February 18

2-18-19 — The Forgotten Cure by Anna Kuchment

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1. Describe the role that locusts, dysentery and war had in the discovery of bacteriophage.

While studying bacterial cultures from sick locusts, d’Herelle discovered clear spots in the bacterial lawns, later to be known as “plaques.” This was his first proof that there was something smaller than known microbes. This was later reinforced when, because of dysentery cases suffered during the war, some patients mysteriously and quickly recovered. d’Herelle recognized the same plaques in there cultures, and began experimenting with the application of this unknown microorganism in order to heal soldiers dying of this disease.

2. Discuss the characteristics of d’Herelle that led him to be a successful scientist. How did he compare to Georgi Eliava?  What happened to the Eliava’s?

d’Herelle had a uniquely selfless drive to study science. He was self-taught, having never attended university, and wasn’t interested in money as much as others. d’Herelle did, however, possess some unfortunate traits. His lack of formal education made him lacking in credibility among the scientific community, and he did make a few assertions that were false that he would’ve understood had he received a real science education. Eliava was born into privilege and became close friends with d’Herelle. They were both incredibly intelligent and had a drive to discover more in their field. Eliava was ultimately executed a few years after Stalin’s rise to power after an officer with a vendetta saw a chance to get back at the scientist for having the traits that he lacked.

3. Discuss the influence war and politics had on the spread of phage therapy.

War initially helped push for the spread of phage therapy, as the need for medicine for soldiers seemed to be solvable by phage usage; however, as the war heated up, more effective alternatives to phage therapy were discovered and popularized. Phage therapy was isolated to small lab groups here and there, but widespread application was halted.

4. What are some of the reasons that the spread of phage therapy failed?

Sulfa drugs were developed that were easy to produce and got consistent results. Using phage therapy, it was difficult to get consistent results. There was also ethical controversy involving control groups and whether it was acceptable to deny a group phage therapy for the sake of having a control group when there was a high likelihood the control group would have significantly more fatalities than the experimental group. Again, d’Herelle’s attitude didn’t gain him any respect in the scientific community, thus pushing readers to his competitors, even if they were incorrect in their assertions.

5. How did the physicists Delbruck and Luria end up as part of the Phage Group? What contributions did they make to phage biology?  Why did phage biology die out in the 70’s?

Both Delbruck and Luria were interested in combining physics and biology, and thus molecular biology was born. They began studying bacteriophages and were able to determine that genetic information was DNA and not proteins. The final nail was hit into phage therapy’s coffin when the rediscovery of penicillin brought a new, first-of-its-kind superdrug to the world. Penicillin wasn’t effective on every illness, some of which having been shown to be susceptible to phage therapy, but it was still preferred. The only places that held on to phage therapy were countries in the Caucasus.

February 15

2-13-19 — Consolidation of Annotations to PhageNotes

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Consolidation of Annotations to PhageNotes

Date: 2-13-19

  • Rational
    • The rational for this lab is to consolidate all annotations into one google sheet for analysis.
  • Procedure
    • The notes section for genes 53-56 in DNAMaster were copied to the class PhageNotes google sheet.
  • Results
    • Annotations for every gene in phage NapoleonB are in one central document.
  • Future Plans
    • The next step is to check the annotated genes and analyze them further.
February 15

2-11-19 Annotation of Phage NapoleonB Genes 53-56

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Annotation of Phage NapoleonB Genes 53-56

