Overall

I was surprised that the person who discovered bacteriophages lacked a formal college education and discovered them through sheer curiosity and persistence.

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

D’Herelle first noticed bacteriophages in locusts that were suffering from a bacterial infection. The outbreak of World War I put a temporary pause on the research of the locusts, but d’Herelle’s research came full circle when he noticed the same phenomenon in the feces of patients infected with dysentery as a result of the war (close quarters and limited supplies/hygiene led to the spread of the infection). The clear spots d’Herelle noticed were plaques similar to those we experienced in lab; the bacteriophages present in the bacterial cultures would kill off the bacteria and leave clear spots, which intrigued d’Herelle. However, he only noticed these clear spots/tubes in locusts/patients that were in recovery, so this led him to believe that the things in the bacteria that appeared clear helped cure the infections.

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 didn’t give up—he was largely doubted by his colleagues, but he persisted nonetheless. Even with publications criticizing him, he remained steadfast in his discoveries from his experiments. His curiosity drove him to continue on in his research. Eliava was similar to d’Herelle in disposition, however Eliava completed a medical degree and formal laboratory training. They both came from wealthy backgrounds, however Eliava lived in Georgia, giving him connections to anti-Soviet people of power. This eventually led to his arrest by the secret police and to his and his wife’s murders when the Soviets purged the intellectual and rich.

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

Like the Cold War generated competition for space travel, World War I created competition between the Soviet Union and the United States for scientific discovery and healthcare. The conditions brought about by the war led to outbreaks of disease from lack of food, close quarters, stress, etc. Thus, it was imperative for each nation to come up with a way to treat its people/troops. The aforementioned rivalry between the two nations led to each experimenting with phage therapy as an easy and cost-effective way to treat infections within their populations.

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

The development of antibiotics overtook the usage of phage therapy; antibiotics were easier to reproduce positive results with, as their genomes did not change and were able to be mass-produced. Additionally, d’Herelle was arguably the largest proponent of phage therapy in the world at this time, and he removed himself from the limelight after the death of Eliava, thereby decreasing the spread of phage therapy because no one was actively promoting it.

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?

Delbruck and Luria created the Phage Group, which united scientists from different backgrounds and fields to come together and study the importance of phages and phage therapy. They generated more interest in the field for a short time and figured out how phage replicated. Unfortunately, they only researched 7 phages, so the spark of interest in the field degenerated fairly quickly.

 13 February 2019 ✷ NapoleonB DNA Annotation pt 2

Procedure

• DNA master was opened and NapoleonB was auto-annotated
• PhageNotes was opened and each aspect of the DNA annotations for genes 22-24 was entered into the program and genes 22-24 and 97 were annotated to completion.
• Blasts for products (protein) were run on PhagesDB, HHPred, and NCBI to ensure accurate and up-to-date blast information.
• Phamerator was opened for AM phages and the synteny of NapoleonB was analyzed compared to genomes of phages in the AM group.
• From PhagesDB, starterator for each gene was opened and compared and all genes annotated agreed with starterator.
• The information was recorded and saved in the google sheet.

Results

the complete annotations for genes 22-24 and 97 as entered into PhageNotes, are as follows:

Conclusion

In genes 22 and 23, there was uncertainty in the function of the gene. The databases couldn’t agree or provide a function, thus it was decided that the function was unknown, or NKF.

Future plans

The annotation of NapoleonB’s genome will be completed, checked, and submitted to phagesdb. The information gathered from the annotation of the genome will lead to the creation of a research question and thus further research into phage biology involving NapoleonB.

 11 February 2019 ✷ NapoleonB DNA Annotation!

Procedure

• PhagesDB was opened and the FastA file for NapoleonB was downloaded
• DNA master was opened and NapoleonB was auto-annotated
• PhageNotes was opened and each aspect of the DNA annotations for genes 21-24 was entered into the program, however, only 21 was annotated to completion and 22 was nearly completed.
• Blasts for products (protein) were run on PhagesDB, HHPred, and NCBI to ensure accurate and up-to-date blast information.
• Phamerator was opened for AM phages and the synteny of NapoleonB was analyzed compared to genomes of phages in the AM group.
• From PhagesDB, starterator for each gene was opened and compared (this was only done for genes 21 and 22 so far) and both agreed with starterator.
• The information was recorded and saved in the google sheet.

