Rationale: correct elesar annotations from the previous class, enter it into elesar annotation class spreadsheet, ans check a classmates annotations

Process:
1. reexamined the the RBS data to correct mistake

2. used NCBI BLAST to get data in addition to phagesdb BLAST

3. used CDD from NCBI to determine SIF syn

4. Entered data into spreadsheet

5. Checked Shepard Saabye’s annoation

Results:

• Gene 6: SSC:3685 – 4974, CP:Yes, SCS:Both, ST:SS, BLAST-Start:Aligns with Nandita gp7 NCBI BLAST q1:s1 0.99 0, Aligns with Nandita gp7 PhagesDB BLAST q1:s1 0.97 0, Gap:16bp gap, LO:Yes, RBS:Kibbler7 and Karlin Medium 3.215 -2.059 Yes, F:portal protein, SIF-BLAST:portal protein Supported by NCBI BLAST Nandita gp7 AYN58629.1 0.95 0, portal protein Supported by PhagesDB BLAST Nandita gp7 MH834621 0.97 0, , SIF-HHPred:portal protein supported by COG gpNA COG4695 0.86 , SIF-Syn:portal protein, upstream gene is terminase, downstream gene is capsid maturation protease, just like in phage Nandita
• Gene 7: SSC:4974 – 6221, CP:Yes, SCS:Both, ST:SS, BLAST-Start:Aligns with Nandita gp8 NCBI BLAST q5:s1 0.98 0, Aligns with Nandita gp8 PhagesDB BLAST q1:s14 0.93 0, Gap:1bp overlap, LO:NA, RBS:Kibbler7 and Karlin Medium 1.979 -4.696 No, F:capsid maturation protease, SIF-BLAST:capsid maturation protease Supported by NCBI BLAST Nandita gp8 AYN58630.1 0.89 0, capsid maturation protease Supported by PhagesDB BLAST Nandita gp8 MH834621 0.93 0, Caseinolytic protease supported by a CD found from the CDSEARCH databse NA gpNA cd07016 NA 2.98E-67, , SIF-HHPred:capsid portal protein supported by COG gpNA COG5518 0.91 , SIF-Syn:capsid maturation protease, upstream gene is portal protein, downstream gene is NFK, just like in phage Nandita

Future Steps:

Start annotating NapoleanB!

Rationale: correct elesar annotations from the previous class and complete supporting information

Process:
1. reexamined the gap, RBS data, LO to correct mistakes

2. used HHPred to analyze predicted functions of the gene for supporting information

3. used phagesdb blast for the same purpose

4. Completed Annotation (Results):

• Gene 6: SSC: 3685-4974 CP: yes SCS: both ST: NA BLAST-Start: matches with Nandita, 7, phagesdb, q1:s1, 97%, 0.0 Gap: 16 LO: yes RBS: Kibler7, Karlin Medium, 3.215, -2.059, yes F: portal protein SIF-BLAST: portal protein, phagesdb, Nandita_7, MH834621, 97%, 0.0 SIF-HHPred: Phage Portal Protein BeeE, COG, BeeE, COG4695, 86%, 99.89 SIF-Syn
• Gene 7: SSC: 4974-6221 CP: yes SCS: both ST: NA BLAST-Start: matches with Nandita, 8, phagesdb, q1:s14, 93%, 0.0 Gap: 1 bp overlap LO: no RBS: Kibler7, Karlin Medium, 1.823, -6.049, yes F: capsid maturation protease SIF-BLAST: capsid maturation protease, phagesdb, Nandita_8, MH834621, 93%, 0.0 SIF-HHPred: Bacteriophage capsid portal protein , COG, NA, COG5518, 91%, 99.8 SIF-Syn

Future Steps:

Figure out how to use SIF syn and fix any new mistakes

Rationale: Annotated genes 6 and 7 of elesar

1. Gene 6
   SSC: 3685-4974
   CP: yes
   SCS: both
   ST: NA BLAST-Start: matches with Nandita, 7, phagesdb, q1:s1, 97%, 0.0
   Gap: 16
   LO: yes
   RBS: Kibler7, Karlin Medium, 3.215, -2.059, yes
   F: portal protein SIF-BLAST: portal protein, phagesdb, Nandita_7, MH834621, 97%, 0.0
   SIF-HHPred:
   SIF-Syn

2. Gene 7
   SSC: 5025-6221
   CP: yes
   SCS: neither
   ST: NA
   BLAST-Start: matches with Nandita, 8, phagesdb, q1:s14, 93%, 0.0
   Gap: 50
   LO: no
   RBS: Kibler7, Karlin Medium, 1.823, -6.049, no
   F: capsid maturation protease
   SIF-BLAST: capsid maturation protease, phagesdb, Nandita_8, MH834621, 93%, 0.0
   SIF-HHPred:
   SIF-Syn

Results/Conclusion:

Annotated 2 genes. Gene 6 I determined to be an RBS because it had the best values for Z score and F score. I changed the starting codon on gene 7 based on the ORF map suggested value and concluded that it was not an RBS.