Date: 2-11-19

  • Rational
    • The rational for this lab was to begin annotation of phage NapoleonB, starting with genes 53, 54, 55, 56.
  • Procedure
    1. DNA Master was opened.
    2. File > Open > Archived DNA Master File was used to open the NapoleonB FastA file from PhagesDB.
    3.  Gene 53 was selected through the listing of predicted genes, and the Product was copied.
    4. The amino acid sequence was loaded into the BLASTp feature on the NCBI website and the BLAST results were analyzed and recorded on the PhageNotes google sheet.
    5. The amino acid sequence was loaded into HHPRED, a program that evaluated the three-dimensional protein structure of the gene region and the results were analyzed and recorded.
    6. The starterator map on PhagesDB was evaluated by choosing gene 53 from the NapoleonB gene list and the information was recorded.
    7. Steps 3-6 were repeated for genes 54-56.
    8. Ctrl + Alt + F brought up the Frames of the genome, ORFS was selected to show the locate open reading frames, and RBS was selected to determine the ribosome binding site for the each gene.
    9. Genemark was used in order to determine if coding potential was covered.
    10. Using the acquired information, the 53rd-56th genes were annotated.
  • Results
    • The following annotations were made for gene 53:
      • Original GeneMark call @bp 33165
        SSC: 33165 – 33263, CP: Yes, SCS: Both, ST:SS, BLAST-Start: Aligns with Arthrobacter Phage Tribby gp53 NCBI BLAST q53:s1 1 5E-13, Aligns with Arthrobacter Phage Tribby gp53 PhagesDB BLAST q53:s1 1 1E-12, Gap: 4bp overlap, LO: Yes, RBS: Kibbler7 and Karlin Medium 3.263 -2.524 No, F: NKF, SIF-BLAST: NKF, SIF-HHPred: NKF, SIF-Syn: NKF

An image of the starterator report

An image of the RBS choices

An image of the PhagesDB BLAST results

An image of the NCBI BLAST results

An image of the HHPRED results

An image of the GeneMark results

  • The following annotations were made for gene 54:
    • Original Glimmer call @bp 33273 has strength 10.13
      SSC: 33273 – 33962, CP: Yes, SCS: Both, ST: SS, BLAST-Start: Aligns with Arthrobacter Phage Circum gp55 NCBI BLAST q54:s1 1 4E-167, Aligns with Arthrobacter Phage KeaneyLin gp52 PhagesDB BLAST q54:s2 1 1E-128, Gap: 9bp gap, LO: Yes, RBS: Kibbler7 and Karlin Medium 1.993 -4.759 No, F: NKF, SIF-BLAST: NKF, SIF-HHPred: NKF, SIF-Syn: NKF

An image of the starterator results

An image of the RBS choices

An image of the PhagesDB BLAST results

An image of the NCBI BLAST results

An image of the HHPRED results

Images of the GeneMark results

  • The following annotations were made for gene 55:
    • Original Glimmer call @bp 33955 has strength 0.38
      SSC: 33949 – 34173, CP: Yes, SCS: Both-CS, ST: SS, BLAST-Start: Aligns with Arthrobacter Phage Circum gp56 NCBI BLAST q55:s1 0.98 3E-44, Aligns with Arthrobacter Phage KeaneyLin gp53 PhagesDB BLAST q55:s2 0.98 7E-36, Gap: 14bp overlap, LO: No, RBS: Kibbler7 and Karlin Medium 1.391 -5.257 No, F: NKF, SIF-BLAST: NKF, SIF-HHPred: NKF, SIF-Syn: NKF

An image of the starterator results

An image of the RBS choices

An image of the PhagesDB BLAST results

An image of the NCBI BLAST results

An image of the HHPRED results

An image of the GeneMark results

  • The following annotations were made for gene 56:
    • Original Glimmer call @bp 34191 has strength 0.52
      SSC: 34191 – 34316, CP: Yes, SCS: Both, ST: SS, BLAST-Start: Aligns with Arthrobacter Phage Circum gp57 NCBI BLAST q56:s1 1 1E-20, Aligns with Arthrobacter Phage Circum gp57 PhagesDB BLAST q56:s3 1 2E-16, Gap: 17bp gap, LO: Yes, RBS: Kibbler7 and Karlin Medium 2.053 -5.205 No, F: NKF, SIF-BLAST: NKF, SIF-HHPred: NKF, SIF-Syn: NKF

An image of the starterator results

An image of the RBS choices

An image of the PhagesDB BLAST results

An image of the NCBI BLAST results

An image of the HHPRED results

An image of the GeneMark results

  • Conclusion
    • Phage NapoleonB has no known functions for genes 53-56.
  • Future Plans
    • The next step is to consolidate the gene annotations to the PhageNotes google sheet.
February 8

2-6-19 — Copying Genes 12 and 13 to PhageNotes

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Further Annotation of Phage Elesar Genes 12 and 13

