Rationale: 

The purpose of today’s lab was to take all of the tools given to us in our bioinformatics toolkit and finally apply them to the annotation of NapoleonB.

Tools:

• DNA Master
• NCBI BLASTp
• HHPred
• Phamerator
• Starterator
• PhagesDB BLASTp
• Genemark (pdf)

Procedure:

• Lab began with a review of all the tools in the bioinformatics toolkit.
• Genes for NapoleonB were split up and assigned to be annotated.
• Genes 61-64 were assigned to me to be fully annotated.
• Ran genes 61 and 62 through a series of BLASTp searches in both NCBI and PhagesDB, searched for protein folding structure in HHpred, analyzed the starterator analysis through PhagesDB, and checked for any supporting information of synteny by examining similar phages in the AM cluster with NapoleonB.

Results:

• Final Annotation for Gene 61:
  • SSC:36690 – 37253, CP:Yes, SCS:BothGL, ST:SS, BLAST-Start:Aligns with Arthrobacter Phage Mudcat gp57 NCBI BLAST q2:s1 0.99 3E-17, Aligns with Mudcat gp57 PhagesDB BLAST q2:s1 0.99 1E-108, Gap:14bp overlap, LO:NA, RBS:Kibbler7 and Karlin Medium 3.118 -2.454 No, F:NKF, SIF-BLAST:NKF, SIF-HHPred:NKF, SIF-Syn:NKF
• BLASTp Results:
  • 
  • PhagesDB BLASTp Result matched with the same phage and gene as the BLASTp result
• HHpred Result:
  • 
• Phamerator Result (Top gene is NapoleonB in comparison to bottom gene from Nason):
  • 
• Final Annotation for Gene 62:
  • SSC:37322 – 41224, CP:Yes, SCS:BothGM, ST:SS, BLAST-Start:Aligns with Arthrobacter Phage KeaneyLin gp60 NCBI BLAST q1:s1 0.99 0, Aligns with KeaneyLin gp60 PhagesDB BLAST q1:s1 0.99 0, Gap:68bp gap, LO:Yes, RBS:Kibbler7 and Karlin Medium 2.905 -3.803 No, F:DNA primase/polymerase, SIF-BLAST:DNA Polymerase Supported by NCBI BLAST KeaneyLin gp60 AXH44198.1 0.99 0, DNA Polymerase Supported by PhagesDB BLAST KeaneyLin gp60 0.99 0, Phage or plasmid associated DNA primase supported by a CD found from the COG databse prophage gp COG3378 2.9E-13, , SIF-HHPred:DNA Polymerase Delta supported by KOG gp KOG0969 0.8194 , SIF-Syn:NKF

• BlastP Result:
  • 
  • PhagesDB BlastP result was identical
• HHpred Result:
  • 
• Phamerator Synteny (Mudcat Top, NapoleonB bottom):
  • 

Conclusions:

• For Gene 61:
  • Despite yielding a BLASTp result with high probability, there was not enough supporting information to help declare a function. Due to this, a function of NKF was called for this hypothetical protein. Calling the start was difficult, but decided to agree with the glimmer start to pull the gene back as far as possible to give the longest open reading frame.
• For Gene 62:
  • There was a significant amount of information to support the calling of a function for the protein examined. NCBI BLASTp and HHPred both gave the function as a DNA Primase/Polymerase, and the synteny from examining Arthrobacter Phage Mudcat seems to support the call. HHPred called a DNA Polymerase Delta found in Eukaryotic cells, which also supports the assumption that the structure of the protein coded in gene 62 is almost identical to DNA Polymerase Deltas found in eukaryotic cells.

Next Steps:

The next steps for this experiment are to continue with the annotation of NapoleonB genes and finish the final two genes in the following lab.

Rationale:

The purpose of today’s lab was to go over the use of Phamerater and tRNA searching to add on to the toolkit used for annotation. Also genes were assigned for us to check to further practice our annotation skills.

