I really enjoyed the public deliberation in class and wish we had more time to go further in depth on the topic. I appreciated the emphasis on respecting other people’s views and not trying to out-fact other people but really hone in on what people value most. For those reasons I think it was more of a productive discussion than debates I have been a part of previously.

On the topic of climate change, I didn;t really agree with a lot of the suggestions listed on the sheet we were given. I think that they really took away from the freedom of the American people. That being said, I think that the public has to become more educated on the issues, and it should come from factual sources not doomsday headlines that are trying to sell papers so maybe it should be a greater part of school curriculum.

Rationale: Complete final version of abstract and continue to accumulate data

Process:

• Lathan had commented that the RMSD (root mean square deviation) values for comparing tertiary protein structures were not very meaningful so I looked into another way to mathematically analyze the differences between the tape measure proteins (TMP)
• All of the structures had been folded by RaptorX so I started aligning them all in comparison to the NapoleonB TMP

Results:

• I found a paper that said that the TM-score is the best way to compare protein structures because it is independent of protein length. The tape measure proteins vary greatly in size so this is necessary to reduce error.
• TM-align also gives a TM-score with its results (it is actually the main poi for the software) so it was an easy switch over.
• Began to organize results in a tabular manner:

phage name	residues	residues in common	RMSD of common residues	TM-score	notes	image
Anansi	1432	1356	78.945	0.1524	random structural similarity
Andrew	787	787	40.957	0.1597	random structural similarity
Atraxa	616	616	29.188	0.1309	random structural similarity
BeautusComedenti	1029	1029	28.36	0.2266
BossLady	876	876	48.243	0.1695
Cheesy	1492	1492	49.672	0.2379
Chubster	1180	1180	41.169	0.2402
Constance	1298	1298	56.792	0.1247	random structural similarity
Corgi	555	555	34.341	0.0913	random structural similarity
DrManhattan	744	744	31.479	0.1715
EdgarPoe	1113	1113	27.514	0.2428
ElephantMan	1463	1427	24.285	0.368
Elesar	1607	1493	47.103	0.1432	random structural similarity
Faja	1348	1348	48.361	0.1552
Galaxy	793	793	32.454	0.1618
Gorgeous	1432	1356	79.112	0.1528
Hestia	1325	1325	43.792	0.153
Joann	853	853	29.797	0.1764
KellEzio	1029	1029	44.416	0.1805
KitKat	1029	1029	28.965	0.2266
Laila	665	665	34.138	0.1063
Laroye	946	946	25.419	0.2367

Next Steps:

Continue aligning stuctures and then use the data to create a figure.

Question #2

Intralytix tried and failed to pass phage therapy through the FDA in 2002. The process of passing a drug through the FDA is challenging for any small startup company because it is typically very expensive and time consuming. In addition to expenses in time and money, Intralytix had to battle the country’s general aversion to phage treatment. At the time, American scientists had tunnel vision for antibiotics and drugs creating added resistance to the emergence of phage therapy. When the FDA was reviewing the product they found a lot of problems including the accuracy of separating lytic and lysogenic phages, a less than desirable animal model that had been used, and unknown rated at which the phages within a cocktail would mutate and affect the experimental animal. Finally, there was not sufficient evidence showing that the results of phage therapy would be different from the results of antibiotics. Intralytix  concluded that testing and revaluating each of these issues would be much too costly and time consuming so the project was put on hold.

From my experience working at a pharmaceutical company, the FDA can be used as a resource, not just a merciless judge. The best and fastest results are achieved by working directly with the FDA and approaching them in the beginning stages and asking for guidance instead of just viewing the FDA as an end location. With FDA guidance, phage therapy funds can be more focused and precise to test the most necessary components. I think it is also important to show how phage therapy has advantages over antibiotics.

