November 7th, 2018
Shepard Saabye

Objectives and Rationale:
Find and amplify the titer of Justin’s lysate in order to mark the phage in TEM.

Previous Results: Cooper and Michael used a 10^-4 titer lysate, and counted 27 plaques on an agar plate that slipped. The plaques were .4 millimeters in radius on average, and the agar that remained on the plate was 35 millimeters in radius. This means the overall titer of the lysate was 2.7*10^7, just shy of a medium titer.

• Procedure:
• Found total number of plaques to web plate (1914),
• Divided plaques needed by the titer, (1914/2.7*10^7) to find the lysate required to make enough plaques to web the plate (7.09 uL of 10^-2)
• Added 8mL LB Broth and 90 uL CaCl to a 50 mL tube
• Collected 3 vials containing .5 mL Arthrobacter
• Added 7.09 uL 10^-2 Lysate to the first vial
• Added 14.18 uL 10^-2 Lysate to the second vial
• Added 2.1 uL 10^-1 Lysate to the third vial
• Added 10 mL 2X TA to the 50 mL tube
• Pipetted 4.5 mL from the 50 mL tube to each vial containing arthro
• Poured all three arthro tubes and the remaining control 50 mL tube to their respective pre-marked plates
• Let sit and placed in incubator at 27 degrees overnight

Analysis and Interpretations: The titer needs to be very high for TEM, and it takes a lot of time to get a high titer. Its a very intensive process to properly web a plate.

Future Plans: Either repeat amplifying or run the TEM again after finding the titer.

Title: Titer Calculations

Date: 7 November 2018

Rationale: After the dilutions were run, the titer of the flooded lysate can now be calculated. The titer must be at the 10^8 level or above in order to be considered adequate or else another plate will be flooded in order to try and increase the titer and amplify the lysate.

Results/Calculations:

• The first picture is the plaque assay of the 10^-1 lysate, which did not yield countable plaques
• The second picture is the plaque assay of the 10^-2 lysate, which yielded many plaques but too many to reasonably count.
• The third picture is the plaque assay is the plaque assay of the 10^-4 lysate, which yielded 27 countable plaques. *NOTE: the top agar did not set correctly on many of the plates (including plates shown and not shown) and the radius of the plate was taken into account when a webbed plate calculation was done.)
• The fourth picture is the clean top agar control plate.

Titer: (27 pfu/10 uL)*(1000uL/1 mL) = 2.7 x 10^7 (the titer is a medium titer and is not considered high enough, therefore another webbed plate will be done and flooded.)

Webbed plate calculation: Aplate/Aplaque = plaques needed to web –> 1225*pi(short radius from top agar shift used)/(0.8)mm^2*pi = 1914 plaques to completely web a plate

1914/2.7×10^7 = .00070889 mL to web a plate or .070889 microliters to web a plate (7.09 of 10^-2 lysate was used in order to more accurately measure this amount)

Procedure: Under an aseptic zone, 3 plaque assays + a control will be made in order to ensure a webbed plate. One plate will be done with the calculated web amount, another plate will be done with twice the calculated amount, and a third plate will be done with three times the calculated amount.

The following recipe was used for the plaque assays:

• 8 mL LB Broth
• 10 mL 2x Top Agar
• 90 microliters CaCl2

~ 4.5 mL transferred into a test tube containing 0.5 mL Arthrobacter + 7.09 uL/14.18 uL/2.127 uL(*) lysate. The plates were left to cool and then placed into the incubator to grow.

(*)Ran out of 10^-2 lysate so 2.127 of 10^-1 lysate was used instead to maintain the integrity of the concentration.

Conclusions: The titer from the previous experiment was too low to be considered acceptable, so the webbed plate will then be flooded in an attempt to amplify the titer of the lysate and hopefully achieve a titer of 10^8 or greater.

Title: Titer Dilutions

Date: 5 November 2018

Rationale: The plate from the previous experiment lysed completely and the bacteria began to regrow (picture in results/observations section). Dilutions will be done of the lysate and plaque assays will be run in order to determine the titer of the lysate.

Procedure: Under an aseptic zone,

• 10 microliters of the flood lysate was transferred into a microcentrifuge tube containing 90 microliters of phage buffer. The resulting lysate was marked as “10^-1” to represent a 10^-1 serial dilution.
• 10 microliters of the 10^-1 lysate was then transferred into another microcentrifuge tube containing 90 microliters of phage buffer. The resulting lysate was then marked as “10^-2” to represent a 10^-2 serial dilution.
• This process was repeated until a 10^-6 dilution was made and 6 different lysates were available.

