Gene 32 and Gene 41 Annotation

Rationale: Finish the annotations of Elesar gene 1 and begin Annotations for gene 32 and gene 41

Procedure:

• DNA Master was opened and Elesar FASTA file opened
• Reading frames were opened and genes 32 and 41 were observed
• Both genes were blasted through NCBI Protein Database
• Elesar gene was pulled up on Genemark
• SSC: CP: SCS: BLAST-Start: Gap: LO: RBS were the categories that were annotated

Results:

Blast results shown above for gene 32 and 41 are both from the NCBI Protein database. The images above also show results for each query of the two genes.

Gene 32

SSC: 23223 26093 CP: yes SCS: Both-Both Blast-Start: hypothetical protein PBI_BRIDGETTE, 30, NCBI, Q601: S62, 81%, 0.0 Gap: 7 LO: yes RBS: Kibler 7, Karlin Medium, 2.117, 4.852, no

Gene 41

SSC: 31001 31219 CP: yes SCS: Both-Both Blast-Start: Mycobacterium virus Brujita, 35, NCBI, Q7: S2, 60%, 4e-13 Gap: 607 LO: yes RBS: Kibler 7, Karlin Medium, -6.128, -8.014, no

Conclusions:

Annotations of genes 32 and 41 were not bad after the TA of the class showed the class what each letter meant under the annotation key. Observing the blast was easy while observing genemark results were hard, but with the help of the lab TA’s, it was possible.

Next Steps:

Start annotations for NapoleonB.

Elsar BLAST Practice 1/28/19

Rationale: Learn basic annotation tools, and become familiar with BLAST NCBI database.

Procedure:  

• DNA Master was opened
• Elsar FASTA file opened and auto annotated
• The reading frames were examined and adjusted
• Genes 1 and 2 were selected to BLAST
• Results from BLAST were examined and used to start annotation
• Gene 14 of Elsar was chosen at random to BLAST-Start the gene in the notes
• the results of Gene 1 and 14 were posted on DNA Master

The procedure of the bLAST was used as a practice BLAST to familiarize with the NCBI BLAST results. These results allow one to compare the query DNA sequence to similar sequences of DNA, and potentially allows identification of conserved DNA in multiple genome.

Gene 14

SSC: CP: SCS: ST: BLAST-Start: hypothetical protein [Arthrobacter sp. ok362], gene 14, NCBI, Q :S, coverage, 5e-58 Gap: LO: RBS: F: SIF: SIF-Syn

Results: 

Conclusions: The video played in the lab helped a great deal on understanding the results of a BLAST gene. With the results of the BLAST, BLAST-Start was done on gene 14. The results from BLAST allow one to compare the query DNA sequence to similar sequences of DNA as one means of verifying one gene or multiple genes. This helps researchers understand the function of the gene and each protein.

Future plans: Learn the basics of annotation through the SEA: PHAGES website and from the lab TAs. BLAST all of the genes of Napoleon B and compare the results of the BLAST with different databases such as Genemark and the auto-annotation results.

Rationale:

The purpose of today’s lab was to be introduced to the process of gene annotation. Elsar genome was annotated as an example for today’s lab to familiarize with the DNA Master program.

Procedure: 

DNA Master opened and preferences was opened > local settings > new feature. Checked “insert template into notes during annotation” and pasted: SSC: CP: SCS: ST: BLAST-Start: Gap: LO: RBS: F: SIF-BLAST: SIF-HHPred: SIF-Syn. Elesar.fasta was opened and was auto-annotated.

Results:

Conclusions:

At first, all of the information was overwhelming, but once the TA’s cleared the confusion, work was done. The reading frames lines were easy to understand after an explanation of how DNA Master presents the reading frames.

Future plans: 

Learn more about DNA master and to begin annotation the genome of Napoleon B.

Purpose: Present phage therapy presentations to peers and learn the basic functions of DNA Master.

Procedure: Presented phage therapy presentations in front of the class, and showed how Vibro parahaemolyticus can help with multiple-antibiotic-resistant O3: K6 cure mice. DNA Master was downloaded and created a guide on how to set up DNA Master. The Fasta file for Elesar was obtained from PhageDB and imported to DNA Master. Auto-annotation was performed on Elesar, and saved onto a computer.

