Title: Annotation of Phage Elesar

Date: 4 February 2019

Rationale: After learning how to operate and use HHPred, an almost-complete annotation of phage Elesar can be made and posted to DNA Master.

Tools: 

• Microsoft Surface Pro 5 Tablet
• DNA Master Computer Software
• HHPred Bioinformatics Tool

Procedure:

• Started DNA Master and loaded previous file “ExtractedFromFastALibraryElesar.fasta”
• Opened HHPred Bioinformatics Toolkit on a different tab
• Due to no significant BLAST hit for genes 16 and 17, the SIF-BLAST annotations were both marked as “NKF”
• The product sequences for genes 16 and 17 were loaded into the input box of HHPred
• Due to no significant HHPred results, the SIF-HHPred annotations were both marked as “NKF”

Results/Observations:

• Below is a screenshot of the HHPred results for gene 16, showing the <90% probability and e-value of 39:

• Below is a screenshot of the HHPred results for gene 17, showing a >90% probability but an e-value of 0.12, which is not an acceptable value:

• Below is a screenshot of the final annotations entered for genes 16 and 17:
• 

Conclusions/Next Steps: In order to finally finish the annotation, the Starterator and Synteny entries must be made, but first the skills must be learned. Once the training annotations are finally completed, the necessary skills will have been learned to finally move onto annotating NapoleonB’s genome.

Title: Exploring DNA Master

Date: 16 January 2019

Rationale: The purpose of this lab was to explore DNA Master and begin the process of learning how to annotate phage genomes. With the skills needed to navigate and use DNA Master, many different conclusions can be drawn from the output including protein function, nucleotide sequence, and more.

Tools:

• Microsoft Surface Pro Tablet
• DNA Master Software

Procedure: A presentation on a specific bacterial infection treatment by means of phage therapy was done before installing and running DNA Master for the first time. Due to technical difficulties, DNA Master did not run perfectly the first time. The program was uninstalled and reinstalled, as well as restarted multiple times to ensure that all necessary updates were completed and the necessary procedures could be run. Finally, after a reinstalling of the program,DNA Master began to run reliably. Once the program was running, the FastA file for bacteriophage Elesar was downloaded and run by the program. Once the FastA displayed the nucleotide sequence for the phage, an auto-annotation was selected and run. A BLAST was unable to be completed at the time of writing.

Observations/Conclusions:

• Elesar yielded 42,954 nucleotides and 67 possible genes
• 66 of the recognized possible genes were classified as ORF’s (open reading frames) while the one remaining feature was classified as “RNA”
• The program lists all of the recognized features and color codes them as to distinguish whether or not it is a forward or reverse reading frame
• Below is a screenshot of the listed genes that DNA Master recognized
• 

• A conclusion that can be made about this lab is that DNA Master has many different features and potential methods to map out and recognize characteristics of a genome, once the necessary skills are learned to navigate the program.

Next Steps: 

• The next step after learning how to use DNA Master is to analyze bacteriophage NapoleonB in order to discover its sequence and protein function.
• Continue to monitor and troubleshoot the DNA Master program for any future problems.

Title: Webbed Plates and Lysate Amplification

Date: 19 November 2018

Rationale/Previous Results: A spot titer was done on the previously flooded lysate and countable growth was seen on the spot for a 10^-8 concentration of the previous flood lysate (“flood lysate 8”). 22 plaques were counted and the titer was calculated to be 2.2 x 10^11, which is considered a very high titer. Pictures of the spot titer are attached. Now that a high titer has been achieved, archiving, characterization, and investigation will begin. A certain amount (>42.8 mL) of lysate is needed for this to happen, so more lysate will have to be made by webbing and flooding plates. Therefore, plates will be webbed and flooded in order to have a high enough volume of lysate to continue the characterization process. (Note: the picture indicates 15 plaques counted, however this was an error and the plaques were counted a second time and the correct number was found to be 22 plaques.)

Procedure:

Under an aseptic zone:

• the following recipe was used to make 6 webbed plates (+control):
  • 14 mL LB broth
  • 17.5 mL 2x Top Agar
  • 157.5 uL CaCl2

~ 4.5 mL transferred to tube containing Arthrobacter culture + 10 uL “flood lysate 8” (high titer). The mixtures were plated, solidified, and incubated for ~24 hours.

Conclusions/Next Steps: A new lysate with a different titer will be made with these webbed plates. They will be flooded with 8 mL of Phage buffer each, and stored until the titer can be confirmed. The lysate will also be filtered in order to assure phage purity, and once enough lysate is acquired, DNA extraction, characterization, and archiving can occur.

Title: Dilutions for a Flooded Plate

Date: 14 November 2018

Rationale: The webbed plates (30 uL and 10uL) webbed completely and were flooded before this lab entry. The flood lysate will be diluted and a titer calculation done in order to determine of the lysate is a high enough titer.

Procedure: Under an aseptic zone,

• The plates containing the flood lysate were filtered through a 22-micron top filter
• Once the lysate was filtered, 10 uL of the lysate (“Flood Lysate 7”) was transferred to a micro-centrifuge tube containing 90 uL phage buffer to create a 10^-1 concentration diluted lysate
  • 10 uL of the 10^-1 was then transferred to a second micro centrifuge tube containing 90 uL of phage buffer
    • This process was repeated until a 10^-4 concentration lysate was created
• Four plaque assays were then done (+ a control) to determine the titer. The following recipe was used:
  • 10 mL LB Broth
  • 12.5 mL 2x Top Agar
  • 112.5 uL CaCl2

~ 4.5 mL transferred to tube containing 0.5 mL Arthrobacter + Plate 1) 10 uL 10^-2 lysate

Plate 2) 10 uL 10^-3 lysate

Plate 3) 10 uL 10^-4 lysate

• The plates were then placed into the incubator

Results/Conclusions: Below are the results of the previous experiment. The titer of the previous flood lysate was too low to be acceptable, and the control showed a growth, but it likely resulted from a mistake in the incubator, as it was clean when checked the previous day. If the titer of the plates done in this experiment do not meet the acceptable value, more plates will be webbed and flooded.

