April 20

April 17, 2018 – Beginning Again

Rationale/Purpose: Today, we FINALLY came up with a procedure which should give us a proper question.

Tools/Procedure: PhagesDB, Phamerator, Yass, DNAMaster

Results: We were originally going to compare the genomes of all 4 phages, but it might be more realistic to compare genes of only DrManhattan and Maureen instead.

Conclusion: We’ve decided to compare the genomes of DrManhattan and Maureen to determine what genes have similar functions. Because these phages infect different hosts, as long as they don’t also infect each other’s, any genes they have with the same functions are thus likely to not be associated with host specificity.

Future Work: We will use Phamerator to determine which genes have high similarity. Then we will look at the functions of those genes and see if they are the same (or similar). Finally, we will use tools to fold the hypothetical proteins with high similarity to determine if their structures are the same, even if their functions haven’t been determined.

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April 20

Motif Analysis

Terra Morris

4/18/18

Continued Motif Analysis

Purpose

In the previous lab, analysis of Arthrobacter globiformis phages for the motifs  “TCAAG” and “GTCAAG”  began using MEME motif finder to possibly determine the presence of stoperators. In today’s lab, analysis of motifs and potential stoperators will continue with the phages that have not yet been examined.

Tools

  • Phages DB
  • MEME Motif finder
  • Google Docs

Procedure

  1. Log onto all above listed tools.
  2. Go to phages DB and search each of the phages being used in the research. Locate on each phage’s profile whether its classified as a lysogenic or lytic phage.
  3. Record Results
  4. Use MEME motif finder on all phages that were determined to have an integrase gene.

Results

All 20 of the Arthrobacter globiformis phages with an integrase gene were determined to have no possible stoperators ending in either “GTCAAG” or “TCAAG”

“TCAAG” motif chart

Conclusion

In today’s lab, the 12 Arthrobacter globiformis phages that had not been searched for a motif ending in “GTCAAG” or “TCAAG” were searched motif ending in those sequences. All 20 phages were determined to not have the potential to have a stoperator ending in “TCAAG”.

Future Work

 

In the future, we will look into potential stoperators sequences for Arthrobacter globiformis phages by analyzing the other motifs that the MEME motif finder determined to exist.

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April 20

Forgotten Cure Part 3

Forgotten Cure Part 3
While there have been many hurdles when it comes to the success of phage therapy, a major component has been the difficulty of getting FDA approval for phage cocktails. Because phage cocktails are technically biological, they must undergo additional studies before becoming available to the public. This is twofold: first of all, we need to be sure phages don’t pose too great a risk to human health and safety. In addition, we need to be sure that the benefits associated with phage therapy is greater than any risk they might have. According to the FDA, FDA approval of a drug means that “data on the drug’s effects have been reviewed by CDER, and the drug is determined to provide benefits that outweigh its known and potential risks for the intended population” (Development). Because there are risks and (hopefully) benefits associated with all drugs, it is important to be sure the risks don’t outweigh the benefits.
While all this is very important, this has made it difficult for phage therapy to be supported because there is so little evidence supporting its benefits despite possible risks. Each phage in the cocktail must be individually studied in order to determine any possible effects. Because phage cocktails can be immensely complex with many different phages, this can be especially difficult because each phage must be studied individually.
There is, however, an alternative. The FDA’s Accelerated Approval process allows drugs to be sped through the process if they are considered “promising therapies that treat a serious or life-threatening condition and provide therapeutic benefit over available therapies” (Development). Due to the current availability and widespread success of antibiotics, it is possible that phage therapy has never been considered an immediate need when it comes to treating bacterial infections. However, as antibiotics become decreasingly effective, alternative therapies will increase in importance. This may allow phage therapy to qualify as a ‘therapeutic benefit over available therapies’ and thus helping it gain FDA approval.

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April 20

April 16, 2018 – Redesigning Our Experiment

Rationale/Purpose: Today, we realized that a lot of the work we’ve been doing is either not scientifically relevant, or not properly answering our question. The goal today was to redesign our question and make it something more testable.

Tools/Procedure: We used Phamerator to identify some genes that are similar between the three A. globiformis phages in the AZ cluster but different from DrManhattan. Then we found the CDD hits for those phages, as seen in Table 1. This was done to search for genes whose function could show why DrManhattan has a different host.