Observations: With presentations, look over the material and practice the presentation before class so when presenting in front of an audience, it should be easy. DNA Master is a great platform for gene annotation and with the help of DNA Master, the annotation of Napoleon can start.

Conclusions: With the start of the semester with presentations, it gave the class as to where we are right now at the start of the semester since the class will have to present multiple times throughout the semester. Prepare for a presentation before the actual presentation. DNA Master was successfully installed, and the next step will be to learn how to annotate.

Rationale: Calculate titer from spot titer experiment. Webbed plates made to yield more lysate so each member who contributed to obtaining a high titer lysate could use 10mL of lysate.

Procedure:  Before the experiment was performed, the workspace was cleaned with both Cidecon and 70% Ethanol. Top agar solution made in a 50mL vial using the formula below (6 plates for experiment and one for control):

• 2mL LB Broth (x7)
• 2.5mL 2X TA (x7)
• 22.5µL CaCl2 (x7)
• lysate
  • 10µL of Flooded lysate x8

4.5mL of this solution was added to a test tube containing 0.5mL Arthrobacter phage + 10µL of high titer lysate. This solution was quickly poured on to a plate, sat for 15 minutes and quickly placed in the incubator for 24 hours at 27 degrees Celsius.

Observations: Spot titer experiment performed on 11/16 during open lab results were used to help calculate titer. The formula used below to calculate titer below:

(22pfu/10µL) x (1000µL/1µL) x 10^8 = 2.2 x 10^11

Leaf for ML soil B was obtained to characterize tree. Tree classified as Quercus palustris.

Conclusions: High titer obtained, but the lysate was used to perform webbed plates. The experiment performed on 11/19 will be flooded on 11/20 and titer will have to be recalculated on 11/26 since the new obtained flooded lysate x9 has different phage concentrations.

Rationale: Flood 3x plate performed 11/7. A new flood lysate will be created (flood x4) and diluted to calculate titer. A secondary plate will also be webbed with flood lysate x3 to try to obtain a high titer.

Procedure: Before the experiment was performed, the workspace was cleaned with both Cidecon and 70% Ethanol. Plates were obtained from the incubator. 5mL of PB was added to the webbed plates to flood plates and shook for an hour. This solution was made to be x4 original flooded lysate. The formula below was used to make new plates that will be used to calculate titer (three plates for control and one for control):

• 2mL LB broth (x4)
• 2.5mL 2XTA (x4)
• 22.5µL CaCl2 (x4)
• Lysate
  • 1.15µl x 10^0
  • 10µL x 10^10-2
  • 10µL x 10^-3

Webbed plate using flooded original lysate x3 was used to make webbed plates for 11/14 if the experiment using x4 lysate failed, the same procedure would be used to create a newly flooded lysate using x3 flooded original lysate. The formula below was used to perform the experiment:

• 2mL LB Broth (x3)
• 2.5 2X TA  (x3)
• 22.5 CaCl2 (x3)
• Lysate x3 original lysate
  • plate 1 10µL
  • plate 2 20µL
  • plate 3 30µL

4.5mL of TA solutions were poured into test tubes containing 0.5mL Arthrobacter phage + lysate, which were quickly poured onto plates. Plates sat for 15 minutes and placed in the incubator at 27 degrees Celsius for 24 hours.

Observations: Control performed on 11/7 showed contaminations. Webbed plates x1 and x2 from 11/7 experiments showed contaminations, and x3 did not show contaminations. Webbed plate was used and flooded to create newly flooded lysate x4 original lysate. The lysate made 11/12 was used to create an x4 webbed plate. A secondary experiment was performed using flooded lysate x3 to be used as a back up if experiments from 11/12 fail.

Conclusions: If experiments performed 11/12 fail, flood x3 lysate plates created on 11/12 to make an x5 lysate. If experiments from 11/12 are successful, calculate titer. High titer will result in the next step of the procedure, and a failed experiment would result in recalculations and repeated experiments to obtain a high titer.