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1 Bacteriophage Lab: Eradicator123 SEA-PHAGES: Bacteriophage Lab: Eradicator123 Biology 112 Section 5 Fall 2020 Ms Erin Tyll 12/1/20 Abstract:

2 Bacteriophage Lab: Eradicator123 The discovery of a novel bacteriophage resulted from multiple experiments following DNA isolation, purification, amplification, and extraction. The phage was named “Eradicator123.” “Eradicator123” is a Siphoviridae phage, as it contains a long, flexible, and noncontractile tail. The novel phage discovered from Woodland Park has a head diameter of 12.7 nm, a tail length of 50 nm, and a 260/280 of 1.865. This number indicated that the phage was close to being pure. Introduction: The purpose of the Sea-Phages project was to isolate an unprecedented bacteriophage from an environmental sample. Bacteriophage analysis provides a better understanding of the way life works, as it gives an insight of certain organismal responses to these bacteria. Analyzing bacteriophages also provides useful information on different ways to control bacteria rather than using the standard antibiotics as a means of controlling diseases. Analyzing the DNA of the bacteriophages from these environmental samples helps detect the functional differences among the bacteriophages being studied. (Proxleitner, Pope, Jacobs-Sera, & Hatfull, 2018). Bacteriophages, which were first discovered around a century ago by Frederick W. Twortz and Felix D’ Herelle, are viruses that infect bacteria. Viruses can only be activated when they are present in a “host” cell. Viruses have many different structures, but the most common structure of viruses is a single stranded DNA surrounded by a protein called a capsid. This makes the “head” of the virus. Attached to the virus’s “head” is a “tail,” which is where the DNA of the virus gets injected from into the bacterial host. Phages can only bind to and infect certain bacterial hosts. (Proxleitner, Pope, Jacobs-Sera, & Hatfull, 2018). In order for a bacteria to be in a phage's host range, the phage has to recognize the receptors found in the cell wall of the bacteria.While the phage is on the cell, it has to work closely with the translation and transcription networks present. (Proxleitner, Pope, Jacobs-Sera, & Hatfull, 2018). The bacteria being worked with during this lab is mycobacterium smegmatis . Mycobacterium smegmatis is found mostly in soil, water, and plants. It is not a disease causing phage and is classified as a

3 Bacteriophage Lab: Eradicator123 saprophyte. M.smeg is 6,988,209 nucleotides long, and it consists of 67% guanine and cytosine and 33% adenine and thymine. There has been one case in which this virus became virulent after someone’s infection. In this case, the infected person had two mutant alleles in his genome. Apart from this, mycobacterium smegmatis is generally considered nonpathogenic. (Mycobacterium smegmatis, 2011) Method and Materials: The following provides a general list of materials that were used frequently throughout the lab procedures. The Sea Phage Discovery Guide, however, provides all the materials needed to perform each specific lab procedure. 1. Host Bacteria 2. Top Agar 3. Environmental Sample 4. Microcentrifuge Tubes 5. Pipettes 6. Pipettors 7. Bunsen Burner For each of the following activities, the aseptic technique was used to clean the bench before and after the lab activity was performed. Collecting Environmental Samples and Direct Isolation-5.1, 5.2 The soil was collected and brought to the lab. The following conditions were recorded: who the soil was collected by, the date discovered, the sample type, the temperature, the GPS reading, the general location, and the sample site and description. When the sample is brought to the lab, the direct isolation