Date: 2-4-19

  • Rational
    • The rational for this lab was to annotate the function data for two genes from phage Elesar in order to gain practice in annotation before transitioning to phage NapoleonB.
  • Procedure
    1. DNA Master was opened.
    2. File > Open > Archived DNA Master File was used to open the same Elesar FastA file from before.
    3. Gene 12 was selected through the listing of predicted genes.
    4. The annotation notes were analyzed and copied to the PhageNotes Google Sheets page.
    5. Gene 13 was selected through the listing of predicted genes.
    6. The annotation notes were analyzed and copied to the PhageNotes Google Sheets page.
    7. Using the acquired information, the 12th and 13th genes were annotated.
  • Results
    • The following annotations were copied for Elesar gene 12 :
      • SSC:8224 – 8568, CP:No There is an overlap with the previous gene, SCS:Both, ST:SS, BLAST-Start:Aligns with Nandita gp13 NCBI BLAST q12:s1 0.87 5E-58, Aligns with Phage Ryan gp13 PhagesDB BLAST q12:s2 0.87 1E-48, Gap:7bp overlap, LO:NA, RBS:Kibbler7 and Karlin Medium 2.463 -3.67 No, F:head-to-tail stopper, SIF-BLAST:head-to-tail stopper Supported by NCBI BLAST Nandita gp13 AYN58635.1 0.87 5E-58, head-to-tail stopper Supported by PhagesDB BLAST Ryan gp13 MH834627 0.87 1E-48, SIF-HHPred:NKF, SIF-Syn:head-to-tail stopper, upstream gene is head-to-tail adaptor, downstream gene is NKF, just like in phage Ryan
    • The following annotations were copied for Elesar gene 13 :
      • SSC:8565 – 8840, CP:Yes, SCS:Both, ST:SS, BLAST-Start:Aligns with Nandita gp14 NCBI BLAST q13:s1 0.76 3E-38, Aligns with Phage Nandita gp14 PhagesDB BLAST q13:s2 0.76 7E-31, Gap:4bp overlap, LO:NA, RBS:Kibbler7 and Karlin Medium 1.137 -7.054 No, F:NKF, SIF-BLAST:NKF Supported by NCBI BLAST gp, SIF-HHPred:NKF, SIF-Syn:NKF
  • Conclusion
    • All the genes present in Phage Elesar have been copied to a single document in order to share the data.
  • Future Plans
    • The next step is to continue annotating more genes on Elesar for practice before transitioning to NapoleonB.
February 8

2-4-19 — Further Annotation of Phage Elesar Genes 12 and 13

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Further Annotation of Phage Elesar Genes 12 and 13

Date: 2-4-19

  • Rational
    • The rational for this lab was to annotate the function data for two genes from phage Elesar in order to gain practice in annotation before transitioning to phage NapoleonB.
  • Procedure
    1. DNA Master was opened.
    2. File > Open > Archived DNA Master File was used to open the same Elesar FastA file from before.
    3.  Gene 12 was selected through the listing of predicted genes, and the Product was copied.
    4. The amino acid sequence was loaded into the BLASTp feature on the NCBI website and the BLAST results were analyzed.
    5. The amino acid sequence was loaded into the BLAST feature on the PhagesDB website and the BLAST results were analyzed.
    6.  Gene 13 was selected through the listing of predicted genes, and the Product was copied.
    7. The amino acid sequence was loaded into the BLASTp feature on the NCBI website and the BLAST results were analyzed.
    8. The amino acid sequence was loaded into the BLAST feature on the PhagesDB website and the BLAST results were analyzed.
    9. Using the acquired information, the 12th and 13th genes were annotated.
  • Results
    • The following annotations were made for Elesar gene 12 :
      • F: The function was determined to be a head-to-tail stopper. This was supported by the following fields.
      • SIF-BLAST: head-to-tail stopper, NCBI BLAST, Arthrobacter Phage Nandita gp13, AYN58635.1, 87%, 5e-58
      • SIF-HHPred: NKF, Pfam-A, DUF3599, PF12206.8, 100%, 99.09
      • SIF-Syn: head-to-tail stopper, upstream gene is head-to-tail adaptor like in arthrobacter phage Ryan, downstream gene has NKF
    • The following annotations were made for Elesar gene 13 :
      • F: NKF. This was deternined because there no significant blast hits pointing to a function.
      • SIF-BLAST: NKF
      • SIF-HHPred: NKF
      • SIF-Syn: NKF
  • Conclusion
    • Phage Elesar gene 12 has a probably function of being a head-to-tail stopper due to its similarities to other head-to-tail stopper genes as well as its synteny.
  • Future Plans
    • The next step is to continue annotating more genes on Elesar for practice before transitioning to NapoleonB.
February 1

