February 17

The Forgotten Cure: Chapters 1-4

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The Forgotten Cure was a fascinating book that introduced topics and background to discoveries that I had not known to exist. World War I was consuming the countries, as many families had loved ones who were being put in unsanitary and unsafe conditions throughout the years. Dysentery had begun to impact many involved in the war, leading to a greater need for a cure to be found. The book revealed that the outbreak of dysentery caused by close quarters and a lifestyle susceptible to the transmitting of diseases allowed for the breakthrough to be made on bacteriophage discovery. Felix d’Herelle had previously seen “plaques” in his own experiments concerning locusts; however, when he began researching dysentery, the presence of plaques occurred again. d’Herelle began to realize the importance of his discovery when it was observed that phage presence in the body began to cure the effects of dysentery. This allowed for the world to turn an eye to the presence and discovery of the bacteriophages.

It was evident that d’Herelle possessed a thirst for knowledge and a passion for the sciences, rather than using it was a means for a career. He took up an unpaid position at a point in his life because he enjoyed the field, which showed that he was not swayed by other motives. d’Herelle displayed dedication and drive for his passions, which was evident in his works. Georgy Eliava was also a scientist that expressed drive and hard work for what he was passionate about. Eliava seemed to conduct himself in a more outgoing manner than d’Herelle however. The Elivia’s ultimately faced death due to the regime of the Soviet Union.

War and politics ultimately played a large role in the spread of phage therapy. War dramatically increased the number of diseases and problems present in the world. With the increase of problems came an increase in the need for a solution in troubling times. The discovery of bacteriophages and phage therapy during the time period allowed for their application and presence on the minds of others. War increased the use and demand for phage therapy to combat the problems being presented. However, while the presence of phage therapy was spread, little was done to advance the knowledge on the topic. Politics during the time period was very tense in all aspects. Many different countries and sides wanted an edge on the newest innovation and technology. I would imagine that scientific phage research was another aspect that many countries were trying to grasp and understand in order to not be behind in the race to discovery.

In my opinion, a major contributor as to why the spread of phage therapy failed was the lack of understanding of the topic. Its presence was known, as well as its potential uses, however, it is not nearly to the extent that it is today. Due to a large number of unknowns, alternatives to phage therapy were used instead to attempt to combat the diseases present, such as the use of antibiotics. While there were some means of spreading new knowledge about phage therapy, there was also a lack of media present during the time. In the present day, new scientific discoveries are extremely accessible to anyone who so chooses to find it. Back during that era, it was significantly harder to communicate and spread knowledge on the topic.

Delbruck’s and Luria’s work and collaboration enabled them to be introduced to the world. While searching for organisms to research, the two physicists came across bacteriophages and became integrated into the Phage Group. The pair, along with the collaborations with Hershey, won a Nobel Prize for their work. They became known for their strides in bacteriophage mutations and their genetic structure. Phage biology began to die out due to the lack of work being conducted on the subject. Delbruck and Luria ultimately moved on from their work on phages and other scientists devoted their time elsewhere.

February 15

Continuing Annotating Genes 86-88 of NapoleonB 2/13/19

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Rationale

Today we will finish the annotations assigned for the genome of NapoleonB.

Tools

  • NCBI
  • HHPred
  • Phamerator
  • PhagesDB
  • Genemark
  • DNA Master
  • Phage Notes

Procedure

  • DNA master was opened and NapoleonB’s FASTA file was autoannotated.
  • The protein sequences were run through NCBI Blast, HHPred, and PhageDB.
  • The remaining tools were utilized to complete the rest of the annotations of Phage Notes. Phamerator was utilized to determine the synteny, while genemark displayed the coding potential.

Results

The results for genes 86-88 are shown below.

Conclusions/Next Steps

Next, we will ensure that no errors were detected in the annotated genome. The annotations will then be submitted to PhagesDB.

February 15

Annotating Genes 85-88 of NapoleonB 2/11/19

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Rationale

Today we started annotating the genome of NapoleonB.

Tools

  • DNA Master
  • NCBI Blast
  • HHPred
  • PhagesDB
  • Genemark
  • Phamerator
  • PhageNotes

Procedure

  • NapoleonB’s FASTA file was downloaded and opened in DNA Master.
  • The genome as auto-annotated.
  • Gene 85 was located and DNA Master, NCBI Blast, HHPred, and PhagesDB were utilized to help annotate the genome on PhageNotes. Genemark and Phamerator were utilized as well.

