February 17

The Forgotten Cure: Chapters 1-4

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The Forgotten Cure: Chapters 1-4

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

Locusts, dysentery, and war played a unique role in the discovery of bacteriophage. During World War I, troops struggled as dysentery plagued their ranks as they lived in incredibly tight quarters with shared space, food, and water. This created a sense of desperation surrounding the situation of dysentery, as troops were suffering major losses and were unable to perform at their peak capacity. Felix d’Herelle, a microbiologist of the time, was working with bacteria cultures when he noticed strange clearings that appeared “randomly” on the plates. Upon further investigation, d’Herelle developed methods to consistently obtain these clearings and he concluded that there was a specimen so small that could be considered invisible that was infecting the bacteria and killing them. When he proved that this specimen was capable of curing individuals rather rapidly of dysentery, the discovery was lauded and the new specimen gained immense popularity, which led to many more people becoming interested and studying bacteriophage.

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?

Felix d’Herelle consistently displayed that he was devoted to the field of science above all else. This dedication allowed him to gain experience in a variety of ways, such as d’Herelle taking an unpaid position at a prestigious scientific organization. His curiosity and drive to learn provided a large basis of his skills that made him a successful scientist. Furthermore, d’Herelle was also very confident and extraordinarily particular about how his lab work was performed. These qualities made for a scientist who was suited for publishing new concepts, as his confidence allowed him to display resilience even when critical articles or other roadblocks were in his way. Georgi Eliava was similar to d’Herelle in regard to their upbringing and interests, but was different in their mannerisms. Both shared very privileged upbringings, which provided the initial resources to explore science and develop interests in such fields. These interests were very strong in both of their lives, and it led to their bond as scientists who worked passionately in the same field. Their mannerisms were described to be very different, as Eliava was said to be smooth, with pomaded hair and a way with women whereas d’Herelle was said to be particular and very serious. Eventually, the Eliava’s met their demise at the hands of the Russian regime, as Eliava fell on the wrong side of a Russian political leader, which led to his imprisonment and execution.

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

War had a very important influence on the spread of phage therapy. Due to the magnitude of the diseases that occurred, an effective and sustainable solution was needed to cure the troops of the disorders that they encountered. This led to the birth of phage therapy, and also the development as the technologies were refined as they were put into practice. Politics had an arguably more influential effect on the spread of phage therapy. Joseph Stalin felt that the Russians were far behind the rest of the developed nations in terms of science and technology – he estimated that they had fallen behind between fifty and one hundred years, and if they were not to make up that difference in the next ten years, they would be rather permanently at a disadvantage. This led to Stalin looking favorably upon the establishment of a phage institute in Georgia, which was established and run by Georgi Eliava. As resources were funneled into this field, more and more discoveries were made that enhanced the understanding of bacteriophages and furthered them as a possible treatment.

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

Two reasons why the spread of phage therapy ultimately failed were the development of other technologies and a low level of comprehension. As the technology spread to the Western world, other technologies would be developed each time a swell of interest in phage therapy would occur. The development of sulfa drugs and penicillins both were considered to be more important and effective methods to treat bacterial infection despite evidence that both, especially penicillins, could result in antibiotic-resistant bacterial strains. While even phages share this problem (CRISPR-Cas9 bacterial defense system), they are more adaptable and present a lower risk due to their specificity. Despite this, the western world prioritized alternative methods of treating bacterial infections. Therefore, resources were not placed into phage research, which stunted its development. Alternatively, a lack of understanding of bacteriophages resulted in a halt of their spread. A series of unfavorable reviews from JAMA (Journal of American Medical Association), described phages as enzymes and proteins, which clearly is not the case. It also suggested that they had limited effectiveness in treating conditions. Moreover, studies about phages were conducted rather haphazardly, as scientists eager to save each patient lacked control groups in their study, which compromised the results that displayed encouraging feedback. Therefore, poor levels of understanding along with the development of other technologies and interests led to the deterioration of the spread of phage therapy.

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?