1-30-19 — Annotation of Phage Elesar Genes 12 and 13

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Annotation of Phage Elesar Genes 12 and 13

Date: 1-30-19

  • Rational
    • The rational for this lab was to annotate two more genes from phage Elesar in order to gain practice in annotation before transitioning to phage NapoleonB.
  • Procedure
    1. DNA Master was opened.
    2. File > Open > Archived DNA Master File was used to open the same Elesar FastA file from before.
    3.  Gene 12 was selected through the listing of predicted genes, and the Product was copied.
    4. The amino acid sequence was loaded into the BLASTp feature on the NCBI website and the BLAST results were analyzed and recorded on the template.
    5.  Gene 13 was selected through the listing of predicted genes, and the Product was copied.
    6. The amino acid sequence was loaded into the BLASTp feature on the NCBI website and the BLAST results were analyzed and recorded on the template.
    7. Ctrl + Alt + F brought up the Frames of the genome, ORFS was selected to show the locate open reading frames, and RBS was selected to determine the ribosome binding site for the first gene
    8. Using the acquired information, the 12th and 13th genes were annotated.
  • Results
    • The following annotations were made for Elesar gene 12 :
      • SSC: 8224, 8568
      • CP: yes
      • SCS: both
        • Glimmer and GeneMark both called the gene at bp 8224 and this choice was selected.
      • BLAST-Start: Arthrobacter phage Nandita, gp13, NCBI, Query 12 to Subject 1, 87%, 5e-58
      • Gap: 7 bp overlap
      • LO: yes
      • RBS: Kibler7, Karlin Medium, 2.463, -3.670, no
    • The following annotations were made for Elesar gene 13 :
      • SSC: 8565, 8840
      • CP: yes
      • SCS: both
      • BLAST-Start: Arthrobacter phage Nandita, 14, NCBI, Query 13 to Subject 1, 76%, 3e-38
      • Gap: 2 bp gap
      • LO: yes
      • RBS: Kibler7, Karlin Medium, 1.137, -7.054, no
  • Conclusion
    • Phage Elesar genes 12 and 13 are similar to a hypothetical protein and a head-to-tail stopper in the genome of Arthrobacter Phage Nandita.
  • Future Plans
    • The next step is to continue annotating more genes on Elesar for practice before transitioning to NapoleonB.
February 1

1-28-19 — Annotation of Phage Elesar Gene 1

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Annotation of Phage Elesar Gene 1

Date: 1-28-19

  • Rational
    • The rational for this lab was to annotate the first gene in phage Elesar in order to gain practice in annotation before transitioning to phage NapoleonB.
  • Procedure
    1. DNA Master was opened.
    2. Export > Create New Sequence from This Entry Only > Genome > Auto-Annotate was used to auto-annotate the phage genome.
    3.  Gene 1 was selected through the listing of predicted genes, and the Product (the results of the amino acids coded for) were copied.
    4. The amino acid sequence was loaded into the BLASTp feature on the NCBI website and the BLAST results were analyzed and recorded on the template.
    5. Ctrl + Alt + F brought up the Frames of the genome, ORFS was selected to show the locate open reading frames, and RBS was selected to determine the ribosome binding site for the first gene
    6. Using the acquired information, the first gene was annotated.
  • Results
    • The following annotations were made for Elesar gene 1 :
      • SSC: 45, 353
        • The start location was moved from bp 84 to bp 45 to achieve the longest open reading frame.
      • CP: yes
        • GeneMark was used to compare the coding potential to the annotation. The selected species was “Arthrobacter_aurescens_TC1” and the results were as follows:
      • SCS: both-cs
        • DNAMaster reported “Original Glimmer call @bp 84 has strength 8.51,” so both Glimmer and GeneMark called the gene at bp 84, but the starting base pair was instead decided to be bp 45.
      • BLAST-Start: no significant BLAST alignments
        • The e value for the BLAST of gene 1 returned as 1.11, outside of the range of accurate matches.
      • Gap: first gene
      • LO: yes
      • RBS: Kibler7, Karlin Medium, 1.222, -6.751, no
        • The total score was not the best for this RBS, but it was decided that this was still the best location for it.
  • Conclusion
    • It is important to declare which database (NCBI or PhagesDB) is being used, as each BLAST can return different results depending on the database.
  • Future Plans
    • The next step is to continue annotating more genes on Elesar for practice before transitioning to NapoleonB.
January 25

