Annotation of Phage NapoleonB Genes 53-56

Date: 2-11-19

• Rational
  • The rational for this lab was to begin annotation of phage NapoleonB, starting with genes 53, 54, 55, 56.
• Procedure
  1. DNA Master was opened.
  2. File > Open > Archived DNA Master File was used to open the NapoleonB FastA file from PhagesDB.
  3.  Gene 53 was selected through the listing of predicted genes, and the Product was copied.
  4. The amino acid sequence was loaded into the BLASTp feature on the NCBI website and the BLAST results were analyzed and recorded on the PhageNotes google sheet.
  5. The amino acid sequence was loaded into HHPRED, a program that evaluated the three-dimensional protein structure of the gene region and the results were analyzed and recorded.
  6. The starterator map on PhagesDB was evaluated by choosing gene 53 from the NapoleonB gene list and the information was recorded.
  7. Steps 3-6 were repeated for genes 54-56.
  8. Ctrl + Alt + F brought up the Frames of the genome, ORFS was selected to show the locate open reading frames, and RBS was selected to determine the ribosome binding site for the each gene.
  9. Genemark was used in order to determine if coding potential was covered.
  10. Using the acquired information, the 53rd-56th genes were annotated.
• Results
  • The following annotations were made for gene 53:
    • Original GeneMark call @bp 33165
      SSC: 33165 – 33263, CP: Yes, SCS: Both, ST:SS, BLAST-Start: Aligns with Arthrobacter Phage Tribby gp53 NCBI BLAST q53:s1 1 5E-13, Aligns with Arthrobacter Phage Tribby gp53 PhagesDB BLAST q53:s1 1 1E-12, Gap: 4bp overlap, LO: Yes, RBS: Kibbler7 and Karlin Medium 3.263 -2.524 No, F: NKF, SIF-BLAST: NKF, SIF-HHPred: NKF, SIF-Syn: NKF

An image of the starterator report

An image of the RBS choices

An image of the PhagesDB BLAST results

An image of the NCBI BLAST results

An image of the HHPRED results

An image of the GeneMark results

• The following annotations were made for gene 54:
  • Original Glimmer call @bp 33273 has strength 10.13
    SSC: 33273 – 33962, CP: Yes, SCS: Both, ST: SS, BLAST-Start: Aligns with Arthrobacter Phage Circum gp55 NCBI BLAST q54:s1 1 4E-167, Aligns with Arthrobacter Phage KeaneyLin gp52 PhagesDB BLAST q54:s2 1 1E-128, Gap: 9bp gap, LO: Yes, RBS: Kibbler7 and Karlin Medium 1.993 -4.759 No, F: NKF, SIF-BLAST: NKF, SIF-HHPred: NKF, SIF-Syn: NKF

An image of the starterator results

An image of the RBS choices

An image of the PhagesDB BLAST results

An image of the NCBI BLAST results

An image of the HHPRED results

Images of the GeneMark results

• The following annotations were made for gene 55:
  • Original Glimmer call @bp 33955 has strength 0.38
    SSC: 33949 – 34173, CP: Yes, SCS: Both-CS, ST: SS, BLAST-Start: Aligns with Arthrobacter Phage Circum gp56 NCBI BLAST q55:s1 0.98 3E-44, Aligns with Arthrobacter Phage KeaneyLin gp53 PhagesDB BLAST q55:s2 0.98 7E-36, Gap: 14bp overlap, LO: No, RBS: Kibbler7 and Karlin Medium 1.391 -5.257 No, F: NKF, SIF-BLAST: NKF, SIF-HHPred: NKF, SIF-Syn: NKF

An image of the starterator results

An image of the RBS choices

An image of the PhagesDB BLAST results

An image of the NCBI BLAST results

An image of the HHPRED results

An image of the GeneMark results

• The following annotations were made for gene 56:
  • Original Glimmer call @bp 34191 has strength 0.52
    SSC: 34191 – 34316, CP: Yes, SCS: Both, ST: SS, BLAST-Start: Aligns with Arthrobacter Phage Circum gp57 NCBI BLAST q56:s1 1 1E-20, Aligns with Arthrobacter Phage Circum gp57 PhagesDB BLAST q56:s3 1 2E-16, Gap: 17bp gap, LO: Yes, RBS: Kibbler7 and Karlin Medium 2.053 -5.205 No, F: NKF, SIF-BLAST: NKF, SIF-HHPred: NKF, SIF-Syn: NKF

An image of the starterator results

An image of the RBS choices

An image of the PhagesDB BLAST results

An image of the NCBI BLAST results

An image of the HHPRED results

An image of the GeneMark results

• Conclusion
  • Phage NapoleonB has no known functions for genes 53-56.
• Future Plans
  • The next step is to consolidate the gene annotations to the PhageNotes google sheet.