Rationale:  In order to have a completely annotated genome for NapoleonB by the end of the week I worked on annotating the rest of the genes assigned to me. These were genes 31,32, and 94.

Tools: PhageNotes, DNAMaster, NCBI, PhagesDB, Phamerator, HHPred, Genemark

Procedure:

1. First I found that all the genes were already at the largest open reading frame and covered all the coding potential so I noted that in my annotations.
2. I blasted all the genes’ products and they all turned up with no known function.
3. In HHPred none of the results had a decent probability to call a function.
4. I recorded the RBS values and calculated the gap.
5. I typed up all my findings in PhageNotes.

Results:

Final PhageNotes annotation

Conclusion and Future Work: Now that the genome is almost completely done being annotated we can proofread and get feedback. For future work we will continue iterating the annotation until it is as complete and accurate as possible.

Rationale: Now that I have learned how to use each Biotech tool I will begin annotating the genome of the phage I discovered the previous semester, along with the help of my classmates. I was tasked with annotating genes 29-32 and 94.

Tools: PhageNotes, DNAMaster, NCBI, PhagesDB, Phamerator, HHPred, Genemark

Procedure:

1. First loaded the FastA file of NapoleonB into DNAMaster and auto-annotated the genome.
2. Checked the Frames view in DNAMaster to see if gene 29 was marked at its LORF. Opened the Genemark file for NapoleonB and found that the gene was covering all the coding potential at that area. Noted the start and stop locations and that the coding potential was completely covered.
3. Using PhagesDB I found that the starterator for gene 29 doesn’t have enough annotated calls to get any information from.
4. I then BLASTed the product of the gene through the NCBI and PhagesDB databases. Both returned the same result as the best hit. However, the hit didn’t have any result for the function. I then ran the product through HHPred and it didn’t return any hits with a decent probability.
5. I then calculated the gap of the gene and recorded the RBS results.
6. I started working on gene 30 and using the Phamerator comparison with other phages in the same cluster, the BLAST results of the gene, and the fact that the gene is a reverse gene in the middle of a forward cassette to determine that the gene needs to be deleted.
7. Recorded all my findings in PhageNotes.

Results:

BLAST for Gene 29 and Gene 30

Conclusion and Future Work: I found that gene 29 had no known function, and  gene 30 was not really a gene. In the future I will continue working on annotating the genes assigned to me so that we can have a fully annotated genome by the end of the week.

Rationale: In order to practice more annotation and to help out others in the group we are proofreading each others annotations. I will be proofreading the annotation for genes 6 and 7. I will also learn how to use starterator and phamerator.

Tools: NCBI BLASTp, HHPred, PhagesDB, Phamerator, DNAMaster, PhageNotes

Procedure:

1. Uploaded the existing annotation for genes 28 and 29 to PhageNotes.
2. Annotated genes 6 and 7 myself using the template on the SEA-PHAGES website. I blasted the  genes and used HHPred to support my function calls.
3. Compared the annotation I made to the annotation for the genes in PhageNotes and told my partner about corrections that needed to be made.
4. Used Phamerator to look at the genes in NapoleonB and looked at the Starterator of a sample gene in NapoleonB.

Results:

Phamerator for NapoleonB and 2 other AM cluster phages

Starterator for NapoleonB gene 60

Conclusion and Future Work: Now that I’ve learned all the techniques for annotating genes I can start working on NapoleonB’s genome. PhageNotes is also a very useful tool for annotating as it makes it much easier to pool our work into one spot then copy over our annotations afterwords.

Rationale: I’m continuing to practice annotation by annotating certain genes of the Elesar genome. Today I will be learning and practicing using HHPred and BLAST to discover the function of the gene.

Tools: BLAST, HHPred, DNAMaster

Procedure:

1. Opened up the DNAMaster file from last lab time containing the incomplete annotations for  genes 28 and  29.
2. Copied the amino acid product for each gene and ran it through HHpred using the COG_KOG_v1.0 and Pfam-A_v32.0 databases.
3. Recorded the results under SIF-HHPred. Gene 29 did not get a significant hit so I recorded it as No Known Function(NKF).
4. Next I ran the amino acid product through NCBI BLASTp and recorded those results under SIF-BLAST.
5. Both of the genes returned to be Minor Tail Proteins so I recorded that in my annotation.