Six plaque assays + a negative control were run in order to determine titer, and the following recipe was used:

• 14 mL LB Broth
• 157.5 microliters CaCl2
• 17.5 mL 2x Top Agar

~4.5 mL transferred into test tube containing 0.5 mL Arthrobacter + 10 microliters of 10^-1/-2/-3/-4/-5/-6 (depending on plate label). The plates were set for 15 minutes and placed into the incubator to grow.

Results/Observations: The following picture is a picture of the plates from the previous experiment:

Conclusions: The plates from the previous experiment likely lysed completely because they were left in the incubator for too long, so the results are inadmissible. In the next lab, the dilutions will be inspected and a titer calculated.

11.7.18 Plaque Assay for High Titer

Rationale: Since the results from Monday (11/5) showed inconsistent results and problems with slipping top agar overlays, it was found necessary to redo the procedure to get five new plates that will be used to begin to raise the titer of the lysate.

Procedure:

Conclusions and Next Steps:

• The next step for this procedure will be to flood the plates and use the new lysate of higher concentration to run another plaque assay. This will likely result in a high-titer lysate, which is the end goal of the procedures that have been run. Based on results from Monday, the inconsistent amounts of LB Broth did have a negative effect on the ability of Top Agar overlay to set properly on the plate. This will be accounted for and be done more carefully in future experiments using the Plaque Assay procedure.

10/05/18

Objective:

• To make a webbed plate from adopted lysate
• To pick a plaque and make another plaque assay

Procedure:

Webbed Plate:

1. Add 300 μl of lysate to 0.5 ml of arthrobacter culture and allow the sample to enrich for 15 minutes
2. 1 top agar is made for 2 lab partners.
3. While the tube enriches, add 4.5ml of LB broth to a conical vial
4. Add 67.5 μl of CaCl2 to the vial.
5. After the 15 minutes have passed, add 4 ml of TA mixture to the vial with arthrobacter and lysate.
6. pour contents of the vial onto an agar plate.
7. pour 4 ml of TA mixture onto another Agar plate for a control plate,
8. Allow the plates to rest for 15 minutes and then place them inverted in the incubator.

Purification:

1. Pick a plaque of the latest plaque assay using a micropipette and dip the tip in a microcentrifuge tube with 70μl of phage buffer.
2. Vortex microcentrifuge tube for 15 seconds.
3. Transfer 30μl of this phage extract to a 0.5 ml of arthrobacter and allow the sample to enrich for 15 minutes.
4. 1 plaque assay is made using the same LB broth and 2X TA as before.
5. Meanwhile, add 2 ml of LB broth to a conical vial.
6. Add 22.5μl of CaCl2 to the conical vial.
7. After 15 minutes have passed, add 2.5 ml of 2X TA to the conical vial.
8. Transfer all the contents of the conical vial to the test tube with the Enriched Arthrobacter.
9. Pour the contents of the test tube onto an Agar plate.
10. Allow TA to solidify for 15 minutes and then place the plate inverted in the incubator.

Analysis and Conclusion;

A webbed plate and a 4th purification run was done so as to preserve time. If this procedure does not result in a webbed plate, same procedures will be repeated for the new phage extract, titer will be calculated and another webbed plate will be attempted. The current lysate has a low titer so further purification might me needed, even if webbed plate is flooded.

November 5th, 2018

Shepard Saabye

Objectives and Rationale: Identify Phage using TEM in a lysate of unknown titer capable of lysing a plate. Identifying this phage will help in the classification of the phage and its characteristics.

Procedure:

• Calibrated TEM
• Allocated Petri Dish and Parafilm
• Placed a strip of parafilm securely inside petri dish top
• Placed a drop of lysate, 2 separate drops of water, and a drop of Uranyl less on the parafilm
• Using precision tweezers, placed a copper grid, shiny side down, on the lysate for 5 minutes
• Moved copper grid to the first drop of water for 2.5 minutes
• Moved copper grid to the second drop of water for 2.5 minutes
• Placed copper grid on uranyl less (UL) for 1 minute, and quickly blotted an excess UL once removed
• Used tweezers to move copper grid into TEM Grid Holder
• Moved Copper Grid to the preliminary Vacuum chamber in the Machine
• Closed Vacuum and observed the grid

Results:

No phages were found.