Results

the complete annotation for gene 21 and the partial annotation for gene 22, as entered into PhageNotes, are as follows:

Conclusion

There was some confusion regarding the function of gene 22 of NapoleonB, and time was limited so the annotation wasn’t completed. Further research needs to be completed in order to make an informed decision on the final function of gene 22 because the databases do not agree on the function–PhagesDB and NCBI say it aligns with a tail protein, but HHPred and the conserved domain information both suggest an unknown function related to botox.

Future plans

The function of gene 22 will be further analyzed and researched and the annotation will be finalized. Then, genes 23, 24, and 97 will be annotated.

6 February 2019 ✷ DNA Annotation (cont.)

Procedure

• DNA master was opened and Elesar was auto-annotated
• Phagenotes was opened and each aspect of the DNA annotation for genes 2 and 3 were entered into the program; blasts were run again to ensure accurate and up-to-date information.
• Genes 2 and 3 were updated and annotations were checked by another student to verify accurate information.
• Genes 18 and 19 were also checked in order to verify the quality and accuracy of annotations.

Results

the updated annotations for each gene, entered into phagenotes, are as follows:

Conclusion

Programs like PhageNotes are helpful in annotating genes because coding the annotations is a very specific process. Simple issues from incorrect formatting can result in an annotation being rejected, so becoming familiar with PhageNotes will help improve the quality of annotations.

Future plans

This same method will be applied to Napoleon B, a phage isolated in 2018, in order to analyze its genome.

4 February 2019 ✷ DNA Annotation

Procedure

• The FASTa file for Elesar was opened and auto-annotated.
• The “features” tab was opened and within the “notes” box, the code for genes 2 and 3 were annotated
  • errors from the prior lab were corrected; for gene 2, the “blast” command was corrected (the phage name and protein number were combined to fix the error). For gene 3, the “blast” command was corrected by swapping the order of the % match and the ratio, RBS was redone, and CP was adjusted from “yes” to “no” because it was incorrect originally.
• The SIF-BLAST and SIF-HHPred commands were annotated:
  • The product codes were run through phagesdb blast and HHPred (+ COG and PFam databases) and that information was added to the annotation as indicated by NCBI.
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• PhagesDB results:
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Results

the updated annotations for each gene are as follows:

Gene 2

SSC:[346,690] CP:[yes] SCS:[both] ST: BLAST-Start:[Andrew_1, phagesdb, 1:1, 62%, 3e-17] Gap:[8bp overlap] LO:[no] RBS:[Kibler7, Karlin Meduim, 3.167, -2.177, no] F:[] SIF-BLAST:[terminase large sububit, phagesdb, Andrew_1, MH834595, 62%, 3e-17] SIF-HHPred:[Phage Terminase, PFam, n/a, PF03354.15, 95%, 98.68] SIF-Syn:[]

Gene 3

SSC:[954,1352] CP:[no] SCS:[Glimmer] ST: BLAST-Start:[Ryan_4, phagesdb, 1:1, 100%, 6e-70] Gap:[263bp gap] LO:[yes] RBS:[Kibler7, Karlin Medium, 2.853, -3.115, no] F:[] SIF-BLAST:[DNA binding protein, phagesdb, Ryan_4, MH834627, 100%, 6e-70] SIF-HHPred:[Transposase, COG, n/a, COG4584, 95%, 98.75] SIF-Syn:[]

Conclusion

Annotation is useful in research in comparing different genes within differing species that potentially contain the same gene. Determining the function of genes based on other’s research and recorded data is useful in speeding up the process of studying a genome.

Future plans

This same method will be applied to Napoleon B, a phage isolated in 2018, in order to analyze its genome.

30 January 2019 ✷ DNA Master + BLAST

Procedure

• The FASTa file for Elesar was opened and auto-annotated.
• The “features” tab was opened and within the “notes” box, the code for genes 2 and 3 were annotated.
• The “frame” was pulled up and the ORF was viewed to see if it could be elongated. The start and stop coordinates and gap/overlap were looked at.
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• The genes were each blasted with phagesdb in order to find a similar gene. This information was included in the annotation.
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Results

the annotations for each gene are as follows:

Gene 2

SSC:[346,690] CP:[yes] SCS:[both] ST: BLAST-Start:[Andrew, 1, phagesdb, 1:1, 62%, 3e-17 ] Gap:[8bp overlap] LO:[no] RBS:[Kibler7, Karlin Meduim, 3.167, -2.177, no]

Gene 3

SSC:[954,1352] CP:[yes] SCS:[both] ST: BLAST-Start:[Ryan_4, phagesdb, 1:1, 6e-70, 100%] Gap:[263bp gap] LO:[yes] RBS:[Kibler7, Karlin Medium, 2.573, -3.709, no]

Conclusion

Annotation is useful in research in comparing different genes within differing species that potentially contain the same gene. Practicing this specific skill will be important because the process was somewhat confusing, but it is surely an important method for genetic research.

