Rationale

Create a rough draft for an abstract as a group and complete gene annotation and discuss any hard calls made.

Procedure

1. Created abstract by combining other group members’ abstracts
2. Fixed gene annotations as a class

Results/Next Steps
Certain genes were reannotated and different calls were made. Next time, we will make a final draft for the abstract and start designing a poster.

1. D’Herelle had took samples from patients from the war who suffered from dysentery, took the bacteria cultures and tested them out onto agar plates. He noticed that there were clear spots on the plate not just from dysentery patients, but also from locusts. He later made a filtrate, a test tube containing bacterial cells from his samples. During these experiments he saw that the dysentery bacteria “dissolved like sugar in water.” He later found out that there could be something that can infect bacteria. He also used this discovery to prove that his dysentery patient can recovered from it, which one patient did in fact, led to a wide discovery and a hunt for more bacteriophages from different places in the world.
2. D’Herelle and Eliava were both hard workers that laid their foundation for the future bacteriophage institute. The co workers said that D’Herelle was very hard working and would always arrive on time each day. They said that he was a “virtuoso” and insisted on doing everything with his own hands. This shows that he is very diligent in his work and is a perfectionist. Eliava was hardworking like D’Herelle in terms of the research. Sadly Eliva and his wife supported the wrong side of the Russians and were eventually executed.
3. War encourage the hunt for bacteriophages because many of the soldiers suffered from diseases and plaque spread among the people. Soldiers are needed to continue the war and the government needed a strong army, a cure is needed. Since the discovery of bacteriophages and its effects on bacteria cells, phage therapy was created for treatments. Politics had its pros and cons about phage therapy. In Elivas’ casem he used politics to help fund research, but also got him executed.
4. Antibiotics came around and eventually won favor over bacteriophages. Due to new technology and research, everyone started to use more antibiotics than phage therapy.
5. Delbruck and Luria worked on x-ray crystallography which lead to their interest in bacteriophages. They later joined Phage group. They also discovered the replication mechanism and the structure of phages with other members. The group members’ interest in phages started to fade away as they wanted to examine larger organisms. Therefore, the phage biology started to die out.

Rationale

Start performing annotations on NapoleonB to understand certain functions for genes and provide further research to be conducted afterwards.

Procedure

1. Opened DNA master with NapoleonB FASTA file
2. Opened DNA –> Frames –> ORF —> RBS (for genes 89-92)
3. Blasted each genes’ products on NCBI, phagesdb, HHPred, and DNA master for preparation
4. Filled out template for each gene using information from above listed sites
5. Opened gene mark for genes 89-92 and phamerator
6. Saved as new file and uploaded findings on class google sheets

Observations/Results

Gene 89

Original Glimmer call @bp 51379 has strength 12.26
SSC:51379,51708 CP:yes SCS:both ST:SS BLAST-Start:Xenomorph_Draft,83,phagesdb,query 1 to subject 1,100,7e-62 Gap:1 LO:NA RBS:Kibler7,Karlin Medium,2.290,-5.154,no F:NKF SIF-BLAST:NKF,Circum_88,ALY08771.1,99%,2e-73 SIF-HHPred:Signal transduction histidine kinase,COG,COG4585,COG4585,100%,87.68 SIF-Syn:NKF

Gene 90

Original Glimmer call @bp 51709 has strength 6.33
SSC:51709,51894 CP:yes SCS:both ST:SS BLAST-Start:Xenomorph_draft,84,phagesdb,query 1 to sequence 1,96,7e-28 Gap:0 LO:NA RBS:Kbler7,Karlin Medium,3.023,-2.566,no F:NKF SIF-BLAST:NKF,Sea_Arcadia_87,ASR80050.1,93.55%3e-35 SIF-HHPred:ribosomal protein L20A (L18A),COG,RPL20A,COG2157,73%,91.48 SIF-Syn:NKF

Gene 91

Original Glimmer call @bp 51891 has strength 7.86
SSC:51891,52247 CP:yes SCS:both ST:SS BLAST-Start:Nason,88,phagesdb,query 1 to sequence 1,95,2e-65 Gap:4 LO:NA RBS:Kibler7,Karline Medium,2.546,-3.590,no F:NKF SIF-BLAST:NKF,Sea_Arcadia_88,ASR80051.1,94.92%,4e-78 SIF-HHPred:FG-GAP repeat,FG-GAP,PF01839.23,34%56.96 SIF-Syn:NKF

Gene 92

Original Glimmer call @bp 52247 has strength 7.98
SSC:52247,52492 CP:yes SCS:both ST:SS BLAST-Start:Xenomorph_draft,86,phagesdb,query 1 to sequence 1,100,2e-41 Gap:1 LO:NA RBS:Kibler7,Karlin Medium,3.269,-2.064,no F:NKF SIF-BLAST:NKF,Mudcat_86,YP_009300775.1,93,67%,1e-48 SIF-HHPred:Agrobacterium tumefaciens protein,Atu,Atu4866,PF11512.8,93%,70.95 SIF-Syn:NKF

For all genes, there wasn’t a huge gap or overlap of >10 bps. All gaps/overlaps ranges from 0-4 bps. I did not had to correct the ORF or start codon as I agreed with starterator for all genes. All genes had NCBI, phagesdb, and DNA master Blast state “hypothetical protein” or “unknown function.” However, HHPred had its functions stated. I made the call that none of the genes had a functions. Also, none of the genes had a conserved domain provided, so on the google sheet CDD was filled out as “no.” Two of the genes after BLAST compared itself to NapoleonB_draft, so I chose to use the BLAST data with the one right below NapoleonB_draft.