 

Results

The results of the final annotation of gene 85 is shown below. The function of gene 85 was reported to be HNH endonuclease.

Conclusion/Next Steps

Next, the annotations of genes 86-88 will be conducted to finish the assigned genes of Napoleon B. The annotations will allow for further research on NapoleonB,

February 8

Continuing of Gene Annotation of Genes 42 and 43 of Elesar 2/6/19

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Rationale

Today we will continue to annotate the genes of 42 and 43 of Elesar and check the work of the gene annotations for gene 16 and 17 of Elesar.

Procedure

  • Phamerator was introduced and an account was made for the database.
  • DNA Master was opened and additions to the previous annotations were made in order to complete the Phage Notes.
  • The final annotations were entered into Phage Notes.
  • The gene annotations of gene 16 and 17 were checked.

Results

The final gene annotations are shown below.

Conclusions/Next Steps

After practicing on Elesar, gene annotations on NapoleonB will be ready to be made.

February 8

Elesar Gene Annotation of Genes 42 and 43 2/4/19

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Rationale

Today we will continue to annotate the genes 42 and 43 of Elesar for practice.

Procedure

  • Genes 42 and 43 were previously annotated, however, corrections were made accordingly.
  • DNA Master was opened and the protein sequence for genes 42 and 43 was obtained.
  • The protein sequence was run through NCBI Blast, as well as HHPred and Gene Mark.

Results

Shown below are the NCBI Blast results from genes 42 and 43 respectively.

Shown below are the HHPred results from genes 42 and 43 respectively.

Final Annotation for Gene 42: SSC: 31255, 31935 CP: yes SCS: both-cs BLAST-Start: hypothetical protein PBI_MAJA_36, 36, NCBI, q46:s2, 2e-46 Gap: 3 overlap LO: N/A RBS: Kibler 7, Karlin Medium, 2.325, -3.883, no SIF-BLAST: NKF SIF-HHPred: Mitoribosomal protein mL52 , HHPred, n/a, PF18699.1, 47.9, 32.7 SIF-Syn: NKF

Final Annotation for Gene 43: SSC: 31932, 32198 CP: no SCS: Both BLAST-Start: hypothetical protein PBI_RYAN_46, 46, NCBI, q1:s1, 2e-53 Gap: 3 overlap LO: N/A RBS: [Kibler 7, Karlin Medium, 2.214, -4.470, no] SIF-BLAST: NKF SIF-HHPred: Secretin_N ; Bacterial type II/III secretion system short domain, HHPred, n/a, PF03958.17, 73.2, 86.81 SIF-Syn: NKF

Conclusion/Next Steps

Some parts of the gene annotations are still missing, however, the additions will be made at a later date. Once all the skills are learned to fully annotate a gene, we will move on to the annotation of NapoleonB.

January 31

Annotation of Elesar 1/30/19

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Rationale

Today we will finish annotating Gene 1 of Elesar and will independently annotate other genes in the genome.

Procedure

  • The work done on the annotation of Gene 1 was completed by re-opening the FASTA file and annotations.
  • Genes 42 and 43 were assigned to annotate for practice, and the following categories were completed with respect to the genes assigned: SSC:, CP:, SCS:, ST:,BLAST-Start:, Gap: LO:, RBS:

Results

Blast results are shown above for Gene 42 and 43. The results show the NCBI database and the matching results for each query.

Gene 42: SSC: 31255, 31935, CP: yes, SCS: both-cs, BLAST-Start: [hypothetical protein PBI_MAJA_36, 36, NCBI, 46, 2, 2e-46], Gap: 3 overlap, LO: N/A, RBS: [Kibler 7, Karlin Medium, 2.325, -3.883, no]

Gene 43: SSC: 31932, 32198, CP: yes, SCS: Glimmer, BLAST-Start: [hypothetical protein PBI_RYAN_46, 46, NCBI, 1, 1, 2e-53], Gap: 3 overlap, LO: N/A, RBS: [Kibler 7, Karlin Medium, 2.214, -4.470, no]

Conclusion/Next Steps

The practice of annotating the genes can be beneficial for future use. Annotations will occur with NapoleonB and it is imperative that an understanding DNA Master is possessed.

January 31

Basics of Blast 1/28/19

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Rationale

Today we will learn more about the BLAST feature in DNA Master by BLASTing genes in the Elesar genome.