Delbruck joined the Phage Group in somewhat of a roundabout way. As a physicist, he attended a presentation where it was suggested that life was so complex that learning all of the intricacies would be nearly impossible. This challenge was interesting to him, and the idea that there may be a few laws that could govern all life motivated him to find a way to study biology. This led him to the radiation experiment on fruit fly genes, which mutated them and allowed Delbruck and the group studying the flies to conclude that genes are the basic unit of life – a belief held to this day. The knowledge of this experiment interested Luria, and he also became interested in biology. As Delbruck and Luria were looking for a new organism to examine the genome of, they turned to phages and ended up joining the Phage Group. Through this group and a series of experiments featuring Hershey (Hershey and Chase experiment), they studied the mechanism of bacteriophages and learned much more about their structure. However, after they had learned so much about phages, their interest was whittled down to seven specific types of bacteriophage, and then later it was changed to the examination of other organisms. Therefore, the examination of bacteriophages fell away from the forefront of science, and phage biology began to die out in the 70s.

Other Note:

The culture of the Phage Group sounded very fun and exciting. It sounded like the ideal summer camp – working hard to learn science during the day and enjoying swimming and play reenactments later at night! It would be fun to have a summer opportunity like this at Baylor, with a phage study group over the summer with a cohort similar to the one selected for BEARS in the SEA.

February 13

2.13.19 Annotations of Genes 51 and 52

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2.13.19 Annotations of Genes 51 and 52

Rationale: Since the first two assigned genes had been processed and annotated on Monday, today it was found to be pertinent to finish annotating the remaining two assigned genes. Therefore, genes 51 and 52 were completed during today’s lab time.

Tools/Procedure:

  • DNA Master
  • HHPred
  • BLAST (NCBI)
  • BLAST (PhagesDB)
  • Genemark
  • Phamerator
  1. Opened DNA Master
  2. Annotated genes 51 and 52 using the above tools

Results:

Gene 51

SSC:32825 – 33016, CP:Yes, SCS:Both, ST:SS, BLAST-Start:Aligns with Arthrobacter Phage Mudcat gp47 NCBI BLAST q1:s1 0.98 3E-39, Aligns with Arthrobacter Phage Mudcat gp47 PhagesDB BLAST q1:s1 1 3E-32, Gap:0bp gap, LO:NA, RBS:Kibbler7 and Karlin Medium 2.611 -3.374 Yes, F:NKF, SIF-BLAST:NKF, SIF-HHPred:NKF, SIF-Syn:NKF

Gene 52

SSC:33040 – 33168, CP:Yes, SCS:Both, ST:SS, BLAST-Start:Aligns with Arthrobacter Phage Circum gp53 NCBI BLAST q1:s6 0.9756 9E-21, Aligns with Arthrobacter Phage Circum gp53 PhagesDB BLAST q1:s1 0.97 2E-17, Gap:23bp gap, LO:Yes, RBS:Kibbler7 and Karlin Medium 3.263 -2.524 Yes, F:NKF, SIF-BLAST:NKF, SIF-HHPred:NKF, SIF-Syn:NKF

 

Conclusions: Since all signs point to no function being found for either gene 51 or 52, it can officially be concluded that these genes can be considered to have no function. Annotations have officially been completed for all four genes that had been assigned in class for NapoleonB.

Next Steps: The next steps will be checking the annotations for the whole class to ensure that all genes had been called properly with the correct format. After, the annotations will be able to be imported to the DNA Master system from PhageNotes.

February 12

2.11.2019 Annotations of Gene 49 and 50 of NapoleonB

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2.11.2019 Annotations of Gene 49 and 50 of NapoleonB

Rationale: Since the procedures and skills for annotating genomes had been developed through extensive practice with phage Elesar, annotations for Napoleon were finally ready to begin. Therefore, genes were assigned and genes 49 and 50 were annotated.

