Phage

PhageBy: Ethan and NoahPurposeTo collect, recreate and analyze phage from the environmentTo sequence DNA from phage

What is a phage?VirusSmaller than bacteriaFound almost anywherePhage TherapyUsing known phage to cure bacterial infectionsSometimes involves reprogrammingMy PhageCollected from base of tree in River Falls, WITaken from moist soil conditionsAbout 44mm below surfaceNamed Lola13

Enrichment of Environmental SamplesMaterials: Soil, Sterile water, Sterile 10 x LB medium, calcium chloride, and bacteriaProcedure:Soil was collected around HudsonThe soil was weighed out and added to a flaskAseptically, water, LB medium, CaCl2 and bacteria was added, and then incubated at 37C for approximately 48 hoursHarvesting and Preparing the Enriched SampleMaterials: Soil sample prepared, phage buffer, microcentrifuge tubes, conical tubes, top agar, and agar plates\Procedure:Pour part of the prepared soil sample into a conical tube and centrifugePour the supernatant into a filter unit, making it the 100 sampleThen take four microcentrifuge tubes and add phage buffer to eachTransfer part of your sample into the first tube, and vortex itContinue this for the rest of the tubes and get tubes of the host bacteriaTransfer your sample into a culture tube and allow to sit in room temperatureThen pour top agar into each culture tube and then transfer the solution onto agar plates and allow them to solidify and then incubatePhage Titer, and Streak PlateMaterials: Sterile inoculating loops, agar plates, and top agarPhage Titer:Count the number or plaques on a countable plateUse the following equation to determine phage titer:# pfu X 1000 ul x 1 = pfu/ml 50 ul 1ml dfStreak Plate:Grab an agar plate and divide into a T and number 1-3Using an inoculating loop, pick one plaque and spread it into one areaUsing a clean inoculating loop, spread a little in area #1 and go into area #2Using a clean inoculating loop, spread into area #2 and go into area #3Take TA and spread onto the least concentrated area first and then incubate

Phage PurificationMaterials: Inoculating loop, phage buffer, host bacteria, and top agarProcedure:Get microcentrifuge tubes and fill with phage bufferUsing an inoculating loop, pick one plaque and place into a microcentrifuge tube and vortex itThen transfer part of your sample into the next microcentrifuge tube and vortex itContinue this for the rest of your tubesTransfer part into culture tubes and allow to sitAdd top agar to each culture tube and spread out evenly over the agar platesRepeat this procedure two more times to get a good, purified phageHarvesting a Phage LysateMaterials: Agar plates, phage buffer, conical tubes, filter system, microcentrifuge tubes, top agar, host bacteria, and inoculating loopsProcedure:Figure out which plate represents a web plate and add phage buffer to it and swirl it gently and wait for an hourCollect the phage buffer into a conical tube and label as unfilteredFilter the sample into a microcentrifuge tube and label as 100Grab more tubes and put phage buffer into them and perform dilutions through 1010Perform a spot test by dividing up an agar plate, and taking part of each sample in each microcentrifuge tube and putting it on the plate and spreading top agar over itThen transfer some of each sample into culture tubes and wait.Add top agar to each tube and spread out onto agar plates evenly

Phage Titer and Web PlatesMaterials: Top agar, phage buffer, agar plates, microcentrifuge tubes, MTLPhage Titer:Count the number of plaques on a countable plateUse the following equation to determine phage titer:# pfu X 1000 ul x 1 = pfu/ml 10 ul 1ml dfWeb Plates:Pick which plate looked the most like a web plateSet up enough dilutions to get to that plateThen take enough of your sample into ten host bacteria tubes and waitAdd top agar to each tube and then spread out evenly over the ten agar platesHarvest Web PlatesMaterials: Phage buffer and vacuum filterProcedure:Add phage buffer to each web plate and allow to sit for an hourPipette the mixture into a conical tube and centrifugeThen using a vacuum filter, filter the sample into another tube and properly label both tubesIsolate and Purify Genomic DNAMaterials: Phage buffer, Nuclease Mix, HTL, phage precipitant, top agar, and host bacteriaProcedure:Transfer some of the filter-sterilized phage lysate into an Oak Ridge Tube and store the remaining lysate at 4CAdd Nuclease Mix and mixIncubate for 30 minutes and then let sit at room temperature for an hourPipette phage precipitant solution to the nuclease treated lysate and capMix through inversion and let sit overnightPhage Titer ProcedureAdd phage buffer to enough microcentrifuge tubes and do enough dilutionsAdd to culture tubes and let sitAdd top agar and spread out over the agar plates and incubateIsolate and Purify Genomic DNAMaterials: Microcentrifuge tubes, Clean Up Rosin, distilled water, isopropanol and TEProcedure:Centrifuge the Oakridge tube and decant the supernatantAdd distilled water to the pellet and resuspend itAdd Clean Up Resin and swirl itAttach 1 column to a syringe and apply the mixture through the columnAdd isopropanol to the column and push through itCentrifuge the column and apply pre-warmed TE to the resin and let it sit and then centrifugeRepeat for a second column and then combine the DNA samples into a single tube and store at 4CPhage Titer:Count the number or plaques on a countable plateUse the following equation to determine phage titer:# pfu X 1000 ul x 1 = pfu/ml 10 ul 1ml df

