d’Herelle first discovered bacteriophages when he observed plaques in the bacterial cultures from sick locusts he had grown, however he was unable to reproduce this, and it was unpursued for a few years. He was able to eventually study bacteriophages when he was studying an outbreak of dysentery in the troops at Maisons-Laffitte. While studying the bacteria the clear spots or plaques once again appeared and d’Herelle was able to observe and study the cause. When he noticed that the plaques only appeared in the samples from patients that were recovering he started to study the possibility of using these bacteriophages as they would be called to help with the recovery of patients with dysentery and other bacterial infections or in other words phage therapy. d’Herelle’s discovery was aided not only by his initial observation when he was studying locusts, but also by the ability to study many samples brought by outbreaks of dysentery often found in troops at the time.

One of the major characteristics that lead to d’Herelle becoming a successful scientist were probably the fact that he was not afraid of criticism, his dedication to his discovery, and that he was outspoken when it came to his discovery. His discovery while accepted by some was challenged by others. If he had been afraid of controversy or had not been as outspoken and willing to defend his discovery when it was challenged, then then the study of phage therapy may not have gone as far or maybe not anywhere at all. He was also very dedicated to the idea of phage therapy and he performed many studies on the effectiveness of this procedure. If he had left more of this to other scientist, it’s likely that it would not have gone as far as the success of his studies are what help spark the interest of other scientists to also perform experiments on phage therapy.

One major way that politics influenced was in Russia. Lenin’s push for public health helped Eliava establish a lab where he could further study bacteriophages and phage therapy. d’Herelle on several occasions also traveled there to assist Eliava which further pushed the study of phage therapy. However, the politics in Russia also halted Eliava’s studies in Russia as well as his and d’Herells’ cooperation when those who had been educated before the revolution including Eliava began being arrested and executed by Stalin. I this way specifically using Russia as an example of how politics helped push the research of phage therapy as well as halted it in Russia.

I major reason was that the results of phage therapy were often inconclusive. This was because scientists often did not have a control to compare their results against. This cause them to be unable to tell the reason behind patients’ recovery especially when more than one procedure was used. One example that was mentioned was that patients with boils would often have them lacerated and cleaned before the phage were added. Without a control it was impossible for anyone to tell whether a patient’s recovery was the laceration of the boils, the phage therapy, or both. A major reason that control groups were neglected was because the scientists often did not wish to deny patients something that could help them of save their lives. This mindset is definitely understandable, however this prevented the successfulness of phage therapy from being confirmed and it was eventually abandoned.

2/15/19

Rational:

To annotate genes 11 and 12 for NapoleonB and enter them in PhageNotes

Procedure:

• Dowloaded the fastA and auto-annotated NapoleonB
• Entered the gene call after genes 11, 12 and 98 in the home tab for PhageNotes
• Opened the genemark for NapleonB
• Checked to see if the coding potential was covered by the current start and that it was the ongest ORF
• Entered the start and stop codon
• Entered who the start was call and chosen by
• Checked phagesdb to see whether the gene’s start matches with the most called start (starterator)
• BLASTed genes 11 and 12 through NBCI and Phagesdb
• Ran the genes through HHpred
• Entered the required information for the NBCI and phagesdb hit in PhageNotes
• Calculated the gap/overlap for the gene and its upstream gene
• Selected the gene to get the information needed for RBS
• Entered the information for the hits in NBCI (BLAST and CDD), Phagesdb, and HHpred for the function
• BLASTed the surrounding genes and looked at Phamarator to look for for SIF-Syn

Observations:

• The genemark showed that the current start does not cover the coding potential for gene 11, but it is the LORF
• The coding potential is covered for gene 12 and it was the LORF
• Both genes were LORF
• Gene 12 had a gap of more than 10 bp
• Genes 11 and 12 both agreed with the starterator
• For gene 11 the NBCI hit was Arcadia and phagesdb was Xenomorph
• For gene 12 the NBCI hit was Arcadia and phagesdb was Xenomorph
• Gene 11 had a overlap of 17 and gene 12 had an gap of 19
• For both genes the SD score for the chosen start was not the best
• The funtion for genes 11 and 12 is capsid maturation protease, however this was only supported by the NBCI BLAST for both genes

Fig.3 – The teal block shows gene 11 and the yellow and green blocks representing genes 10 and 12 for the phage KeanyLin do not show signs of synteny as they do not match the function of the genes 10 and 12 in NapoleonB.

Conclusion:
The genes 11 and 12 were finished and their function was found to likely be a a capsid maturation protease for both. Note the only source that supported this was the NBCI BLAST the Phagesdb, Phamerator, and HHpred did not. Next lab I will finish the annotation of  98 and help check any mistakes made.