Tools:

• DNA Master
• Phamerator
• Aragorn
• PhageNotes

Procedure:

• Aragorn and tRNAscan-SE were introduced to practice searching for tRNA folding in phage annotation.
• Phamerator accounts were set up to compare annotated genomes of phages in the same cluster as phage Elesar.
• Genes 30 and 31 were revised by checking the Starterator and Synteny regions of annotation notes.
• Was assigned genes 4 and 5 to check over to see if annotations were correct, so genes were annotated fully to check for any errors.

Results:

• Final Annotation Notes for Gene 4:
  • SSC:1349, 1984, CP:Yes, SCS:Both, ST:SS, BLAST-Start:Aligns with Nandita gp5 NCBI BLAST q1:s1 0.95 3E-122, Aligns with Nandita gp5 PhagesDB BLAST q1:s1 0.95 3E-122, Gap:overlap 4, LO:, RBS:Kibbler7 and Karlin Medium 2.23, -4.085 ,no, F:terminase, small subunit, SIF-BLAST:terminase small subunit Supported by NCBI BLAST Nandita gp5 AYN58627 0.95 3E-122, terminase small subunit Supported by PhagesDB BLAST Nandita gp5 0.95 3E-122, supported by a CD found from the databse gp , , SIF-HHPred:terminase small subunit, COG, prophage, gpNA, COG3747, 0.75, 99.89, SIF-Syn:
  • 
  • 
• Final Annotation Notes for Gene 5:
  • SSC:1971 ,3668, CP:Yes SCS:Both, ST:SS, BLAST-Start:Nandita, gp6, NCBI BLAST, q4:s5 0.99 0, Aligns with Nandita gp6 PhagesDB BLAST q4:s5 0.99 0, Gap:13bp overlap, LO:Yes, RBS:Kibbler7 and Karlin Medium 1.724 -5.684 No, F:terminase, large subunit, SIF-BLAST:terminase large subunit Supported by NCBI BLAST Nandita gp6 AYN58628 0.99 0, terminase large subunit Supported by PhagesDB BLAST Nandita gp6 0.99 0, supported by a CD found from the databse gp , , SIF-HHPred:terminase large subunit supported by Terminase_1 gpNA PF03354.15 0.87 99.8, SIF-Syn: 
  • 

Conclusions:

The main proteins called by both NCBI BLASTp and HHpred seemed to call both small and large subunit phage terminases in genes 4 and 5 respectively. It can be assumed based off the supporting data that these are the proteins coded by genes 4 and 5 as both probabilities were above 90 percent in both cases.

Next Steps:

The next steps for this experiment are to practice searching for synteny within the genome and also practice further with annotating genes. Now that phamerator have been introduced, gene comparison between phage Elesar and similar phages in the cluster.

Rationale:

The purpose of today’s lab was to go over and correct the previous annotations of Elesar genes 30 and 31, and practice using additional methods of protein sequence analysis.

Tools:

• DNA Master
• HHPred
• NCBI BLAST and BLASTp

Procedure:

• Began the lab with information regarding HHPred use and protein domains.
• Once complete, was given feedback regarding previous annotations of Elesar genes 30 and 31
• Updated gene annotations of genes 3o and 31 by adding SIF BLAST and SIF HHPred information regarding protein function.

Results:

• Updated Annotation Notes for Gene 30:
  • SSC:22118, 22477 CP:yes SCS:Both BLAST-Start: Nandita gp29, NCBI,  q1:s1, 78%, 9E-62 Gap:overlap 1bp LO: yes RBS: Kibler7, Karlin Medium, 2.408, -3.770, yes F:NKF SIF-BLAST:NKF, no putative conserved domains have been detected SIF-HHPred: NKF, no matches with a probability above 90% SIF-Syn
  • 
• Updated Annotation Notes for Gene 31:
  • SSC:22487, 23215 CP:yes SCS:Both ST: BLAST-Start:Ryan, gp30, NCBI, q1:s1, 83%, 2e-139   Gap:9 LO:yes RBS: Kibler7, Karlin Medium, 2.117, -4.325, yes F:NKF SIF-BLAST:NKF, no putative conserved domains have been detected  SIF-HHPred:NKF, no matches with a probability above 90% SIF-Syn
  • BLASTp Protein Domain results yielded no conserved domains
  • HHPred result yielded a viral coat protein hit, but the probability was too low to call.
  • 