1-23-19 — Gene Annotation Intro and Template Setting

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Gene Annotation Intro and Template Setting

Date: 1-23-19

  • Rational
    • The rational for this lab was to add a template to DNAMaster for autoannotation and understand the counting and significance of gaps and overlaps between gene sequences.
  • Procedure
    1. DNA Master was opened.
    2. A template was added by going to File > Preferences > Local Settings > New Feature. ‘Insert template into notes during autoannotation’ was selected and a template code was pasted in the text box.
    3. The code “SSC: CP: SCS: ST: BLAST-Start: Gap: LO: RBS: F: SIF-BLAST: SIF-HHPred: SIF-Syn” was added with the following meanings:
      • SSC: Start/Stop Coordinates
      • CP: Coding Potential
      • SCS: Start choice source
      • ST: Starterator
      • BLAST-Start: The best BLAST match for the gene
      • Gap: Gaps or overlaps
      • LO: Longest ORF, for genes with ~10 bp between start codon and upstream stop codon
      • RBS: Ribosome binding sites
      • F: Gene function
      • SIF-BLST,-HHPred,-Syn: Supporting information for the function
    4. Elesar was opened and autoannotated with this template saved.
  • Results
    • The template appeared in the notes section of the autoannotated Elesar.
  • Conclusion
    • Using the template allows for more accurate presentation of annotation information in the notes section.
  • Future Plans
    • This template will be used for future annotations. As gene annotation experience is gained, it will be easier to recognize and analyze trends as well as correctly manually annotate gene sequences.
January 18

1-16-19 — DNA Master Setup

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DNA Master Setup

Date: 1-16-19

  • Rational
    • The rational for this lab was to set up DNA Master for future use and to start with the annotation of phage Elesar.
  • Procedure
    1. DNA Master was downloaded and opened.
    2. Preferences were changed for multiple fields. Colors of certain sequences were changed in order to match the rest of the classmates and their annotations.
    3. The option to use PBI server for SEA-PHAGES courses was selected.
    4. A fasta file of a phage (in this case, phage Elesar) was downloaded and opened in DNA Master.
    5. The fasta file was auto-annotated by going to File > Open > FastA Multiple Sequence File and selecting the desired file.
  • Results
    • The result of this lab was becoming comfortable with using the program and opening files.
  • Conclusion
    • DNA Master can be difficult to get running. Preferences must be checked before working to make sure that everything will run well.
  • Future Plans
    • In following labs, the goal will be to more thoroughly go through annotated gene sequences, editing and recording information over time.
November 30

11-28-18 — High Titer Plaque Assay Second Attempt

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Date: Wednesday, November 28th, 2018

Title: High Titer Plaque Assay Second Attempt

Rationale: The purpose of today’s lab is to calculate the titer of the lysate using a previous plaque assay and then to attempt to web a plate using the titer calculations.

Class Question: Is there a difference in bacteriophage presence or type in soil samples taken from live oaks vs those from red oaks?

Procedure:

  1. An aseptic zone was set up.
  2. Plates from last lab were evaluated and found to have negative results.
  3. A new 10^-7 dilution was made using the positive lysate.
  4. 20 μL of 10^-7 diluted lysate was added to a culture tube containing .5 mL arthrobacter and left to infect for 15 minutes.
  5. Top agar solution was made using the following recipe:
    1. 4 mL LB Broth
    2. 5 mL 2x top agar
    3. 45 μL 1M CaCl2
  6. 4.5 mL of TA solution was added to a top agar control plate.
  7. 4.5 mL of TA solution was added to the culture tube and pipetted to mix.
  8. The contents of the culture tube were poured into a plate.
  9. The plates were left to sit for 15 minutes before being inverted and placed in an incubator for the next 48 hours.

Observations: The plates from last lab yielded negative results. This is most likely due to the original 10^-7 dilution losing its infectivity. The newly made dilution should produce results, as it has a high titer.

Results: This experiment yielded a top agar control and a plaque assay that, according to titer calculations, should be a webbed plate.

Next Step: The next step is to evaluate the plates and flood it with phage buffer.