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4 Bacteriophage Lab: Eradicator123 protocol is performed. Combine m.smeg with 3 mL of TA. Mix by drawing into the pipette. Pour on the plate,label with table #, section, and date. After agar solidifies, spot 3 mL. Transfer 1.4 mL of enrichment into the microcentrifuge tube. Centrifuge to pellet the bacteria, and transfer the supernatant to a clean microcentrifuge tube. Preparing Phage Filtrate, Plaque Assay, Enriched Isolation-5.2,5.3,5.5 In direct isolation, a 15 mL conical tube was filled ⅓ to ½ way with soil. The tube was capped and inverted several times to mix thoroughly. After this, the tube was incubated while shaking for 1-2 hours. The sample sat for 20 minutes. 2 mL were removed from the syringe from the top of the liquids sample. The syringe was fastened to the top of the filter, and the top of the filter was taken out of the package quickly to prevent contamination. The filter was then screwed tightly. 0.5 mL of the filtrate was dispersed into the microcentrifuge tube, making sure the filter was not clogged. The 50 uL of phage filtrate was dispersed into the 250 uL sample of m.smeg using a micropipette. The combination of the phage and host bacteria was mixed by gently tapping the tube The sample sat undisputed for 10 minutes. The filter was then attached to the vacuum filtration unit. The enriched sample was poured into the filtrate. The provided plate was labeled with section, table number, and “Direct Isolation. 3 mL of TA in inoculated tube was drawn using the 5 mL pipette. The mL of top agar was sucked into and transferred onto a plate. After the enriched sample was filtered, the filtrate was capped. 0.5 mL of the host bacteria m.smeg was sucked into the 1 mL pipette and transferred into the filtrate. The bacteria mixed with the filtrate will shake for 2-5 days. Spot Test- 5.6 Combine 250 uL of m.smeg with 3 mL of TA. Mix by drawing into the pipette. Pour on the plate. Label with table #, section, and date. After agar solidifies, spot 3 mL. Transfer 1.4 mL of enrichment into the microcentrifuge tube. Centrifuge to pellet the bacteria. Transfer the supernatant to a

5 Bacteriophage Lab: Eradicator123 clean microcentrifuge tube Picking a Plaque, Serial Dilutions, Plaque Assay for Purification- 5.4, 6.1, .2 Place the tip on the P200 micropipette. Hold the pipette to the agar surface and gently stab the agar. Place the end of the tip into the phage buffer in the corresponding microcentrifuge tube. Tap the tip of the wall of the tube and pipette up and down to dislodge the phage bacteria. Mix by vortexing. Take out 3 microcentrifuge tubes and label 10^-1,10^-2, and 10^-3. Put 90 uL of phage buffer into the microcentrifuge tubes. Combine 10 uL of each dilution with 250 uL of m.smeg. Wait 10 minutes and while waiting, label the provided plates. Add 3 mL of TA into the tube. Draw up the pipettor to mix and pour onto the plate to solidify Collecting Plate Lysates, Spot Titer, Serial Dilutions- 6.3, 6.4, 6.2 The bacterial lawn was prepared. First, a grid was drawn on a webbed plate. Mix 3 mL of TA with 250 uL of m.smeg(10^-3). Gently mix both the pipette and pour on plate.The next step was to harvest a lysate. A 0.22 um filter was prepared. The filter was opened but not removed from the package. A 5 mL syringe was used to suck lysate from the plate. The syringe was attached to the filter. The syringe plunger was depressed and the filtrate was collected in a 15 mL conical tube.The lysate was used as the 0 for the serial dilutions. Take out 8 microcentrifuge tubes. Add 90 uL of phage buffer into each tube. Add 10 uL of the 10^0 into the 10^-1 tube, and keep adding 10 uL of each dilution into the next labeled tube. Spot 3 uL of each sample and pipette onto the labeled plate. Full Plate Titer-6.5 8 microcentrifuge tubes were taken out and labelled from 10^0 until 10^-8. 90 uL of phage buffer was added into each of the microcentrifuge tubes. 10 uL of the 10^0 was added into the first tube, and 10 uL of each dilution was added into the next labelled tube. 3 uL of each dilution was added onto its

6 Bacteriophage Lab: Eradicator123 labelled section on the bacterial lawn. When the Spot Titer is complete, the dilutions with a countable number of plaques will go through a plaque assay. For the plaque assay, a 10^-1 serial dilution was prepared. 3 ul, 5 ul, and 7 ul of the 10^-1 serial dilution was mixed into three separate tubes of 250 ul of m.smeg.After waiting 10 minutes, 3 mL of top agar was mixed into the each of the tubes of m.smeg and phage concentration. Immediately after the top agar was mixed, each mixture was plated on three individually labelled plates. The Titer was calculated using the following formula: Titer (pfu/ml) = (# pfu/ volume used in μl) x (103 μl/ml) x dilution factor* Making Webbed Plates from a Lysate-7.1 The number of plaques were estimated and the volume of lysate was calculated. A plate with a high density of plaques was picked. The amount of plaques in one quadrant were counted. The value for how many more plaques were needed to make a web plate. The volume needed to make a web plate was calculated. The dilutions were then performed, following the Serial Dilutions method. The dilutions were then plated in order to create webbed plates. The proper number of agar plates and host bacterial cultures needed to plate the dilutions. The bacterial host cells were then infected with the volume that was appropriate for it. The mixture was then incubated for phage attachment to happen. Each plate may be diluted two or three times. Without inverting, the plates were incubated. The best webbed plate was then chosen. Phage DNA Extraction- 9.1 The first step of this procedure is to degrade the DNA/RNA in a high-Titer phage lysate. 1 ml of the phage lysate was transferred into a microcentrifuge tube. 5 uL of nuclear is mixed into the lysate. This is then incubated at 37 degrees celsius for 10 minutes. The next step is to denature the protein capsid, which was initially done by adding 2 ml of DNA clean up resin into a 15 mL conical tube. The tube was