Future Steps: Figure out if my annotations were correct and figure out how to successfully use template notes.

Rationale: auto-annotate elesar

Process:

1. genome -> auto-annotate -> annotate
2. auto-annotate with pecaan
   1. entered phage name “elesar”
   2. uploaded fasta file
   3. clicked “process”
3. copied pecaan documentation, pasted it into documentation tab of dna master
   1. removed documentation previously there
   2. clicked “parse”, once directed to a new window, clicked the new “parse” button.

Results/Conclusion:

I could see a bar at the bottom with different colored “gene” arrows.

Future Steps: learn how to view more of the data after auto annotating

Rationale: Download DNA Master and auto annotate elestar genome

Proces

• download DNA Master from download link and go through setup using the install wizard
• open DNA Master directly from Program Files (x86) by right clicking and selecting run as administrator
• download fastfa version of elestar from phage db
• go to DNA Master and select file -> open -> fastfa -> select elsetar
• in the bottom right hand corner of the DNA Master view of elestar, click export -> create sequence from this entry only
• then on the top bar of DNA Master, select genome -> annotation -> auto-annotate

Complication:

• for some reason DNA Master would not auto-annotate because of a “Glimmer Failure”

Next Steps:

• troubleshoot Glimmer Failure and auto annotate elestar

Rationale: There is not enough time to continue to pursue a high titre lysate from Mellissa’s phage. The results for MEL X are shown below.

Control for MEL X plaque assay

MEL X plaque assay. The agar did not solidify correctly so it is not smooth, but there is no evidence of plaque.

Next Steps:

Focus on group data for the essay and give up on finding a phage 🙁

Rationale: Results for the second plaque assay from MEL 2 did not show any plaques so perform a plaque assay for a new plaque picked from Melissa’s plate labeled MEL X

Process:

1. washed table and setup aseptic zone
2. picked plaque and swirled it in 50 µL phage buffer (MEL X plaque lysate)
3. Made LB agar media for 3 plates
   • 6 mL LB broth
   • 68 µL 1 M CaCl2
   • 7.5 mL 2X TA
4. added 50 µL lysate (Mel X plaque) to 0.5 mL arthro and let sit for 15 minutes
5. Poured control plate with ~5mL from the LB agar media
6. Added arthro lysate mixture to  LB agar media
7. Poured plate from tube and let sit for ~15 minutes
8. Incubated for 48 hours at 28 degrees Celsius

Next Steps: Another plaque assay but with alterations to get a higher titer.

Rationale: perform a second plaque assay for lysate collected from “mel plaque”, or the singular plaque found on the first plaque assay from Melissa’s soil

Control for first plaque assay of Melissa’s soil

First plaque assay for Melissa’s soil showing one plaque

Process:

1. washed table and setup aseptic zone
2. picked plaque and swirled it in 70 µL phage buffer
3. Made LB agar media for 2 plates
   • 4 mL LB broth
   • 50 µL 1 M CaCl2
   • 5 mL 2X TA
4. added 50 µL lysate (Mel plaque) to 0.5 mL arthro and let sit for 15 minutes
5. Poured control plate with ~5mL from the LB agar media
6. Added arthro lysate mixture to  LB agar media
7. Poured plate from tube and let sit for ~15 minutes
8. Incubated for 48 hours at 28 degrees Celsius

Next Steps: Another plaque assay but with alterations to get a higher titer.

Rationale: perform plaque assay for the sample enriched from Melissa’s soil and also the enriched sample from R&L experiment, soil sample 2

Process:

1. washed table and setup aseptic zone
2. Made LB agar media for 3 plates
   • 6 mL LB broth
   • 68 µL 1 M CaCl2
   • 7.5 mL 2X TA
3. added 30 µL lysate (Melissa’s soil and R&L SS2) each to 0.5 mL arthro and let sit for 15 minutes
4. Poured control plate with ~5mL from the LB agar media and divided the remaining agar into 2 tubes
5. Added arthro lysate mixture to each LB agar media tube
6. Poured plates from tubes and let sit for ~15 minutes
7. Incubated for 48 hours at 28 degrees Celsius

Next Steps: Another plaque assay but with alterations to get a higher titer.