2/11/19

Rational:

To start annotating the assigned genes 9-12 and 98 for NapoleonB and enter them in PhageNotes

Procedure:

• Dowloaded the fastA and auto-annotated NapoleonB
• Entered the gene call after genes 9 and 10 in the home tab for PhageNotes
• Downloaded the genemark for NapleonB
• Checked to see if the coding potential was covered by the current start and that it was the ongest ORF
• Entered the start and stop codon
• Entered who the start was call and chosen by
• Checked phagesdb to see whether the gene’s start matches with the most called start (starterator)
• BLASTed genes 9 and 10 through NBCI and Phagesdb
• Ran the genes through HHpred
• Entered the required information for the NBCI and phagesdb hit in PhageNotes
• Calculated the gap/overlap for the gene and its upstream gene
• Selected the gene to get the information needed for RBS
• Entered the information for the hits in NBCI (BLAST and CDD), Phagesdb, and HHpred for the function
• BLASTed the surrounding genes to look for for SIF-Syn

Observations:

• The genemark showed that the current start covers all the coding potential for genes 9 and 10
• Both genes were LORF
• Gene 9 had a gap of more than 10 bp
• Genes 9 and 10 both agreed with the starterator
• For gene 9 the NBCI hit was Arcadia and phagesdb was Tribby
• For gene 10 the NBCI hit was Mudcat and phagesdb was Xenomorph
• Gene 9 had a gap of 14 and gene 10 had an overlap of 4
• For both genes the SD score for the chosen start was the best
• The funtion for gene 9 was portal protein and gene 10 was hydrolase

Conclusion:

The genes 9 and 10 were finished and there function were found to likely be a portal protein and a hydrolase repectively. Note the only source that supported gene 10 as a hydrolase was a more specific type of hydrolase. Next lab I will finish the annotation of 11, 12, and 98.

2/6/19

Rational:

To enter the annotations made on genes 20 and 21 for Elesar on PhageNotes in order to share it with the rest of the class. This makes it easier and faster to annotate the entire genome of Elesar. Also to check the annotations of two other genes.

Procedure:

• Entered my name next to the genes I annotated in PhageNotes and added the auto-annotation calls
• Entered the information for SSC, CP, SCS, LO, BLAST-Start (NBCI), Gap, F, SIF-BLAST (NBCI), SIF-HHPRED, and SIF-Syn
• BLASTed genes 20 and 21 in phagesdb and entered the needed information
• For RBS entered only the SD final score, whether it was the best, and the z-score
• Used the BLAST for phagesdb to predict the function of the genes
• Checked genes 8 and 9 by annotating them myself and comparing my annotations with the annotation entered

Observations:

• Information for BLAST-Start and SIF-BLAST using phagesdb was needed
• For gene 9 the start codon was moved back, but the SCS did not support this change
• The rest of the annotations for genes 8 and 9 seemed correct

Fig.2 – This image shows the information found on gene 9 from phagesdb. The information matches the annotations entered in PhageNotes.

Conclusion:

The annotations for 20 and 21 were finalized and entered into PhageNotes. Genes 8 and 9 were also checked for mistakes in the annotations. Next lab I will start annotating NapoleonB along with the rest of the class.

2/4/19

Rational:

To finish the annotaion of genes 20 and 21 in order to continue the annotation of Elesar’s genome. Identify the function of the gene by comparing the functions of other proteins the genes were matched up with.

Procedure:

• First BLASTed gene 20 and found the function of the gene that was the closest match
• Opened HHpred
• For selected databases deleted existing database and entered COG_KOG_v1.0 and Pfam-A_v32.0
• Entered the information about the protein the matched including the function
• Looked for synteny by looking at the possible function of the surrounding genes to look for genes with similar functions
• Entered the predicted funtion of the gene
• Did the same for gene 21 as gene 20

Observations

• The BLAST of gene 20 showed that the possible function of the gene could be a tail protein
• HHpred also indicated that gene 20 was a phage tail protein
• The BLAST of gene 21 indicated it was a minor tail protein
• HHpred showed no known function for gene 21
• Both genes 20 and 21 are likely minor tail proteins

Fig.1 – This image shows the hits for gene 20 that were found using HHpred. The top bar is the protein that matches most closely to gene 20.

Conclusion:

The last of the annotations for gene 20 and 21 were finished and entered into the notes section of the gene in DNA Master. The function of these genes was also predicted using BLAST, HHpred, and synteny. Next lab I will annotate other genes in the same way that genes 20 and 21 were annotated.

1/30/19

Rational:

To continue annotation and learning how annotate gene 1. Annotated what has been learned thus far for gene 20 and 21.