Conclusions:

No known function could be determined from the information given from the BLASTp and HHPred results. The few hits that were given had incredibly low probabilities and did not contribute to function determination. Could be a possibility that there is very few data on this given gene, or that it actually does not have a function.

Next Steps:

The next steps are to check with annotated phage genomes in the same cluster and determine if they have a similar gene that codes for the same protein to help determine protein function. Also further annotation practice is required as Starterator and synteny have yet to be used in the annotation notes.

Rationale:

The purpose of today’s lab was to practice the actual process of manually annotating a gene of Arthrobacterphage Elesar using the guiding principles and annotation rules set by SEA-PHAGES.

Tools:

• DNA Master
• Genemark
• NCBI BLAST

Procedure: 

• Lab began with the reviewing the guiding rules of gene annotation as well as examining the requirements for notes written when manually annotating a gene.
• Once complete, Genemark was opened to understand the information given and how it is able to be applied to manual gene annotation.
• Began the manual annotation of Gene 1 of Arthrobacterphage Elesar.
• Gene 1 was BLASTed for the protein sequence

Results: 

• Final annotation notes for Gene 1
  • SSC:45,353
  • CP:yes
  • SCS:Both-CS
  • ST: BLAST-Start:no significant BLAST alignment
  • Gap:first gene
  • LO:yes
  • RBS: Kibler7, Karlin Medium, 1.222, -6.751, no
• BLASTp results for Gene 1
  • 
  • no significant hits were recorded for the protein sequence coded.

Conclusions:

The gene annotation did not begin at the longest ORF, so it was decided to move the start of the gene up to nucleotide 45, as Genemark noted there was coding potential beginning to that base pair. Unfortunately, no significant BLAST alignments were found for both the NCBI database and Phagesdb, meaning there is a possibility this protein can be something that has yet to be uploaded into any database used. Also, the RBS was not determined by DNA master to be the strongest site, but was still noted as any other recommended RBS would have shortened too much of the gene.

Next Steps: 

The next steps for this lab are to continue practicing manual annotation under the guiding principles reviewed in lab to further prepare for the annotation of Arthrobacterphage NapoleonB.

Title: 1/23/19 Annotation Intro

Rationale: 

The purpose of today’s lab was to be introduced to the process of gene annotation, finalize the set preferences for DNA master, and learn about analyzing information given in auto annotation with phage Elesar.

Tools: 

• DNA Master Auto Annotate Function
• FASTA file “Elesar”

Procedure:

• Opened DNA master and uploaded template “SSC: CP: SCS: ST: BLAST-Start: Gap: LO: RBS: F: SIF-BLAST: SIF-HHPred: SIF-Syn” to begin auto annotating Elesar.
• After uploading the template, the genome was auto annotated.
• The ORFs of the genome were examined and identified by using DNA –> Frames –> ORFs

Results

• The majority of the genome contains forward ORFs, with gene number 19 being among some of the longest.
• Auto annotation only revealed approximately 11 reverse ORFs, making up only 16.67% of the genome.

Conclusions:

The genome of Elesar’s lack of reverse ORFs can also indicate a lack of primers that begin coding in the reverse direction. Also, the size of the ORFs, regardless of direction, could have a correlation between the size of proteins produced and/or the number of proteins produced within that gene.

Next Steps:

Need to continue with the familiarization of DNA master so it can be fully utilized to manually annotate genomes. Further practice is needed to begin manually annotating NapoleonB and discovering which proteins are coded within the genome.

