Rationale: Annotate genes assigned on Arthrobacter NapoleonB

Materials:

• DNA Mastering
• PhagesDB Blast
• NCBI Blast
• Starterator
• NCBI Domains Website
• Computer

Procedure:

• Opened up DNA Mastering on computer
• Downloaded NapoleonB file from PhagesDB database
• Opened the file on DNA Mastering and auto-annotated
• Was assigned to annotate genes 37-40, placing results on NapoleonB PhageNotes
• After working on annotations, saved annotated NapoleonB file

Observations:

Genes of NapoleonB!

NCBI Blast results of gene 39

The unfinished annotations of genes 37-40 of Arthrobacter NapoleonB

Conclusion/Next Steps:

• It’s very exciting to finally start annotating Arthrobacter phage NapoleonB! Although the day was filled with confusion and a little but of chaos, the annotations are on their way to being finished. The next steps would be to continue annotating the genes and then have them be checked.

Rationale: Finish annotating the genes assigned in class. Post annotations to master data sheet (PhageNotes) for checking.

Materials:

• DNA Mastering
• PhagesDB
• NCBI Blast
• Computer
• Phamerator
• PhageNotes

Procedure:

• Load up DNA Mastering
• Opened up PhagesDB and NCBI BLAST scanning website
• Learned about annotating reverse genes
  • Upstream gap is reversed
  • Indicate direction on phage notes
• Learned about Phamerator and PhageNotes
• Added annotations into PhageNotes

Observations:

Finalized annotation for gene 61

Finalized annotation for gene 24

Conclusion/Next Steps:

• After this practice, will be ready to annotate NapoleonB. The practice with PhageNotes and DNA Mastering will allow continual and smooth annotations throughout the rest of the semester.

Rationale: Learn how to annotate specifically the functionss and SIF-BLAST, SIF-HHPred, and SIF-Syn

Materials:

• Computer
• DNA Mastering Application
• Annotation Key
• HHPred
• PhageDb
• NCBI BLAST

Procedure:

• Booted up DNA mastering and revised QTM 5
• Added annotations to gene 24 and 61 (Assigned last class)
  • Specifically annotated SIF-BLAST, SIF-HHPred, and SIF-Syn
• Utilized SIF-HHPred to predict protein structure and shape
• Uploaded annotations into spreadsheet

Observations:

The hits of the predictions. The first hit has the lowest evalue, which indicates a probable match to the query gene

The prediction of gene 61

The names of hits off the database which can be used to determine the function of the gene

The prediction of gene 24

Next Steps/Conclusion:

After this lab, the sequencing of NapoleonB may start. These previous labs have proven as practice for annotating, and will show when annotating of NapoleonB begins.

Rationale: Practice annotations of Elesar. Assigned two genes to completely annotate

Materials:

• Computer
• NCBI Database
• PhagesDB Database
• GeneMark
• DNA Mastering

Procedure:

• Opened saved DNA Mastering from previous lab
• Annotated genes 24 and 61 for Elesar
• Focused on SSC, CP, SCS, BLAST-Start, GAP, LO, and RBS
• Used GeneMark to determine if the gene had coding potential

Observations:

Coding potential of gene 61 of Elesar

Coding potential of Gene 24 of Elesar

Annotations of Gene 61

Annotations of Gene 24

The annotations of Genes 24 and 61, completed for QTM 5

Conclusion/Next Steps: This practice allowed as an example for when NapoleonB is annotated.

Rationale: Practice BLASTING on the PhagesDB (Protein) Server as well as the NCBI (Protein) Database.

Materials:

• Computer
• DNA Mastering Application
• NCBI Database (Protein)
• PhagesDB Database (Protein)
• Annotation Key
• GeneMark

Procedure:

• Opened DNA Mastering on the computer and opened Elesar file
• Auto-annotated and opened up frames view of the genes
• Opened up PhagesDB and NCBI Databases on separate tabs
• Copied the protein sequence from the genes (This case Gene 1) and inputted into the BLAST in both NCBI and PhagesDB
• Recorded the annotations needed (SSC: CP: SCS: ST: BLAST-Start: Gap: LO: RBS: F: SIF-BLAST: SIF-HHPred: SIF-Syn)

Observations:

• This screenshot shows the annotations in the notes box for the gene. Not all annotations are filled in because time ran out

Conclusion/Next Steps:

• Learned more about DNA Mastering and the process needed to completely annotate and document the gene. With this new information, will be able to apply to the annotation of NapoleonB

Rationale:

Learned how to evaluate reading frames, as well as how to calculate the gap or overlap between genes.

