Rationale:

The purpose of today’s lab was to practice the actual process of manually annotating a gene of Arthrobacterphage Elesar using the guiding principles and annotation rules set by SEA-PHAGES.

Tools:

• DNA Master
• Genemark
• NCBI BLAST

Procedure: 

• Lab began with the reviewing the guiding rules of gene annotation as well as examining the requirements for notes written when manually annotating a gene.
• Once complete, Genemark was opened to understand the information given and how it is able to be applied to manual gene annotation.
• Began the manual annotation of Gene 1 of Arthrobacterphage Elesar.
• Gene 1 was BLASTed for the protein sequence

Results: 

• Final annotation notes for Gene 1
  • SSC:45,353
  • CP:yes
  • SCS:Both-CS
  • ST: BLAST-Start:no significant BLAST alignment
  • Gap:first gene
  • LO:yes
  • RBS: Kibler7, Karlin Medium, 1.222, -6.751, no
• BLASTp results for Gene 1
  • 
  • no significant hits were recorded for the protein sequence coded.

Conclusions:

The gene annotation did not begin at the longest ORF, so it was decided to move the start of the gene up to nucleotide 45, as Genemark noted there was coding potential beginning to that base pair. Unfortunately, no significant BLAST alignments were found for both the NCBI database and Phagesdb, meaning there is a possibility this protein can be something that has yet to be uploaded into any database used. Also, the RBS was not determined by DNA master to be the strongest site, but was still noted as any other recommended RBS would have shortened too much of the gene.

Next Steps: 

The next steps for this lab are to continue practicing manual annotation under the guiding principles reviewed in lab to further prepare for the annotation of Arthrobacterphage NapoleonB.