Results:

HHPred and BLASTp results for genes 28 and 29.

Conclusion and Future Work: I learned how to use HHPred and determine the function of a gene. Next I will learn how to use synteny to support my call for the function as well as supporting my start choice with the starterator.

Rationale: Continuing to practice annotating I will be annotating genes 28 and 29 of Elesar by myself.

Tools: NCBI, PhagesDB, DNAMaster

Procedure:

1. Opened the saved file from the previous lab, which had already be auto-annotated and had the template loaded.
2. Recorded the SSC, CP, SCS, GAP, and LO for gene 28.
3. Blasted the product of the gene using both the PhagesDB and NCBI databases. Recorded results under Blast-Start.
4. Highlighted ORF in the frame viewer and used a Kibler7 Scoring Matrix and a Karlin Medium Spacing Matrix to find the Z score and Final Score.
5. Repeated steps 2-4 for gene 29.
6. Posted and saved file.

Results:

Blast results for Gene 28

Blast Results for Gene 29  (Blast results were the same for NCBI and PhagesDB so only NCBI shown)

Conclusions and Future Work: I was able to succeed in annotating the gene by myself. I noticed the importance of blasting using both databases as they could have different results such as in the case of Gene 28. In the future I will continue working on annotating Elesar as well as filling out the rest of the required parts of the annotation.

Rationale: In order to learn how to annotate a genome I practiced annotating the first gene of Elesar.

Tools: NCBI BLAST, DNAMaster

Procedure:

1. Began by auto-annotating the genome of Elesar using the template set-up from last lab.
2. Opened the frames view and looked at the open reading frame of gene 1. Due to there being a larger Open Reading Frame(ORF) possible I moved the ORF to the Largest Open Reading Frame(LORF).
3. Noted in the annotation the Start and Stop Coordinates(SSC), the Coding Potential(CP), and Start Choice Source(SCS).
4. Blasted the product using the NCBI database, which turned up no good matches, which I noted under Blast-Start.
5. Noted the fact that this is the first gene for GAP, and noted that this is indeed the LORF.
6. Opened up frames and highlighted the ORF and clicked RBS. Using a Kibler7 Scoring Matrix and a Karlin Medium Spacing Matrix, I recorded the Z Score, Final Score, and whether or not it was the best Final Score.
7. Posted and saved file.

Results:

Results of RBS.

Conclusion and Future Work: I’ve learned the first few steps to annotating a genome. Now I will begin working on annotating genes by myself.

Rationale: In order to begin practicing annotation on the genome of Elesar, the auto-annotation preferences and template were set-up. Also, some practice was done to better understand the necessity and location of gaps and overlaps in reading frames.

Tools: DNA Master, Microsoft Word

Procedure:

1. Went in to preferences and local preferences to paste the template in for auto-annotation.
2. Using an assigned word file, practiced calculating gap and overlap
3. Ran an auto-annotation on Elesar and viewed the reading frames.
4. Helped other students fix DNA master

Results:

Conclusion:

I was able to view the reading frames of Elesar graphically and the template showed up in the annotation notes.

Future Work:

I will practice annotating some genes of Elesar using the template created today.

Rationale: In order to aide in performing annotations this year, the software “DNA Master” was installed onto my laptop. The FASTA file of the phage Elesar was used to test whether the program was installed properly.

Tools:

1. WINE was used in order to help install the Windows version of the software on my mac, as it seemed to be more stable than the mac version.

2. PhagesDB was used to download the executable installer and the FASTA file for Elesar.

Procedure:
1. The executable installer was downloaded from the PhagesDB website as well as WINE.