Materials:

• Laptop
• DNA Mastering Application
• Learning Manual Powerpoint

Procedure:

• Opened DNA Mastering on laptop
• Entered in updated template code from PowerPoint (In preferences tab)
• Auto-annotated Elesar and brought up the Open Reading Frames

Observations:

• The spreadsheet shows indicates the endpoints of the 5′ and 3′, which can be used to calculate the gap or overlap between genes

Conclusion/Next Steps:

• With this lab, learned more about DNA Mastering and how to operate the application. The next step(s) would be to auto-annotate Elesar

Rationale:

The purpose of today’s lab was to learn how to operate DNA Master as well as present the presentation we made on Monday about the articles pertaining to phage therapy.

Materials:

• Laptop / Desktop Computer
• DNA Mastering Program

Procedure:

Began class with presentations regarding the articles on phage therapy. Then installed the DNA Mastering application on laptop. Updated the application and then downloaded the Elesar FASTA file from the PhagesDB website. Elesar was then auto-annotated by DNA Mastering.

Observations:

The frames in green represent a forward ORF, while the red represents a reverse ORF

Although there were a few problems initially, the FASTA file Elesar was successfully annotated

Results/Next Steps:

DNA Mastering auto-annotated Elesar and resulted in ORF’s. Although most of it is confusing and gibberish on the screen, with enough time and exposure/practice, the class’ genome will be able to be annotated. Hopefully, the lab can began to annotate NapoleonB and start determining functions of the genes.

Rationale: Will be running the procedure of pelleting the lysate to obtain the bacteriophage DNA

Procedure:

• Created an aseptic zone to prevent bacterial contamination
• Added 10mL of lysate into a 50mL conical tube
• Pipetted 40μL of nuclease into the tube and inverted the tube around 10 times
• Added in 4mL of PEG and inverted the tube one to two times
• Placed the tube into the shaker for 30 minutes
• Took the tube out of the shaker and allowed to sit in room temperature for 45 minutes
• After the allotted time, moved the tube into a centrifuge and allowed to centrifuge for 20 minutes at 10,000 G
• After centrifuging, decanted the supernatant in the tube and collected the pellet(s)

Observation:

The spot test to confirm the strength of the titer (Was 1 X 10^9)

Conclusion/Next Steps: Will be continuing the next steps of DNA extraction with the hopes of breaking open the capsid of the bacteriophage.

Rationale: Prepared a TEM grid to obtain an image of the bacteriophage in the lysate

Procedure:

• Aseptic zone was set up to prevent bacterial contamination
• Pipetted 100μL of lysate from the original 50mL conical vial into a micro-centrifuge tube
• Traveled to the TEM lab
• Placed a strip of ParaFilm onto/into a agar plate
• Pipetted 20μL of lysate (in a drop) onto the ParaFilm, as well as two separate drops of 20μL DI water
• Pipetted one drop of 20μL Uranyl Acetate onto the ParaFilm strip
• Using precision forceps, placed a copper grid shiny side down into the lysate drop and let sit for five minutes
• After the allotted time (timed with a phone), transferred the grid into the first DI water drop and allowed to sit for two and a half minutes
• After the allotted time, transferred the grid into the other DI water drop and allowed to sir for two and a half minutes
• After the allotted time, transferred the grid into the Uranyl Acetate drop and allowed to sit for one minute
• After the allotted time, immediately extracted the grid from the Uranyl Acetate and wicked away extra liquid with a paper towel
• The prepared grid was put into the TEM and then imaged

Observations:

Image of the phage with dimensions measured

First image of the bacteriophage

• The measurement of the head was .055μL
• The measurement of the tail was .180μL

Conclusion/Next Steps: After getting this image, will be moving towards DNA extraction and the procedures following it, such as nano-drop and PCR.

Rationale: Created webbed plates from the team’s lysate (With titer strength of 2.2*10^11) to obtain enough lysate for all the members of the team.

Procedure:

• Aseptic zone to prevent contamination from bacteria
• Obtained the previous lysate and obtained six agar plates
• Added in 10μL lysate into each 0.5mL arthrobacter vial (Six total)
• Obtained a conical vial and pipetted in 14mL LB Broth, 17.5mL 2XTA and 157.5μL CaCl2
• Immediately pipetted 4.5mL into each arthrobacter+lysate vial and then plated
• Allowed to sit for 15 minutes and then moved to incubation

Observations:

The spot test for lysate #5; not the lysate used to web

The spot test for lysate #6; not the lysate used to web

Conclusion/Next Steps: Will be returning in 24 hours to flood the plates and then filter then to obtain a lysate of equal strength tot he 2.2*10^11