Results:

Table 1

Conclusion: There are a number of phages which are different between these phages, but none whose functions clearly show a strong relationship to host specificity.

Future Work: It looks like we are going to need to take a different approach to this work to identify differences and similarities between these phages.

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April 20

April 11, 2018 – Identifying Differences

Rationale/Purpose: The goal of today’s research was to identify specific genes in Maureen, Yang, DrM, and Liebe which could be helpful in identifying host specificity.

Tools/Procedure: Today we used Phamerator and PhagesDB.

Results: 

DrManhattan infects Arthrobacter atrocyaneus NRRL B-2883

Yang, Liebe, and Maureen infect Arthrobacter globiformis B-2979

Genes 17-20 and 45 have zero similarity between Yang and DrManhattan

Genes 16-20 and 53-58 have zero similarity between Liebe and DrManhattan

Genes 16-22, 30-31, and 53-58 have zero similarity between Maureen and DrManhattan

Genes 17-20 are all not similar between DrManhattan and the rest of the phages that infect glob

Conclusion: We chose to look at genes 17-20 because they show the highest dissimilarity between the phages, according to PhagesDB.

Future Work: The next steps are to identify the functions of these genes and determine if they are relatable to host specificity.

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April 20

The Search for Motifs and Stoperators

Terra Morris

4/16/18

The Search for Motifs and Stoperators

Purpose 

In the previous lab, Arthrobacter globiformis phages were analyzed for integrase and CRO genes. In today’s lab, we will begin to analyze the phages that did have an integrase for the motifs  “TCAAG” and “GTCAAG” using MEME motif finder to possibly determine the presence of stoperators.

Tools

  • Phages DB
  • MEME motif finder
  • Google Docs

Procedure

  1. Log onto all above listed tools.
  2. Go to phages DB and search each of the phages being used in the research. Locate on each phage’s profile whether its classified as a lysogenic or lytic phage.
  3. Record Results
  4. Use MEME motif finder on all phages that were determined to have an integrase gene.

To Use MEME motif finder

  1. Download the desired phage’s FASTA file from PhagesDB. Open the MEME motif finder and upload the FASTA file.
  2. Select “Any number of repetitions” under “How do you expect motif sites to be distributed in sequences?”
  3. Input “100” under “How many motifs should MEME expect to find?”
  4. Select advanced options, then limit the width of the potential motifs to 13 base pairs. Start the search.
  5. Once the search is complete, analyze the results and search for sequences ending in “TCAAG” or “GTCAAG”. If such a sequence exists, notice where it starts in the phage’s genome and check the phage’s gene list on Phages DB to verify that the sequence is in an intergenic region.
  6. Record Results

Results

In the previous lab, 20 Arthrobacter globiformis phages were determined to have an integrase gene:

Andrew, Auxilium, Bridgette, Constance , Coral , Cote, Daob, Elesar, Faja, Hestia, Isolde, Judy, Kepler, Lunar, Melons, Nandita, Polka, Ryan, Richie, and Seahorse.

Eight phages were subjected to MEME analysis, with no positive results. Any sequences ending in “TCAAG” or “GTCAAG” were in the middle of genes, disqualifying their potential to be stoperators.

“TCAAG” motif chart

Conclusion

Our research initially began with the analysis of all 45 Arthrobacter globiformis phages, this number was narrowed down to 20 after 20 of the 45 phages were determined to have an integrase gene in the previous lab. Of the 20 phages we’re currently analyzing, eight of the have been examined so far via MEME motif finder. The motif finder has led to the discovery of motifs ending in “TCAAG”, but none could be considered viable stoperators. Hopefully we can discover a possible stoperator sequence soon.

Future Work

In the next lab, MEME motif analysis will continue on the rest of the 20 phages that we’re analyzing for stoperator potential.

 

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April 20

Abstract Writing – April 13, 2018

Rationale/Purpose: Today’s goal was to write a completed abstract based on our research thus far.

Tools/Procedure: We’ve used Phamerator, NCBI, PhagesDB, and DNAMaster to conduct our research, but we wrote our abstract in Google Docs.