Procedure:

• Opend the auto-annotated Elesar
• Calculated the gap/overlap between the upstream gene from gene 1
• Determined whether the gene was the longest ORF
• Selected the gene and pressed the RBS button to get the Z-score, final score and determine whether the current ORF is the best according to the RBS
• For gene 20 and 21 found the SSC
• Looked at GeneMark to determine if the gene covers the coding potential
• Found the longest ORF
• BLASTed the proteins for gene 20 and 21
• Wrote down the name of the phage name that aligned with the gene and the gene number
• Wrote what database the alignment was found in
• Determined what basepair the query and the subject started matching at
• Found the e-value for the match (shows the likelihood the alignment was random)

Observations:

• Gene 1 is the first gene so in gap 1st gene was written
• The gene was pushed back to an earlier start making it the longest ORF
• Entered the scoring and spacing matrix under the RBS (Kibler7 and Karlin Medium)
• The Z-score was not the best for the current start, but it was determined that it was still the best
• Both alignments for gene 20 and 21 were from NBCI

Conclusion:

The annotations up to RBS were finished for gene 1. Genes 20 and 21 were then also annotated up to the same point. Next lab I will finish learning how to do the rest of the annotations steps and practice on gene 20 and 21.

1/28/19

Rational:

To learn what information to put down and where to find it in order to annotate individual genes.

Procedure:

• Opened the auto-annotation for Elesar
• Put the start of gene one back in order to cover the coding potential and have the longest ORF
• Wrote the start and stop coordinates for gene one
• Used the GeneMark analysis of Elesar to determine whether the current start of the gene covers the coding potential
• Wrote who the start codon was called by as well as who it was selected by
• BLASTed the protein code for gene one to look for significant alignments with other genes

Observations:

• The start codon was called by both GeneMark and Glimmer, but called by me
• The BLAST results showed no significant BLAST alignments
• Under the new start all of the coding potential was covered

Conclusion:

The annotations for SSC (start/stop coordinates), CP (coding potential), SCS (start choice source), and BLAST-start were determined and written in the template in the notes section for the gene. Next lab I will learn how to do the rest of the annotations and continue to annotate gene 1 and other genes.

1/23/19

Rational:

To further annotate the genome of Elesar and starting to check for mistakes in the auto-annotation as well as learn how to calculate gaps and overlaps between genes.

Procedure:

• Clicked preferences under files
• Clicked local setting and new features
• Added SSC: CP: SCS: ST: BLAST-Start: Gap: LO: RBS: F: SIF-BLAST: SIF-HHPred: SIF-Syn into the template notes box
• Calculatted the gap and overlap between some example genes on the questions that matter worksheet
• Calculated the gap by finding the difference between the end of one gene and the start of the next minus one
• Calculated the overlap by finding the difference between the end of one gene and the start of the next plus one

Observations:

• Some of the foward and reverse genes overlap or are too close to be correct (22/24, 24/25, 32/33, and 55/56 are examples of this)

Results:

Some of the genes like 22 (which is reverse) and 24 (forward) are too close and indicate a possible mistake in the auto-annotation. Next lab I will calculate the gap and overlap between genes (especially between forward and reverse genes).

1/16/19

Rational:

To auto-annotate Elesar’s genome in order to practice for the annotation of the phage found in class.

Procedure:

• Downloaded the fastA for Elesar phage
• Opened fast file by clicking file open and fastA multi sequence file
• Clicked export and created a sequence from only that entry
• Clicked genome at the very top of the screen and clicked auto-annotate
• Clicked features in the fastA library to see the geneome info
• Clicked DNA and frames to see ORF analysis
• Clicked ORF button to compare potential start codons
• On fastA library clicked genome and profile
• To create ORF map clicked DNA and export map then draw

Conclusion:

The genome of Elesar was annotated. Next lab I will use the steps taken to annotate Elesar to annotate the phage found in class.

11/28/18

Rational:

To do DNA extraction on lysate 8 in order to obtain a sample of DNA from the phage found in lysate 8.

Procedure:

• Cleaned lab desk
• Added 500 ML of sterile water to phage precipitate
• Added 2 mL of DNA clean up resin and mixed
• Put the mixture into two centrifuge tubes and spun at 12,500 G for 3 min
• Pulled off the supernatent
• Added 1 mL of 80% isopropenol and spun at 12,500 G for 3 min
• Pulled off the supernatent
• Repeated the previous two steps two more times
• Added 1 mL of 80% isopropenol
• Put the solution in a column tube and filtered
• Centrifuged the filtered column at 12,000 G for 5 min
• Put the remaining solution in a new centrifuge tube and added 100 ML of 80 C elution buffer
• Centrifuged for 1 min at 12,000 G
• Ran the nanodrop on the sample

Observations:

• DNA concentration- 417.74 ng/ML

Conclution:

The nanodrop results showed the the DNA concentration in lysate 8 was 417.74 ng/ML. As there is not enough time nothing else will be done with this sample however, next I would have done PCR and gel electrophoresis on lysate 8 since it has a high titer.