Next Steps

Continue with the annotation of the current genes that were assigned to me and edit them along the way. Also to apply the knowledge for future research and presentation.

Rationale

Re-annotation and corrections were made in the lab as well as peer review for other gene annotations. This helped us learned our mistakes and help correct others to prepare us for future annotation of NapleonB

Procedures

• Opened DNA master with Elesar file
• Blasted gene 4 and 5 through NCBI, phagesdb, and HHPred
• Used annotation file to see any mistakes and made correction to template
• Repeated with genes 20 and 21 for peer review

Observations

Gene 4:

Original Glimmer call @bp 1349 has strength 7.44

SSC:1349,1984 CP:yes SCS:both ST: BLAST-Start:Nandita,5,phagedb,query 1 to subject 1,95%,3e-122 Gap:0 LO:NA RBS:Kibler7,Karlin Medium,2.230,-4.085,no F: SIF-BLAST:terminase small subunit,phagesdb,Nandita_5,AYN58627,95%,-122 SIF-HHPred:teminase small subunit,COG,COG3747,75%,99.57 SIF-Syn:

Gene 5:

Original Glimmer call @bp 1971 has strength 13.74

SSC:1971,3668 CP:yes SCS:both ST: BLAST-Start:Nandita,6,phagedb,query 4 to subject 5,99%,0 Gap:14 LO:NA RBS:Kibler7,Karlin Medium,1.724,-5.684,no F: SIF-BLAST:terminase large subunit,phagesdb,Nandita_6,AYN58628,97%,0 SIF-HHPred:terminase large subunit,Terminase_1,PF03354,87%,100 SIF-Syn

Results

Wrote out the e value incorrectly compared to last lab day. The e value changed for gene 4 according to NCBI. Blast results from multiple sites highly suggested that both genes were a terminase.

Next Steps

Continue to re-annotate until no mistakes are made and use those skills to apply to NapoleonB

Rationale

Re-edit template from last lab day and add SIF-Blast and SIF-HHPred

Procedure

1. Opened DNA master with elesar fasta file
2. Opened DNA frames, clicked on ORFs, highlighted target gene, clicked on RBS
3. Blasted gene 4 via phagedb, NCBI, and HHPred
4. Edited template for SSC, CP, SCS, Blast-start, Gap, LO, and RBS and used guidelines and annotation key for assistance
5. Added SIF-BLAST and SIF-HHPred template
6. Repeated same procedures above for gene 5.
7. Clicked post after each gene annotation

Observations

By looking through NCBI, phagesdb, and HHPred, all of them seem to agree that the protein coded is a terminase small/large subunit, and the phage name is Nandita.

Next Steps

Have a better understanding of the templates and hope to correct any mistakes from today’s template.

Rationale

Annotate and update template for genes 4 and 5 from elesar. Practice learning how to annotate each gene.

Procedure

1. Opened DNA master with elesar fasta file
2. Opened DNA frames, clicked on ORFs, highlighted target gene, clicked on RBS
3. Blasted gene 4 via phagedb
4. Filled out template for SSC, CP, SCS, Blast-start, Gap, LO, and RBS and used guidelines and annotation key for assistance
5. Repeated same procedures above for gene 5.
6. Clicked post after each gene annotation and saved as new dnam5 file.

Observations

Both gene 4’s and 5’s scores are not the lowest, which is why RBS has “no” on both of them, but there is still a high chance that both genes coded for Nandita.

Next Steps

Finish the rest of the template and have a better understanding of annotating any gene from elesar to prepare the annotation of NapoleonB.

Rationale

In the beginning of the lab, we learned about lambda phage cycle. We also learned about the purpose of Blast, how to perform Blast, and how to read the results from the NCBI database. Learned how to make the LORF (longest open reading frame), and update SSC, CP, SCS, and the rest of the template

Observations

Next steps

Make LORF for other genes and update template (SSC, CP, SCS, etc) for that gene.

Rationale

Redo auto annotation of Elesar  by changing the preferences.  Learned about locating reading frames and calculating gap and overlaps.

Tools

• personal laptop
• DNA Master
• Elesar file

Procedure

1. Opened DNA master with the Elesar file
2. Changed preferences and added the code to the template
3. re auto annotated elesar

Observations

above are the reading frames and reverse after changing the preferences. It is the same as last pictures from previous lab notebooks.

Next Steps

Blast the genome and learn more about locating the reading frames and possible coding potentials.

Rationale

The purpose of this lab was to get familiar with the DNA Master program and start annotating the genome sequence of Elesar.

Tools

• personal laptop
• DNA Master
• Elesar fastA file

Procedure

1. DNA Master wasd downloaded unto personal computer and updated
2. Elesar file was downloaded and auto annotated through DNA Master
3. Made sure everything on that file had the coordinates and was annotated properly.

Observations/Results

This process took awhile to get the sequence auto annotated correctly. The green colored is forwards ORFs and the red colored are the reverse ORFs. The second pic showed each line as possible start codons.

Next Steps

Learn more and get used to using DNA Master. Soon NapleonB will be auto annotated and learn more about its genome.