Results: 

Bacteriophages that are clustered together usually infect the same host and have similar genetic composition. According to the Phages Database, one exception is the AZ cluster. This study was conducted to determine why the bacteriophage DrManhattan infects Arthrobacter atrocyaneus NRRL B-2883 while bacteriophages Maureen, Liebe, and Yang, also in the AZ cluster, infect Arthrobacter globiformis B-2979. Using Phamerator, the genes that were different between DrManhattan and the other Arthrobacter globiformis B-2979 phages were identified to be 17-20. NCBI BLAST determined the functions of these genes, which include helicases and tail proteins. Gene 17, however, was different between between DrManhattan and the other phages; while DrManhattan codes for a helicase, the others code for tail proteins, which could play an interesting role in the host in infects. Through this research, we hope to understand more about the way phages are organized into clusters.

Conclusion: We definitely have a long way to go, as we can’t really write our research into our abstract yet because it isn’t complete. Hopefully we can complete this soon.

Future Work: The next steps are to finalize our genome comparisons and see if we can identify crucial differences between genomes in the cluster.

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April 20

April 9, 2018 – Collecting Literature and Beginning Research

Rationale/Purpose: The purpose of today’s work was to design an outline for our final project. We found and cited relevant resources and planned our activities for the next few weeks.

Tools/Procedure: Today we used ScienceDirect to search for literature.

Results: 

Title:

Bacteriophage ED1 Cluster Infecting Multiple Hosts

Research Question:

Why do bacteriophages within the same cluster of ED1 infect multiple hosts and what does this have to do with their comparative DNA sequences?

Is there a relationship between genomic differences between the phages of cluster ED1 and the differing hosts they infect?

Background:

The bacteriophages that a part of the ED1 cluster do not infect all of the same hosts. For most bacteriophages within the same cluster, this is different. Most bacteriophages within a cluster are able to effectively take over one host. There is no variety within the cluster.

A phage’s ability to infect a bacteriophage and integrate itself into the host’s genome comes from the genes that it possesses. This includes integrases and other such genes that perform similar functions. Therefore, bacteriophages within the same cluster should have the very similar genomes if they are infecting the same host. If they infect different hosts, it should be understood that the genomes of those bacteriophages are most likely different. However since they are in the same cluster they cannot be too far off. Our goal is to examine the bacteriophages in the cluster ED1 and understand the similarity of bacteriophages within the same cluster that infect different hosts.

Tools:

Phamerator, DNA Master, NCBI Blast

Sources for developing the research question and answering it:

Bacteriophage evolution differs by host, lifestyle, and genome https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5540316/

More Is Better: Selecting for Broad Host Range Bacteriophages

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5014875/

Phages of Staphylococcus aureus and their impact on host evolution

https://www.sciencedirect.com/science/article/pii/S1567134813001718#f0010

Biodiversity of bacteriophages: morphological and biological properties of a large group of phages isolated from urban sewage

https://www.nature.com/articles/srep34338

Phage Morphology Recapitulates Phylogeny: The Comparative Genomics of a New Group of Myoviruses

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3391216/

Schedule of Labs:

4/11 – use phamerator to determine the differences between genomes of ED1 pages

4/11 – Introductory research to support findings

4/16 – Finish comparative genomic analysis

4/18 – check genomic analysis

4/23 – finish poster/presentation

4/25- Practice Presentations

4/27- CURES in Biology Presentations 2-5 pm

All sources in APA Format:

Comeau, A. M., Tremblay, D., Moineau, S., Rattei, T., Kushkina, A. I., Tovkach, F. I., … Ackermann, H.-W. (2012). Phage Morphology Recapitulates Phylogeny: The Comparative Genomics of a New Group of Myoviruses. PLoS ONE, 7(7). https://doi.org/10.1371/journal.pone.0040102

Jurczak-Kurek, A., Gąsior, T., Nejman-Faleńczyk, B., Bloch, S., Dydecka, A., Topka, G., … Węgrzyn, A. (2016). Biodiversity of bacteriophages: morphological and biological properties of a large group of phages isolated from urban sewage. Scientific Reports, 6, 34338. https://doi.org/10.1038/srep34338