2. WINE was installed and the executable was run through WINE to install DNA Master.

3. The FASTA file for Elesar was downloaded from PhagesDB and loaded into DNA Master.

4. An auto-annotation was run on the Elesar genome.

Results: The program successfully auto-annotated the genome of Elesar from the FASTA file provided.

Conclusion: DNA Master was successfully installed properly onto my laptop.

Future Work: Next I will practice annotation on the genome of Elesar using the help of the newly installed program. Once I have sufficiently practiced, I will start annotating the genome of NapoleonB.

Rationale: Now that I have enough lysate to run DNA extraction I can proceed with doing so and now I can view my TEM that I prepared a couple weeks ago.

Procedure:

Wednesday:

1. Pipetted 10mL of lysate into a 50mL conical vial.
2. Added 40µL of Nuclease Mix and gently shook the vial for about a minute.
3. Added 4mL of Phage Precipitant Solution and gently shook once again.
4. Incubated at 37°C for 30 minutes then took out of incubator and left at room temperature for 40 minutes.
5. Centrifuged at 10,000g for 20 minutes.
6. Decanted supernatant and stored pellet in freezer overnight.
7. Since I already prepped the TEM last week I just had to go in and view my phage under  the microscope.

Thursday:

1. Added 500µL of sterile water to pellet and re-suspended by pipetting up and down.
2. Added 2mL of resin to re-suspended pellet and pipetted up and down to stir.
3. Split up the solution into two microcentrifuge tubes and spun at 12.5g for 3 minutes.
4. Decanted the supernatant and added 1mL of 80% isopropanol to each tube. Mixed by flicking tube and gently swirling.
5. Spun at 12.5g for 3 minutes.
6. Repeated steps 4 and 5.
7. Decanted supernatant and added 1mL of 80% isopropanol. Re-suspended pellet.
8. Poured each tube into separate vacuum filters.
9. Added each column to a new microcentrifuge tube and spun at 12000g for 5 minutes.
10. Took column out and placed into another set of new microcentrifuge tubes. Added 100µL of elution buffer to column and let sit for a minute.
11. Spun at 12000g for 1 minute then placed the solution into one microcentrifuge tube. Stored at -20°C overnight.

Observations:

This image was produced using TEM. My phage has a tail length of 100nm.

Conclusions and Next Steps:
Now I need to find out how much nucleic acid is in my sample using the nanodrop machine. From there I will run PCR and gel electrophoresis to find out what cluster my phage is.

Rationale: Since I need at least 12.8mL of lysate for DNA extraction and archiving I need to make some webbed plates to flood. I calculated that I need 8µL of lysate diluted to the 10^-5 to  web a plate, so I will plate three  plates with  that amount.

Procedure:

1. Put parafilm on a plate and pipetted 15µL of lysate onto the parafilm.
2. Pipetted 20µL of water twice onto the parafilm.
3. A TA aliquotted uracyl acetate onto the parafilm
4. Placed a TEM stage from section A10 onto the lysate and let sit for 5 minutes.
5. Transferred stage to water and let sit for 2.5 minutes.
6. Repeated for the second drop of water.
7. Transferred stage to uracyl acetate for 1 minute then took off and placed back into container on the side to be used for TEM.
8. Pipetted 8µL of 10^-5 lysate into 0.5mL of arthrobacter 3 times and let sit for 30 minutes.
9. Made top agar with 8mL of LB broth, 90µL of CaCl2, and 10 mL of 2X TA.
10. Plated control with 4.5mL of solution.
11. Aliquot 4.5mL of solution into each tube of enriched lysate and arthrobacter.
12. Mixed by pipetting  up and down then plated each.
13. After letting solidify inverted and placed into incubator for 24 hours.

Observations:

The plate wasn’t fully lysed because the plaque morphology isn’t uniform. I placed back in the incubator over break in hopes of getting a slightly more webbed plate.

Conclusions and Next Steps: This means that there is more than one type of phage in  my  lysate. However, I do not have time to run another round of purification. This means that I will just have to keep running with my current lysate and acknowledge that there are two phages in my lysate when I extract DNA. From there I can differentiate between the two phages.