Tools/Procedure:

  • DNA Master
  • Starterator
  • HHPred
  • NCBI BLAST
  • PhagesDB Blast
  • Phamerator
  1. Opened PhagesDB and found NapoleonB fasta file. Downloaded file
  2. Auto-annotated NapoleonB on DNA Master
  3. Opened ORF Screen
  4. Began RBS value examination
  5. Annotated genes 49 and 50

Results:

Gene 49-

 

Gene 50-

 

 

 

Phamerator that applied for both genes:

Gene 49: 

SSC:31823 – 32392, CP:Yes, SCS:Both, ST:SS, BLAST-Start:Aligns with Arthrobacter Phage Tribby gp48 NCBI BLAST q1:s1 0.99 3E-132, Aligns with Arthrobacter Phage Tribby gp48 PhagesDB BLAST q1:s1 0.98 e-100, Gap:15bp gap, LO:Yes, RBS:Kibbler7 and Karlin Medium 2.684 -3.22 Yes, F:NKF, SIF-BLAST:NKF, SIF-HHPred:NKF, SIF-Syn:NKF, upstream gene is NKF, downstream gene is NKF, just like in phage Nason

Gene 50: 

SSC:32516 – 32824, CP:No No ORF available would cover all of the potential, SCS:Both, ST:SS, BLAST-Start:Aligns with Arthrobacter Phage Arcadia gp49 NCBI BLAST q1:s1 0.99 2E-65, Aligns with Arthrobacter Phage Nason gp49 PhagesDB BLAST q1:s1 0.98 e-50, Gap:123bp gap, LO:No, RBS:Kibbler7 and Karlin Medium 3.16 -2.293 Yes, F:NKF, SIF-BLAST:NKF, SIF-HHPred:NKF, SIF-Syn:NKF, upstream gene is NKF, downstream gene is NKF, just like in phage Nason

Conclusions: Both of the genes annotated today had fairly straightforward decisions that were made. The questionable decisions came within the coding potential of gene 50 and the gap that preceded gene 50. There was a very slight amount of coding potential that extended past the earliest start codon of the gene, which resulted in the answer that was listed but there was deliberation on whether or not the amount of potential was considered to be significant. Furthermore, the gap between gene 49 and 50 is slightly longer than is normally seen when examining bacteriophage genomes, so there are some questions about whether or not there is a gene that was not detected by the auto annotation.

Next Steps: The gap between genes 49 and 50 will be examined, and genes 51 and 52 will be annotated during the next available lab time!

February 7

2.06.19- Inputting Annotations to PhageNotes & Checking Annotations

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2.06.19- Inputting Annotations to PhageNotes & Checking Annotations

Rationale:

  • In order to organize the information that had been collected over the previous lab sessions, Lathan created PhageNotes to efficiently compile the data into a usable format. Therefore, it was necessary for everyone to submit their gene annotations to the system. Furthermore, it was found to be beneficial to check each other’s genes to ensure that there were few errors.

Tools:

  • DNA Master
  • PhagesDB BLAST
  • NCBI BLAST
  • HHPred
  • Genemark

Procedure:

  • Opened DNA Master
  • Opened files containing previous annotations
  • Checked over all annotations and numbers using Genemark, BLAST softwares, and HHPred
  • Inputted information into PhageNotes
  • Checked PhageNotes & ran annotations for both Genes 10 & 11 to ensure proper notations had been found

Results:

  • Gene 11’s LO was changed, as the database listed it as no when it was the longest possible. The rest of the genes appeared to be correct upon examination.
  • Genes 50 & 51 were inputted to the database with slight alterations on gene 50’s HHPred, as certain fields should have been omitted and the changes were reflected in the official PhageNotes.

Conclusions:

  • The genes were inputted to PhageNotes. Assuming there are no errors that had been missed, the PhageNotes will be able to be used to fill in the DNA Master with correct annotations. Furthermore, the script from DNA Master would be able to be sent to SEA-Phages for processing in the format it is in.

Next Steps:

  • The techniques and skills will be applied to NapoleonB shortly.
February 7

2.4.2019- Corrections of Original Annotation

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Rationale: In lab today, feedback surrounding annotations from Wednesday was given. Therefore, it was found to be pertinent to correct mistakes that had been made, and additional processing techniques, such as HHPred, were learned and utilized.

Tools:

  • DNA Master
  • HHPred
  • NCBI BLAST
  • PhagesDB BLAST

Procedure:

  • Genes 50 and 51 of Elesar were re-annotated, with the LO and RBS categories being fixed. SIF-HHPred and SIF-BLAST was also ran for both genes.

Results:

Photos show results of gene 50 and 51, respectively.