Mavrich, T. N., & Hatfull, G. F. (2017). Bacteriophage evolution differs by host, lifestyle and genome. Nature Microbiology, 2, 17112. https://doi.org/10.1038/nmicrobiol.2017.112

Ross, A., Ward, S., & Hyman, P. (2016). More Is Better: Selecting for Broad Host Range Bacteriophages. Frontiers in Microbiology, 7. https://doi.org/10.3389/fmicb.2016.01352

Table 1: Morphological and physiological characteristics of phages from the collection, and reference phages T4, λ, and T7. (n.d.). Retrieved April 4, 2018, from https://www.nature.com/articles/srep34338/tables/1

Xia, G., & Wolz, C. (2014). Phages of Staphylococcus aureus and their impact on host evolution. Infection, Genetics and Evolution, 21, 593–601. https://doi.org/10.1016/j.meegid.2013.04.022

Future Work: The next steps are to identify differences between the phages, use some comparative genomic tools, and focus on our presentation.

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April 20

Integrase and CRO gene search

Terra Morris

4/11/18

Integrase and CRO gene search

Purpose

In the previous lab, we located primary literature to support our research and began to search for integrase genes in the 45 recorded Arthrobacter globiformis phages in order to determine lysogeny of those phages. In today’s lab, we will continue to search for integrase genes in our phages, search for CRO genes in these phages, and locate the phams that these genes potentially fall under.

Tools

  • Phages DB “Pham finder”
  • Phamerator
  • Google Docs

Procedure

  1. Log onto computer and open all the necessary tools listed above.
  2. Continue to search Phamerator for the presence of Integrase genes.
  3. Go to phagesdb.org and find the ‘phages’ tab. Then click the ‘phams’ tab on the list. Type ‘Integrase’ into the empty ‘search gene notes’ text box to search for integrases.
  4. Go through every pham and look for the clusters listed in the previous lab notebook. If a cluster is found, and the genome is one of the phages being analyzed, record.
  5. Repeat steps 3 and 4 but type in “CRO” to search for CRO genes instead of integrases. Record results.

Results

The CRO gene search in Phages DB produced negative results

The following is a chart of the integrase genes found in the phages we were searching.

Integrase Chart

Integrase Chart Continued

 

The blank areas indicate that no integrase was found.

Conclusion

In conclusion, there were no CRO genes found in any of the phages we are studying, but twenty did have integrase genes which could indicate potential lysogeny.  The work was a little tedious but it gave us very informative results.

Future Work

In the future, we will be analyzing our selected phages’ genomes for stoperators.

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April 20

Independent Research

Terra Morris

4/9/18

Independent Research

Purpose

In the previous lab, we began looking into our approved research question and synthesized a project outline  in order to provide guidance and structure for our projects. During today’s lab, five primary articles were located, reviewed, and recorded. Also, we selected the phages that we will be examining and began to search them for integrase genes to determine potential lysogeny.

Tools

  • Phamerator
  • NCBI protein blast
  • Google Docs
  • NCBI pubmed

Procedure

  1. Log onto computer and open all necessary tools.
  2. Explore NCBI and Pubmed for primary research articles.
  3. Develop a refined research question.
  4. Compile a list of phages that will be used for research, then use Phamerator to investigate the presence of integrase genes in the phages. If an integrase gene is present, the phage is likely lysogenic.
  5. Compile the data for further analysis.

Results

We are working with all phages who have the host Arthrobacter Globiformis, which fall under the following clusters:

AS2, AS3, AU2, AU3, AW, AY, AZ, FA, FC, FD, FE, FF, FG

 

Potential Research questions

  1. How many Arthrobacter globiformis phages are temperate?
  2. Are there potential stoperators in the Arthrobacter globiformis genomes?

We developed a chart to properly record integrase results.

Integrase Chart

Conclusion

In conclusion, our group found primary research articles and developed a more refined research project. One thing I struggled with today was documenting what I was doing as I proceeded with the research. In the next lab I will try to be more efficient by capturing what I’m doing as I’m doing it instead of having to backtrack for my data.

Future Work

In the next lab, we will be continuing to search for integrase in our selected phages and will also search for CRO genes.

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