Analysis and Interpretations:

The titer may have been too low to effectively find phages. In addition, the Uranyl Less may have been ineffective compared to Uranyl Acetate.

Future Plans: We will either repeat the experiment with Uranyl Acetate, or work to increase the titer of the lysate used.

11.5.2018 Plaque Assay for High Titer Lysate

Rationale: Since the plaque assays from Friday (11/2) were visualized and calculated to have a titer of 6.8×10^7, it was found to be necessary to use lysate from Friday to create multiple plates of 10^-2 dilution to be flooded to obtain a lysate of higher concentration.

Procedure:

1. Established an aseptic zone.
2. Added 10μL of 10^-2 lysate to 5 different 0.5mL Arthrobacter. Let sit for 10 minutes.
3. Obtained 5 plates and labeled “CEW 11/5”.
4. Added 10mL LB Broth to conical tube.
5. Added 112.5µL CaCl2 to conical tube. Swished to mix.
6. Added 2mL of solution from conical tube to each tube with 0.5mL arthrobacter and lysate.
7. Added 2.5 mL 2X Top Agar to each tube. Swished and poured onto 5 separate plates.
8. Added 2mL LB Broth, 22.5µL CaCl2, and 2.5mL Top Agar to conical tube
9. Poured all tubes onto 6 separate plates (1 plate was top agar control and was labeled accordingly).
10. Let sit for 10 minutes before placing them in the incubator.

Results:

• The lysate obtained had a concentration of 6.8×10^7 pfu/mL. This was a medium titer. The results of the plates created on Friday (11/2) are shown below.
• There was very slight contamination found on the top agar control.
• 

Observations:

• Two of the five plates had top agar overlays that did not set correctly, which led to the overlay slipping on the plate. This was likely caused by small variations in the 2mL additions from the conical tube to the individual tubes with Arthrobacter. The other three plates were set correctly and should be able to be used as normal.
• Lysate was three days old when it was used. After usage, it was made known by Dr. Adair that it may not have the same effectiveness due to the degradation of phage. This may impact the results that are able to be seen on the plate.

Conclusions/Next Steps:

• Since the lysate did not have a concentration of 10^8 or more, it was not able to be used as a high titer lysate. However, it is getting closer to that value, and with the plaque assays done today it will likely be possible to flood the plates to obtain a lysate with a higher titer. Thus, it will be necessary on Wednesday to flood the functional plates and use the new lysate obtained to create new dilutions that would theoretically contain phage at a higher concentration than previously obtained.

Positive Sample Gel Electrophoresis
October 31st, 2018
Shepard Saabye

Objectives and Rationale: Use Gel Electrophoresis to test weather 10^0 Titer Lysate can be used to find phage, or if PCR is useless in our experiment.

Procedure:

• Retrieved PCR tubes from Freezer
• Added .40 mL 1x TAE to a flask
• Added 8 grams agarose to the flask
• Swirled and heated Agarose solution until dissolved
• Once warm, but not hot, added Ethidium Bromide to the solution
• Poured into the gel apparatus
• Set up electrophoresis machine
• Placed gel in the machine and added TAE until the gel was well submerged
• Added DNA Ladder and the 3 experimental DNAs into wells, as well as 3 positive controls
• Ran Electrophoresis for 40 minutes
• Imaged in UV imaging machine

Results: Found all tested samples to be negative, including the confirmed positive sample.

Analysis and Interpretations: Because the positive sample came out as negative, the whole base of using PCR to identify phages is shaky, even unlikely. We can run more tests to confirm, but based off two results, the 10^0 solution is not adequate for PCR.

Future Plans: I will be adopting Justin’s Phage and working to get to a high titer.

Positive Sample PCR
October 29th, 2018
Shepard Saabye

Objectives and Rationale: Complete PCR on a known sample, to understand if Phage DNA clusters are recognizable at a 10^0 titer.

Procedure:

• Filtered enriched lysate into a microcentrifuge tube
• Boiled Lysate to release DNA from Capsids
• Added 2 microliters DNA to 3 different PCR Tubes, for 3 people
• PCR Tubes contained 12.5 microliters of a 1x Taq Polymerase and dNTP master mix solution
• Added 4 microliters of each primer to its respective tube
• Added 2.5 microliters of DD water to bring total volume of PCR tube to 25 microliters
• Performed PCR on each PCR tube

Results: No results to report.

Analysis and Interpretations: Nothing to Analyze or Interpret.

Future Plans: Perform Gel Electrophoresis on DNA from PCR.