Rationale: write abstract and continue comparing protein structures

Process:

1. wrote abstract
2. selected phages and entered amino acid sequences for their TMPs into raptorX

Results:

Abstract:

Tape measure proteins (TMPs) help determine bacteriophage tail length and facilitate DNA transit into the bacterial cell during infection [2]. Tail length is a key characteristic for classifying a bacteriophage into a tail morphology family (Myoviridae, Siphoviridae, or Podoviridae), and therefore TMPs play a role in determining tail morphology [2]. Arthrobacter phage TMPs are highly conserved in each phage cluster, and it has been noted that arthrobacter Myoviridae phage seem to have an abnormal number of similarities to phages in the Siphoviridae family [1].  This experiment was conducted to determine if Arthrobacter phage TMPs contain any conserved structural or genetic domains within each tail family tested (Myoviridae or Siphoviridae), and to determine if there is any consistent conservation found throughout all Arthrobacter phage TMPs. This was determined bioinformatically by scanning the amino acid sequences of 43 randomly selected Arthrobacter phage TMPs for motifs or other similarities in addition to comparing hypothetically folded TMPs. The distances between the atom backbone of the proteins were used to calculate the root-mean-square distance (RMSD) in order to compare two different structures. In effort to further characterize NapoleonB, each TMP was aligned against NapoleonB’s TMP.*** Preliminary results suggest at least one conserved motif between all TMPs. The protein structures also seem to suggest at least visual similarities between Myoviridae TMPs with more data to be collected. Based on the avaliable data, it appears that TMPs vary widely with a few key areas of similarity that can be further researched.  

***Results are unfinished  

Next Steps:

TM-align all folded proteins with NapoleonB’s structure

Rationale: Create outline for project and compare different protein structures

Process:

1. Lucy and I planned out what we thought we needed to do to complete project
2. began comparing structures using TM-align and chose different phages fold the TMP of
3. began testing to see what the fastest and easiest way to fold lots of proteins is

Results:

Outline:

Title Comparison of Tape Measure Proteins in Siphoviridae and Myoviridae Tail Families Using Arthrobacter Phage

Guiding Question: What distinguishes tape measure proteins across Siphoviridae and Myoviridae tail families in Arthrobacter Phage and are there conserved domains?

Abstract:

• TMPs play a major role in determining phage tail families and how phage infect their hosts
• Amino acid sequences for arthrobacter phage TMPs were compared to find regions of similarity or difference to examine whether or not there is conservation between Myoviridae and Siphoviridae TMPs
• Hypothetical proteins structures were generated to compare the structural differences in TMPs to see if structural components were conserved even if amino acid sequences were not
• ***We don’t have fully fleshed out conclusion or results but we will alter our abstract as needed

List of tools used:

• I-tasser
• Clustal Omega
• MEME Motif
• TM align

Introduction (Background Information):

• Tape Measure Proteins (TMPs) help control tail length/type and how a phage infects its bacterial host
• Arthrobacter phage cluster appear to exist in all three tail types (Podoviridae, Myoviridae, and Siphoviridae) but Podoviridae do not call TMPs
• Previous research suggests that in arthrobacter phage myoviridae and siphoviridae TMPs are more similar than in phage that infect other hosts
• Previous research suggests that structure is conserved more than the specific amino acid sequence
• Protein structures can be compared statistically by finding the root-mean-square deviation (RMSD) or the measure of the average distance between the backbone atoms of superimposed proteins

Types of Data Collected:

• Computer generated folded proteins
• MEME motifs showing conserved motif domains
• Clustal Omega amino acid sequence comparisons
• TM alignments that give numerical comparisons of protein structures

Results (to date):

• Amino acid sequences for TMPs in each cluster appear to be highly conserved (identical or only 2-3 amino acid differences) – found using clustal omega comparisons
• Amino acid sequences for TMPs in each tail family do not appear to be highly conserved but the type of amino acid (as determined by clustal omega color coding) appear to be more conserved – found using clustal omega comparisons
• Have started to model predicted protein structures and myoviridae proteins are visually more similar to each other than to siphoviridae proteins
  • However: NapoleonB (sipovirade) and Chubster (myoviridae) are more mathematically similar than Chubster and Sonny (myoviridae)
• Structures are scoring relatively low in similarity and tend to fall in “random structural similarity” category
• Using MEME motif software several cross cluster but within tail family motifs have been identified in addition to two motifs that seem to appear in both Myovirade and Sipovirade phage