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7 Bacteriophage Lab: Eradicator123 then inverted. After this, the phage DNA was isolated. Two Wizard Kit columns were labelled, and the plungers were removed from two 3 ml syringes and a column was attached to each syringe barrel. The column and syringe were set on a new microcentrifuge tube, and then a pipette was used to transfer 1.5 ml of phage DNA/resin solution to the column. A plunger was put into the syringe and all the liquid was carefully pushed into the colonial tube. The column was then unscrewed from the syringe barrel, and then the plunger was released and the column was set into a clean microcentrifuge tube. The plunger was then removed from the syringe barrel and then the syringe barrel was reattached to the column. After these steps, the salts were washed from the DNA. Firstly, 2 mL of 80% isopropanol was added to each syringe column. A plunger was put into the syringe and all the liquid was carefully pushed through, which collected the flow-through in the used 15 ml conical tube. When each column was transferred into a 1.5 mL microcentrifuge tube, each tube was spun at 10,000 × g for 5 minutes. The tubes were then transferred to new 1.5 mL microcentrifuge tubes and spun for another minute at 10,000 × g. The columns are then removed from the microcentrifuge tubes and placed directly in a 90 degrees Celsius heating block for one minute to evaporate the last traces of isoprenol. Each column is then placed into a new microcentrifuge tube and 50 uL of 90 degree Celsius sterile ddH2O is added into each column. Protocol 10.2- Setting Restriction Enzyme Digests Once seeing the concentration of DNA, the volume of the DNA sample needed to be obtained was calculated. 0.5 ug of DNA was calculated. Six microcentrifuge tubes were prepared and labelled with each of the restriction enzymes. 18.4 ul of the sterile diH20 was added into each microcentrifuge tube. 2.5 uL of the corresponding buffers were added depending on the enzyme going to be used in the microcentrifuge tubes. 3.6 uL of the phage genomic DNA was added into the microcentrifuge tubes. 0.5 uL of the restriction enzymes were added into the microcentrifuge tubes. The enzymes were added last, the uncut

8 Bacteriophage Lab: Eradicator123 DNA had extra 0.5 uL of diH20 rather than an enzyme. All the contents in the tube were mixed gently and quickly spun in the microcentrifuge tubes for less than a minute. Incubate at 37 deg celsius for an hour. Protocol 10.3- Gel Electrophoresis of Restriction Enzyme Digests Before attending the lab, the restriction enzyme digest samples were prepared for the electrophoresis. During the lab activity, the gel was loaded with the proper volume of DNA ladder. 20 ul was pipetted from each RE reaction to the wells. The pipette plunger is slowly depressed to allow the solution into the cell. The pipette is removed from the gel before releasing the plunger. The electrodes are plugged into the appropriate locations on power supplies. The gel is run until the blue dye has migrated about 3.5 inches from the well. This takes approximately an hour. Results: Figure 1 shows the direct isolation sample from 9/14/20, which gives a picture of the possible bacteriophages that can infect the m.smeg . Figure 2 is of the enriched sample. The zones of clearing, or plaques, are caused by the lysis of the bacteria due to the infection and replication of the bacteriophage. Figure 3 is of the spot titer from 9/11.The 10^1 to 10^7 dilutions from this plate were used to perform the Full Plate Titer. Figure 4 is of the sample of plate purification from 9/15. This 10^0 plate has many small clear spots, which allowed for plaques to be picked. Serial dilutions were used to create a well concentrated sample that was used for titer plates. Figure 5 is of the Webbed plate sample during phage amplification. In the displayed picture, the webbed plate at the top was created to help amplify the mycobacterium phage to help make additional lysates for DNA extraction. Figure 6 shows the DNA Extraction Spectrophotometer results. The concentration of phage DNA is determined. This was used to set up Restriction Enzyme Digests. However, the concentration of DNA