 

Procedure

  • Elesar was opened again in DNA Master and the FASTA file was autoannotated.
  • Gene 1 of Elesar was auto-annotated using the details from the annotation key and auto-annotation page.
  • The protein sequence of Gene 1 was copied and inserted into the protein BLAST at NCBI.
  • The BLAST results were then interpreted.

 

Results

Blast results are inserted above for Gene 1. Hypothetical protein A of Arthrobacter sp Hiyo is shown to have the highest score. A gap of two is indicated as well.

 

Conclusions/Next Steps 

A portion of the annotation of Gene 1 was done as a group and will be finished by the next lab. The Gene annotation of Elesar will continue until a better understanding of DNA Master and Blast is reached.

January 25

Elesar Annotation 1/23/19

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Rationale

Today we will learn more about reading frames and explore more of DNA Master using the genome of Elesar.

 

Procedure

  • DNA Master was opened and the template “SSC: CP: SCS: ST: BLAST-Start: Gap: LO: RBS: F: SIF-BLAST:SIF-HHPred: SIF-Syn” was uploaded for future annotations.
  • Elesar was opened using the FastA file and the new template was employed to autoannotate.
  • ORFs were also accessed to be observed and begin the process of learning their functions.

 

Results

More information on gaps and overlaps, as well as ORFs were presented. Knowledge about DNA Master and ORFs are still limited, therefore no notable results were achieved from the day. The new template may be used for future autoannotations.  A better understanding of DNA Master and the different tools in the program was reached instead.

 

Conclusion/Future Steps

The tools being used in DNA Master will allow for the autoannotation of NapoleonB in the future. DNA Master will be explored more using Elesar to become familiar with the program.

 

January 17

DNA Master Installation and Auto Annotation 1/16/19

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Rationale

Today we presented our phage therapy powerpoints, downloaded DNA Master onto our personal computers, and began practicing annotating DNA on the genome of phage Elesar.

Procedure

  • Various groups from the classes presented their articles on phage therapy.
  • DNA Master was downloaded onto the computers.
  • A FastA file was downloaded for Elesar from PhagesDB.
  • Elesar was uploaded to DNA Master and was auto-annotated.

Observations/Results

DNA Master revealed that there are 66 ORFs in Elesar. There are 11 reverse ORF of the 66 documented. There are also 55 forward ORFs. Some tasks on DNA Master took a large amount of time to process, however, each task ran smoothly.

Conclusion/Next Steps

I really look forward to learning more about DNA Master and the different discoveries that can be made while using the program. In the coming months, we will dive more into the program and learn more about phage NapoleonB. We also will attempt to improve our presentation skills throughout the semester.

November 30

DNA Extraction 11/29/18

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Rationale

Today we will conduct DNA extraction on the precipitate previously made.

Procedure

  • Established an aseptic zone.
  • 0.5 µL of sterile water was added to the phage pellet to resuspend.
  • 2 mL of incubated Resin was added to the vial and was shaken to fully coat the DNA.
  • Approximately 1.5 mL of the solution was added to one microcentrifuge tube, and 1.5 mL of the solution was added to another microcentrifuge tube.
  • The two tubes were spun at 12-13k g for 3 minutes.
  • The supernatant was removed. 1 mL of 80% isopropanol was added to each tube and the pellet was resuspended.
  • The two tubes were spun at 12-13k g for 3 minutes.
  • The supernatant was removed. 1 mL of 80% isopropanol was added to each tube and the pellet was resuspended.
  • The two tubes were spun at 12-13k g for 3 minutes.
  • The supernatant was removed. 1 mL of 80% isopropanol was added to each tube and the pellet was resuspended.
  • A column syringe was assembled and attached to the vacuum hood. The contents were placed in the column syringe filter and the DNA/resin mixture was trapped in the column filter.
  • The two column filters were placed in 2 microcentrifuge tubes and were centrifuged at 12,000g for 5 minutes. This process was repeated twice.
  • 100 µL of Elution Buffer was added to the column and was set aside for 1 minute. The two microcentrifuge tubes were centrifuged at 12,000 g for 1 minute. The product of the two tubes were combined and were placed into the -20 C freezer as a result.

Observation

Dr. Adair centrifuged the phage precipitate preparation before lab since the centrifuging swinging bucket was not functioning at the previous lab.

Conclusions/Next Step

Next, the DNA will be analyzed using a Nanodrop and PCR will occur.