Gene 50:

SSC: 36095,36970 CP: Yes SCS: Both BLAST-Start: Nandita, gp50, NCBI, Query 1 to Subject 1, 99%, 0.0. Gap: 40. LO: No RBS: Kibler7,Kibler Medium, 2.097, -4.366, Yes. SIF- BLAST: DNA Polymerase, Nandita gp50, AYN58673.1, 96%, 0.0 SIF- HHPred: DNA Polymerase III Beta, pfam, PF02767.16, 99.48, 5.8e-16

Gene 51:

SSC: 36967,37350 CP: Yes SCS: Both. BLAST-Start: Auxilium, gp 51, NCBI, Query 1 to Subject 1, 96%, 4e-14. Gap: 4bp overlap. LO: N/A RBS: Kibler7, Kibler Medium, 2.496, -3.662, Yes. SIF- BLAST: NKF. SIF- HHPred: NKF.

Conclusions: Since all evidence for the first gene points to one specific function, it can be called that the function of gene 50 of Elesar is to code for DNA Polymerase III. No such call can be made for gene 51, as only a hypothetical protein was called for this sequence, with no substantial support from the SIF-HHPred.

Next Steps:

The next step that will happen is that skills that have been developed through the past few labs will begin to be applied to NapoleonB. The only other skill that needs to be developed is the use of starterator, which will occur at a later date.

 

January 30

1.30.19 Annotating Elesar Genes

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1.30.19 Annotating Elesar Genes

Rationale: Today, two genes were annotated from the Elesar genome to begin practicing the skill of completely annotating a gene from a bacteriophage genome.

Procedure:

  • Opened FASTA file with previous example annotations on Gene #1
  • Answered Gap, LO, and RBS.
  • Assigned genes 50&51.
  • Annotated genes 50&51 from the Elesar genome.

Results:

 

Gene 50

SSC (Start/Stop Coordinates): 36095,36970

CP (Coding Potential): Yes

SCS (Start-choice source): Both.

BLAST-Start: Nandita, gp50, NCBI, Query 1 to Subject 1, 99%, 0.0.

Gap: 40.

LO (Longest ORF): Yes.

RBS (Ribosome Binding Site): Kibler7,Kibler Medium, 2.097, -4.366, Yes.

Gene 51

SSC: 36967,37350
CP: Yes
SCS: Both.
BLAST-Start: Auxilium, gp 51, NCBI, Query 1 to Subject 1, 96%, 4e-14.

Gap: 4bp overlap.

LO: N/A

RBS: Kibler7, Kibler Medium, 2.496, -3.662, Yes.

Conclusions: After performing the annotations for both gene 50 and 51, new skills were developed and a slightly better understanding of gene annotation was achieved. A complete understanding of the process has yet to be obtained, but as more genes are practiced, it will hopefully become more clear in regards to what to do for each step.

Future Steps: In the future, more genes will be practiced on Elesar to develop additional skills, but NapoleonB will be annotated eventually when confidence in the skills has been developed.

January 28

1.28.19 BLAST Analysis

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1.28.19 BLAST Analysis

Rationale: The next step in learning how to accurately and scientifically annotate a genome was considered to be learning BLAST analysis techniques. Therefore, this laboratory session was dedicated to practicing BLAST analysis using the Elesar phage genome.

Procedure:

  • Opened DNA Master
  • Opened FASTA File Elesar
  • Export –> Create Sequence from this entry only
  • Genome –> Auto Annotate –> Annotate
  • Copied Gene 5 protein sequence and inputted to NCBI BLAST database
  • Analyzed Results

Results:

[Elesar, 5, NCBI, Query 1 to Subject 1, 99%, 0.0, terminase large subunit]

Conclusions: Since the query had a 99% coverage and an e value of 0.0, it was concluded that Gene 5 of Elesar was likely coding for the terminase large subunit. This is backed by BLAST evidence, as 97% of the amino acids were a direct match to the NCBI sequence and 98% of the amino acids could be considered positive.

Future Plans: In the next lab session, the full annotation process will be assembled and put together. At this point, it will be possible to practice annotations on Elesar, and finally later on NapoleonB.

January 24

1.23.19 Introduction to Annotation

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1.23.19 Introduction to Annotation

Rationale: Today’s procedure was necessary to continue our understanding of DNA Master and gene annotation. Therefore, participating in the lecture, tweaking our DNA Master settings, and working through the QTM worksheet were prudent steps to being introduced to annotation.