9 Bacteriophage Lab: Eradicator123 was calculated based on the formulas provided in the SEA PHAGES manual. Figure 7 shows the electrophoresis results. From these results, it can be concluded that there was a low concentration of DNA and the DNA did not migrate. Figure 8 are the electron microscopy results, which indicates that Eradicator123 is a Siphoviridae phage, as it contains a long, flexible, and noncontractile tail. The novel phage discovered from Woodland Park has a head diameter of 12.7 and a tail length of 50 nm . Legend: TA: Top Agar(stored at 55 deg Cel) Deg Cel: Degrees Celsius Figure 1: Direct Isolation This plate shows the direct isolation sample from 9/14/20, which gives a picture of the possible bacteriophages that can infect the m.smeg.

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10 Bacteriophage Lab: Eradicator123 Figure 2: Enriched Sample This is the enriched sample. The zones of clearing, or plaques, are caused by the lysis of the bacteria due to the infection and replication of the bacteriophage.

11 Bacteriophage Lab: Eradicator123 Figure 3: Spot Titer The 10^1 to 10^7 dilutions from this plate were used to perform the Full Plate Titer.

12 Bacteriophage Lab: Eradicator123 Figure 4: 10^0 Plate Lysate Purification Figure 5: Webbed Plates- Amplification

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13 Bacteriophage Lab: Eradicator123 Figure 6: DNA Extraction Spectrophotometer The concentration of our phage DNA resulted in 35.6 ng/uL - A-260 wave: 1.746 - A-280 wave: 0.9375 - 260/280: 1.865

14 Bacteriophage Lab: Eradicator123 Figure 7: Gel Electrophoresis

15 Bacteriophage Lab: Eradicator123 Figure 8: Electron Microscopy The capsid diameter is measured to be 1.27 cm(12.7 mm) and a tail length of 3.81 cm(38.1 mm) and a 100 nm size bar to be 1.905 cm(19.05 mm.) Calculation for determining Phage size: (100 nm)/(19.05 mm)=(unknown scaled size)/(38.1) Unknown scaled size tail=50 nm Discussion: The direct isolation activity performed on 9/14 gave a possible picture of all the bacteriophages present in the sample. The following samples could have possibly been contaminated throughout the lab activities. On 9/22, a new soil sample was used from another group. It is concluded that the previous sample was contaminated due to improper pipetting..While performing the Serial Dilution and the Spot Titer, contamination was present in the results and the bacterial lawn.This can be due to not staying in the zone of sterility. Due to the inconclusive results, the protocols had to be repeated several times until there was no contamination in the final plates. On 10/19, the mycobacterium smegmatis was contaminated and had to be regrown. This contamination affected the results of the Full Plate Titer, which had to be repeated. On 11/2, after looking at the gel electrophoresis results, the DNA could have been contaminated, meaning foregin material was present. Furthermore, the enzyme was not able to cut the DNA properly. The smaller fragments should have been towards the positive and the larger fragments should be towards the negative; in our case it was the opposite. In DNA Extraction, the 260/280 ratio indicated that the phage

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16 Bacteriophage Lab: Eradicator123 was close to being pure because The closer the 260/280 ratio of the waves to 1.8 the more pure our phage DNA is. The electron microscopy results showed that Eradicator123 is a Siphoviridae phage, as it contains a long, flexible, and noncontractile tail. These final results were achieved due to performing the lab activities more carefully, which prevented contamination and improper pipetting. References: Proxleitner, M.,Pope, W.,Jacobs-Sera, D., Sivanathan, V. & Graham, G. (2018). SEA-PHAGE Discovery Guide ; Howard Hughes Medical Institute, Chevy Chase, MD https://microbewiki.kenyon.edu/index.php/Mycobacterium_smegmatis#:~:text=Mycobacterium%20 smegmatis%20is%20a%20Gram,of%20different%20species%20of%20bacteria .

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