Future plans

This same method will be applied to Napoleon B, a phage isolated in 2018, in order to analyze its genome.

28 January 2019 ✷ DNA Master + BLAST

Procedure

• The FASTa file for Elesar was opened and autoannotated.
• The protein sequence for gene 1 was opened and copied and pasted into the BLASTp tool on NCBI’s website and run to find matching sequences,

Results

Conclusion

The NCBI BLASTp result was not very effective in locating a similar gene for Elesar Gene 1. This just means that similar genes are not stored in the database as of yet. The E value is really high for each of the results and this means that it is very likely the results are random. The identity percentages are low as well and thus the protein sequences are not very closely related.

Future plans

This same method will be applied to Napoleon B, a phage isolated in 2018, in order to analyze its genome.

23 January 2019 ✷ DNA Master Annotation

Procedure

• DNA Master was  opened and preferences was opened > local settings > new feature
  • checked “insert template into notes during annotation” and pasted “SSC: CP: SCS: ST: BLAST-Start: Gap: LO: RBS: F: SIF-BLAST: SIF-HHPred: SIF-Syn” into box
• Opened Elesar.fasta and auto-annotated it

Results

Conclusion

DNA master is a useful tool in which DNA can be analyzed and genes may be characterized. The processes and the program as a whole are still a bit confusing, but the overall idea of it is logical.

Future plans

This same method will be applied to Napoleon B, a phage isolated in 2018, in order to analyze its genome.

16 January 2019 ✷ DNA Master

Rationale: DNA Master was downloaded onto personal computers in order to practice annotating DNA.

Procedure

• Elesar, a previously discovered phage within PhagesDB, had a recorded DNA sequence file that was downloaded and then annotated through DNA master.
• The preferences on DNA Master were updated to use different colors and base pairs that were to be included were updated; the program was also given permission to use the HHMI database.
• The FastA file for Elesar was opened and run through the program, producing the images seen below.

Observation, Results, Data

The genome of Elesar is larger than I had imagined, given the small size of a bacteriophage. The sheer length of its genetic code is impressive to see written out as it is in DNA master.

Interpretations, Conclusion, Next Steps

In the following lab periods, the genome of phages discovered within the 2018-19 cohort of Baylor Univerity’s SEAPHAGES group will be analyzed and annotated.

28 November 2018 ✷ Pick a Different Plaque

Rationale: Members of group 6 adopted phage from Soil 4 from Melissa (collected 10/5/18) in order to help amplify the phage sample. A plaque was picked from Melissa’s original plate because the prior plates run by members of group 6 came up negative.

Procedure

• The table was cleaned with Cidecon and 70% ethanol and an ethanol lamp was lit.
• Plaque “Y” on the plate from the prior lab session was picked and swirled into 50 µL of phage buffer. 30 µL of this mixture was added to 0.5 mL of arthrobacter and allowed to sit for 15 minutes while the plates were made.
• A plate for 2 plaque assays and a control plate were made with the following concentrations and volumes:

• component	volume	final concentration
  LB Broth	6 mL	–
  2X Top Agar	7.5 mL	1X
  1M Calcium Chloride	68 µL	4.5 µM

• The arthro/lysate mix was combined with 5 mL of the above plate mix and the plate was poured, allowed to harden, and then inverted and incubated at 37 degrees celsius. The remaining lysate was refrigerated.

Observation, Results, Data

The originally positive plaque assay was picked and plated but ended up with negative plaque assays that had weak spots that mimicked plaques, but weren’t actually plaques.

 The spots, when looked at under the microscope, weren’t circular nor clear, as true plaques should be. Thus, when Melissa’s initial plate was looked at again under the microscope, the plaques present were more easily identifiable and were able to be picked and plated.

Interpretations, Conclusion, Next Steps

In the following lab periods, the presence of a webbed plate will mean that the lysate is a high titer and can be studied more closely using TEM. The current titer is very low, which may indicate a weaker phage or phage presence.