Rationale: 

The purpose of today’s lab was to present the phage therapy powerpoint and configure DNA Master and its preferences to begin annotation. FASTA file of phage “Elesar” was loaded and auto-annotated as well.

Tools Used:

• Computer/Laptop
• DNA Master Program

Procedure:

The lab began with presentations which covered an array of topics all focused on the area of phage therapy and the experiments associated with the practice. Once presentations were completed, the classroom discussed various methods to increase our ability to present things scientifically, and how to improve on being a public speaker overall. Once this exercise was complete, the lab was walked through the DNA Master installation process, and the FASTA file for phage Elesar was uploaded and auto annotated.

Observations:

• Several phage therapies, such as phage pVp-1 therapy with Vibrio parahaemolyticus, were successfully tested against animal patients and host to treat human illnesses.
• Enclosing phage used for treatment in liposomes allows the phage to survive longer in the patient’s body, providing a solution to the issue of the immune system of the patient attacking the foreign phage particles.
• The auto annotated results of phage Elesar yielded 66 genes with one RNA and 66 ORFs.
• 

Conclusions:

It can be concluded that the lab has a general knowledge of phage therapy, and what our research means in relation to that. Also, with DNA master installed, it now yields the opportunity to begin to annotate phage NapoleonB, to discover which genes code for which proteins, and the functions behind each of those coded proteins.

Next Steps:

The next steps for this research are to begin manually annotating the genome of Arthrobacterphage NapoleonB to discover the proteins coded by the genes and what the function of those proteins are.

Rationale:

The goal in lab today was to analyze the spot titer to calculate the titer of the lysate. If the titer calculated was high enough, then the beginning procedure for DNA extraction would begin. If not, then another flood and plaque assay would be run to continue amplifying the titer of the lysate.

Results from 11/14/18:

• Despite being set on the bottom rack of the incubator, which is at a different temperature than the rest of the racks, the spot titer yielded visible plaques up to the 10^-6 dilution. It had completely lysed the first few dilutions, but also contained contamination on the top agar control due to unknown contamination.
• 
• The 10^-6 dilution had approximately 5 plaque forming units (pfu) present, so that was used to calculate the titer of the lysate. Titer was calculated using the equation below.
• 
• Titer was 5*10^8 pfu/mL, which was a high titer lysate and DNA extraction could begin.

Materials:

• High Titer Lysate (HTL)
• Conical Vials
• Nuclease Mix
• Phage Precipitant Solution

Procedure:

1. Established an aseptic zone
2. Lysate had to be archived, and began with naming the phage “Kiersten”.
3. After naming, 2.8 mL of lysate was transferred to a conical vial for archiving.
4. 100 µL of lysate was aliquoted to a micro centrifuge tube for prepping a TEM grid next lab.
5. 10 mL of the HTL was then transferred to a separate 50 mL conical vial, and 40 µL of the nuclease mix was added to the HTL. It was then mixed gently to combine.
6. 4 mL of the phage precipitant solution was added as well, and mixed gently to combine.
7. The lysate solution was then left to sit and incubate at 37 degrees celsius for 3o minutes.
8. Vial was then weighed out and a conical vial was weighed with water to balance out in a centrifuge and the lysate solution was stored in the freezer for the next lab.

Results/Data:

• The lysate was prepped for DNA extraction, but no actual DNA extraction took place. With the high titer of lysate, the increased presence of phage particles will allow for a larger pellet to form, which will aid with DNA extraction.

Conclusions:

The prepping of the HTL allows for the pelleting of phage to occur once run through a centrifuge as the nuclease removes any remaining bacterial DNA floating in the lysate, and the phage precipitant solution pulls water molecules out of the lysate to aid pelleting.

Next Steps:

The next steps for this experiment are to prep a TEM grid for microscopy and pellet the phage particles.