Conclusions (if any have been drawn):

1. TMPs are highly conserved
2. Preliminary data seems to suggest that structure is more conserved than amino acid sequences
3. There are motifs that contain regions of highly conserved amino acids or amino acid types suggesting that these maybe the conserved domains (or as close as TMPs get)

Decided to go with raptorX because while it may not be as accurate, it produces foldings at a much higher rate so using it allows us to have actual data to work with.

Next Steps:

Continue folding all 43 proteins and compare them all to NapoleonB and then create a scatter plot of RMSD scores

Rationale: continue working on individual project. More specifically, select the specific phages from arthobacter clusters that we want to look at the tape measure protein of

Process:

• went through and randomly chose ~3 phages per cluster that had an annotated tape measure protein
• started to plug the amino acid sequences into I-Tasser

Observations/Results:

• None of the podoviridae phages have an annotated tape measure protein
  • can we figure out what gene it is?
  • is it that different from the other types of phages?

Nest Steps:

Research podoviridae tape measure proteins

Rationale: Practice presenting the poster

Process:

take turns presenting the poster in pairs to the rest of the class

Results/What I learned:

• Don’t go into too much detail
• know how to describe the figures
• make sure you aren’t talking too fast

Nest Steps:

Present poster at scholar’s day!!

Rationale: Begin a research outline for approved research question

Process:

• Narrow down project to be more specific
• Find software to help analyze research question
• consolidate an outline

Result:

Goals of Project:

• Compare Tape Measure proteins between the Podoviridae (with short noncontractile tails), Siphoviridae (with long noncontractile tails), and Myoviridae (with long contractile tails) tail family

Bioinformatics Tools:

• FASTA searches
• Phamerator
• NCBI BLAST
• PDB
• Clustal Omega
• I-tasser

Methods Outline

• Look in literature to see if we can find any similar experiments from which we can draw data or info
• Figure out what cluster corresponds to what tail type
• Select a number X of phages from each family
• Compare these tape measure proteins (amino acids) to other tape measure proteins in the family and then in comparison to other families
• Use Protein folding software to simulate a tape measure protein (or several) for each family and then look for differences

Next Steps:

I will start experimenting with software and Lucy will search in literature

Rationale: Start brainstorming ideas for different bioinformatics projects

Process:

• Looked through different bioinformatic software and find topics of interest
• Designed 4 different research questions

Results:

Questions:

1. Are there repeats in NapoleonB? Are these segments specific to NapoleonB or found in other phage? Can we find meaning in this information (we hope so)?
2. Are there sections of NapolonB’s genome that have significantly different %GC composition than the genome as a whole? Do these parts match other phage or bacteria (new acquisitions?)? How common is this for Arthrobacter phage?
3. How conserved are phage genes? What genes do NapoleonB and most other AM cluster phage have in common? What about all Arthrobacter phage? How similar are the DNA sequences?
4. Does NapoleonB have any Rho terminators and can we use protein folding software to predict their structure? Are these structures common in other Rho terminators for Arthrobacter phage?

Lathan liked question 3 so we did some refining:

• To refine maybe find several conserved genes for each cluster (or a couple clusters) and compare these
• Also look for areas within clusters where genomes vary significantly
• Does genome length effect similarity
• Will phamerator allow us to compare cross cluster
  • Rachel says we can edit the code so that we can do this
• We can also look and see if any of the known function gene hits didn’t hit to an AM cluster or had other good hits to phage outside the cluster

Next Steps:
Get question approved my Leo and develop research methods

Rationale: Begin brainstorming ideas for individual project

Process:

1. ideas:
   • investigate different plaque morphologies
   • investigate host range
   • create program to analyze pharmerator maps and analyze information in a new way

Conclusion:

• still need an idea because all of the above had different problems of some sort

Next Step:

solidify an idea