

Gene 7

Start: 2886bp Stop: 3269bp FWD GAP: 8bp Overlap SD Final Value: SD Score: -2.904 (Best score) Z-Value: 2.767 CP: The gene is not covered The gene can’t be extended to cover all – approximently SCS: Agrees with Glimmer, Agrees with GeneMark NCBI BLAST: hypothetical protein GORDON_5 [Arthrobacter phage Gordon] Q:6 S:39 E-Value: 2.0e-50 CDD: No good hit PhagesDB BLAST: Gordon_5, function unknown Q:6 S:39 E-Value: 6e-41 HHPred: No good hit LO: Yes ST: We cannot shorten the gene without losing the best z-score, SD value, and longest open reading frame F: NKF FS: NCBI, PhagesDB Notes:

Gene 7 GeneMark Results

Gene 7 PhagesDB Best Results

Gene 7 NCBI Blast Best Results

Gene 7 HHpred Results

Gene 8

Start: 3266bp Stop: 3793bp FWD GAP: 4bp Overlap SD Final Value: SD Score: -3.207 (Best score) Z-Value: 3.047 CP: The gene is not covered The gene can’t be extended to cover all – approximently SCS: Agrees with Glimmer, Agrees with GeneMark NCBI BLAST: hypothetical protein GORDON_6 Q:18 S:1 E-Value: 2e-74 CDD: No good hit PhagesDB BLAST: Gordon_6, function unknown Q:18 S:1 E-Value: 6e-59 HHPred: No good hit LO: Yes ST: We cannot shorten the gene without losing the best z-score, SD value, and longest open reading frame F: NKF FS: NCBI, PhagesDB Notes:

Gene 8 GeneMark Results

Gene 8 PhagesDB Best Results

Gene 8 NCBI Blast Best Results

Gene 8 Starterator Results

Gene 9

Start: 3812bp Stop: 5551bp FWD GAP: 18bp Gap SD Final Value: SD Score: -2.137 (Best score) Z-Value: 3.188 CP: The gene is covered SCS: Agrees with Glimmer, Agrees with GeneMark NCBI BLAST: terminase large subunit [Arthrobacter phage Gordon] Q:1 S:1 E-Value: CDD: Phage terminase-like protein, large subunit, contains N-terminal HTH domain [Mobilome E-Value: 3.99e-49 PhagesDB BLAST: Gordon_7, terminase, large subunit Q:1 S:1 E-Value: 0.0 HHPred: Terminase, DNA packagin E-Value: 1.1e-27 LO: Yes ST: Agrees with Starterator F: terminase, large subunit FS: NCBI, PhagesDB, HHpred Notes:

Gene 9 HHpred Best Results

Gene 9 PhagesDB Best Results

Gene 9 NCBI Blast Best Results

Gene 9 NCBI Blast Conserved Domain Results

Gene 10

Start: 5548bp Stop: 6399bp FWD GAP: 4bp Overlap SD Final Value: SD Score: -2.451 (Best score) Z-Value: 3.043 CP: The gene is covered SCS: Agrees with Glimmer, Agrees with GeneMark NCBI BLAST: hypothetical protein CAPNMURICA_8 Q:1 S:1 E-Value: 0.0 CDD: No good hit PhagesDB BLAST: CapnMurica_8, function unknown Q:1 S:1 E-Value: 1e-152 HHPred: No good hit LO: Yes ST: Agrees with Starterator F: NKF FS: NCBI, PhagesDB Notes:

Gene 10 GeneMark Results

Gene 10 PhagesDB Best Results

Gene 10 NCBI Blast Best Results

Gene 10 HHpred Best Results

Gene 10 Starterator Results

Gene 11

Start: 6396bp Stop: 6977bp FWD GAP: 4bp Overlap SD Final Value: SD Score: -4.241 (Best score) Z-Value: 2.184 CP: The gene is covered SCS: Agrees with Glimmer, Agrees with GeneMark NCBI BLAST: hypothetical protein GORDON_9 Q:5 S:4 E-Value: 1e-79 CDD: No good hit PhagesDB BLAST: Niktson_Draft_8, function unknown Q:1 S:1 E-Value: 6e-75 HHPred: No good hit LO: Yes ST: Agrees with Starterator F: NKF FS: NCBI, Phages DB Notes:

Gene 11 GeneMark Results

Gene 11 PhagesDB Best Results

Gene 11 NCBI Blast Best Results

Gene 11 HHpred Best Results

Gene 11 Starterator Results

Conclusions and Next Steps:

• We skipped over genes 5 and 6 because we thought it would be best to decide as a team what to do. The situation is that neither gene is definitively better than the other, so a decision should be made as a group. The genes we did finish were put us back on track to finish in the two week window. We will look to have another productive day like today to continue with schedule.