Procedure:

  • Opened fasta file “Elesar” on DNA master
  • Adjusted template to be inserted during autoannotation
  • Auto-annotated Elesar genome
  • Used DNA–> Frames –> ORFs to identify all Open Reading Frames

Results:

Open reading frames of Elesar were found and briefly examined. Manual annotation of the genome has yet to be learned, so no further progress or understanding was achieved during this lab session.

Conclusion:

Since it was found to be possible to set up the DNA Master program for Open Reading Frame use, it will later be possible to find the Open Reading Frames on NapoleonB. This will be helpful as the genome is being processed and analyzed.

Future Plans:

Skills and lessons learned through the lecture, DNA Master steps, and QTM will later be helpful in analyzing NapoleonB, which will occur at a later date.

January 18

1.16.18 Starting DNA Master

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1.16.18 Starting DNA Master

Rationale: In Wednesday’s lab session (after the presentations), it was found to be important to set up DNA master and practice the procedure for auto-annotating bacteriophage genomes.

Tools/Procedure: In Wednesday’s lab, only DNA master and its auto-annotation feature was used.

Results: The genome for Elesar was successfully auto-annotated. The program had already been downloaded before the lab had begun and the steps for preparing the program had already been completed. The genome was downloaded from phagesdb.org, then it was auto-annotated. This was all that time permitted during the time in the lab.

Conclusion: The result of the lab is that I successfully learned how to auto-annotate a genome after downloading on phagesdb.org. There were no difficult decisions to be made, as the genome was not processed fully or annotated further than the auto-annotation.

Future Work: In the following labs, I plan to use the auto-annotation technique on NapoleonB to process that genome. I also plan to build on that basis by learning more about manually editing the auto-annotation. This will ideally permit a good understanding of the phage’s genome and genes after using DNA master on the genome.

November 29

11.28.18 Finishing DNA Extraction

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11.28.18 Finishing DNA Extraction

Rationale: Since a pellet had been obtained through the first part of DNA extraction, it was possible to finish the process of DNA extraction. Therefore, the next logical step was found to be the second half of the procedure that ended with measuring the sample using the nanodrop.

Procedure:

  1. 0.5mL sterile water added to thawed phage pellet
  2. Pellet was resuspended
  3. 2mL of resin was added to the pellet, then the tube was inverted many times to mix
  4. Resin and re-suspended phage pellet was added to two distinct microcentrifuge tubes (split evenly)
  5. Tubes were centrifuged at 12,500g for 3 minutes
  6. Supernatant was removed with pipette and placed in waste
  7. 1mL of 80% isopropanol was added to each microcentrifuge tube
  8. Steps 5-7 were repeated twice
  9. 1mL of 80% isopropanol was added to the pellet to re-suspend
  10. Contents of tubes were added to a column in a vacuum to move DNA to the filter
  11. Column was added to microcentrifuge tube, then centrifuged for 5 minutes at 12,000g
  12. Column was moved to new microcentrifuge tube
  13. 100µL of Elution Buffer (85 degrees Celsius) was added to column, then centrifuged for 1 minute at 12,000g
  14. DNA quantified on nanodrop
  15. Cleaned station

Observations

  • Pellet was initially very difficult to see, which led to doubt about whether or not DNA was strongly present
  • Nanodrop was very sensitive and difficult to place drop on platform without missing or creating air bubbles
  • Pellets after centrifuging formed more of a cloud shape rather than solid in bottom of tube, which made it more difficult to remove the supernatant clanly.

Results

  • The DNA concentration present was 65.69µg/µL. The 260/280 ratio was 1.89, and 260/230 was 0.16.

Conclusions

  • Since the concentration of DNA was above 50, the sample theoretically has a chance to work using the PCR procedure to cluster the phages. The ideal number would have been closer to 500µg/µL, but the sample should still be able to proceed. Furthermore, the ratios found in the results section illustrate that the sample is relatively pure and that the isolation was done reasonably accurately, as the 260/280 ratio should have been close to 2, and it was. The next step will be to process this sample using PCR clustering to see whether or not the procedure will work with the lower concentration and to determine specifics about the phage’s DNA.