Title: Webbed Plate and Flooding

Date: 12 November 2018

Rationale: The 3x plate from the previous experiment contained the most plaques, so it will be flooded in this experiment. A new flood lysate will be created and diluted in order to calculate a new amplified titer. A secondary plate will  be webbed in order to immediately flood in case the flood titer fails to reach the acceptable limit.

Procedure: Under an aseptic zone,

• 5 mL phage buffer added to plate and shaken for 1 hour
• Transferred to 15 mL conical vial to create a flood lysate
• Dilutions made by transferring 10 uL flood lysate into a micro-centrifuge tube containing 90 uL Phage Buffer
  • Dilution process repeated until a 10^-3 dilution was created
• 3 plaque assays (+control) were made using the following recipe:
  •  8 mL LB Broth
  • 10 mL 2x Top Agar
  • 90 uL CaCl2

~ 4.5 mL transferred to tube containing 0.5 mL Arthrobacter + Plate 1) 50 uL 10^0 lysate

Plate 2) 10 uL 10^-2 lysate

Plate 3) 10 uL 10^-3 lysate

• 3 more plates were made in order to ensure a plate would be webbed in the next lab in case of titer failure using the following recipe/procedure:
  • 6 mL LB Broth
  • 7.5 mL 2x Top Agar
  • 67.5 uL CaCl2

~ 4.5 mL transferred to tube containing 0.5 mL of Arthrobacter + Plate 1) 10 uL original flood lysate

Plate 2) 20 uL original flood lysate

Plate 3) 30 uL original flood lysate

Conclusions/Results: Only the 3x web calculation plate from the previous experiment yielded a decent amount of plaques (pictured). If the flood lysate fails to yield a high titer, a plate will be webbed and ready to flood to try and get a high titer and in order to save time.

Title: Webbed Plate

Date: 29 October 2018

Rationale: After pairing with a lab partner, different methods will be used in order to assist in obtaining a high titer lysate for an already discovered phage.

Procedure: Under an aseptic zone, 2 plates were webbed using a confirmed positive lysate using the following recipe:

• 5 mL 2x Top Agar
• 4 mL LB Broth
• 45 microliters CaCl2

~4.5 mL pipetted into a test tube containing 0.5 mL Arthrobacter culture + either 20 or 30 microliters of positive lysate (one plate was done with 20 microliters and another done with 30 microliters in order to assure a complete lyse and to maximize chances of obtaining a high titer.) THe plates were solidified for 15 minutes and put into the incubator.

Conclusions: In an attempt to raise the titer of the lysate (currently 5.7 x 10^4), the webbed plates were done with double and triple lysate quantities to fully web plates and provide favorable environments for flooding. Assuming one or both plates web completely, they will be flooded in the next lab and the titer checked after a plaque assay.

Title: Soil Sample 3 Enrichment & Metadata

Date: 17 October 2018

Rationale: A 3rd soil sample was collected from a planted tree in front of Earle Hall, the soil will be washed and enriched as well as the metadata of the soil taken. The resulting lysate will be incubated and tested in the following week.

Procedure: Aseptic zone created by washing lab bench with CiDecon and Ethanol, and by lighting an ethanol lamp

• 2 mL of collected dirt added to 15 mL conical vial
• LB Broth added to ~12 mL mark
• Shaken/vortexed for 10 minutes
• Centrifuged for 5 min @ 3000 G
• Filtered through 22 micron Top Filter
• 0.5 mL of Arthrobacter added to ~7.5 mL of filtered lysate
• Placed into shake incubator

The metadata of the soil was also taken with the following procedure:

• % Water:  a small amount of soil was placed into a weigh boat and massed at 11.26 g, the soil will be left to evaporate and will be weighed after evaporation, the % water composition will then be calculated
• % Sand, Silt, Clay: ~4 mL of dirt added to falcon tube, DI water added to 12 mL mark on tube. 3 drops of Soil Dispersion Liquid was added, the mixture was shaken for 30 seconds, and the mixture was placed under the fume hood to disperse.
• pH: A small amount of dirt was added to a small pH vial with DI water and shaken for 10 seconds. A pH strip was then inserted into the mixture and the pH was then recorded.

Conclusions: With the new soil sample, another attempt at finding phage can be run. A spot test and a plaque assay will both be done to test phage presence, and picked if positive results are observed. The pH of the soil was recorded at 6.5 and the rest of the metadata will be recorded in the next entry.

Title: October 15, 2018

Date: 15 October, 2018

Rationale: The previous soil sample tested negative for phage, and can no longer be tested. Due to the weather conditions at the time, the collection of a third soil sample was not possible. By the next lab, a 3rd soil sample will have been collected and will be ready for enrichment and testing.

Conclusions: Both soil samples thus far have yielded conclusive evidence that phage is not present. A third soil sample from a different location will be collected to hopefully find phage. Attached are the results of the 2 most recent plaque assays, one pick resulted in no growth, while the other yielded a negative result due to contamination (the top agar control was contaminated).