Gel Electrophoresis and DNA QuantificationMaterials: Agarose powder, TAE buffer, gel red, DNA ladder, Loading dye, TEGel Electrophoresis:Add agarose powder to an Erlenmeyer flask and add TAE bufferMicrowave and allow the solution to boil overAdd gel red and pour into the gel electrophoresis plate and allow to hardenPull out the comb and flood with TAE bufferInject a DNA ladder in the first wellIn a microcentrifuge tube add TE, DNA, and loading dye Vortex the solution and inject it into a wellDNA Quantification:Put a sample of your DNA into a spectrophotometer and record the numbers and calculate out the amount of micrograms per microliterDigest Phage Genomic DNA, Set Up GelSolutions Tube Amounts12345610x Reation Buffer2ul2ul2ul2ul2ul2ulPhage Genomic DNA5ug5ug5ug5ug5ug5ug10x BSA2ul2ul2ul2ul2ul2ulBam HI1.5ulCla I2.5ulEco R I1.5ulHae III1.5ulHind III1.5ul Distilled WaterEnough to make 20ul totalMaterials: Reaction buffer, DNA, enzymes, water, agarose powder, and TAE bufferProcedure:First mix your DNA sample by flicking the closed tube and incubatePrepare each tube according to the table and store until readyGel Electrophoresis:Add agarose powder to an Erlenmeyer flask and add TAE bufferMicrowave and allow the solution to boil overAdd gel red and pour into the gel electrophoresis plate and allow to hardenElectrophoresisMaterials: Gel, cut up DNA, loading dyeProcedure:Add TAE buffer to the gel so that it is over floodedAdd dye to the cut up DNALoad the DNA ladder and samples 1-6 into the wellsTurn on to 100 volts Digest Phage Genomic DNASolutions Tube, Amount123410x Reaction Buffer2ul2ul2ul2ulPhage Genomic DNA5ug5ug5ug5ug10x BSA2ul2ul2ul2ulPst I1.5ulBcl I1.5ulNeo I1.5ulEco R V1.5ulDistilled waterEnough to make 20ul totalMaterials: Reaction buffer, DNA, enzymes, water, agarose powder, and TAE bufferProcedure:First mix your DNA sample by flicking the closed tube and incubatePrepare each tube according to the table and store until readyGel Electrophoresis:Add agarose powder to an Erlenmeyer flask and add TAE bufferMicrowave and allow the solution to boil overAdd gel red and pour into the gel electrophoresis plate and allow to hardenGel ElectrophoresisMaterials: Gel, cut up DNA, loading dyeProcedure:Add TAE buffer to the gel so that it is over floodedAdd dye to the cut up DNALoad the DNA ladder and samples 1-6 into the wellsTurn on to 100 volts Setting up Electron MicroscopyMaterials: Bacteriophage, phage buffer, parafilm, grid, distilled water, uranyl acetateProcedure:Transfer some HTL into a sterile microcentrifuge tube and centrifuge for an hourRemove most of the supernatant and add phage bufferRemove the cover paper from a piece of parafilm and place into a petri dishPlace a PELCO tab onto the parafilm and remove the linerRemove 2 grids and place shiny side up so the edge is touching the adhesivePlace part of the phage preparation onto each gridWait and then wick off the excessive fluid and then place water onto each gridWick off and then place more water onto each gridAdd uranyl acetate to each grid and then wick off and allow to air dryTransport the grids to an EM facilityResults (After 3 Enrichments)3 different morphologies developedCreated a streak plate for eachSmaller and bigger morphologies purified very wellTitrated each out separately with 4 dilutions

Results (After discovering morphologies)Smaller morphology had plaques to 2nd dilutionBigger had plaques to 4th dilutionSmall titer:4.6x10^-4 pfu/mLBigger titer:5.4x10^-6 pfu/mLRepeat purificationResults (after repeating purification)Smaller morphology disappeared. Bigger morphology purified to all plates (8 in 10^-2, 2 in 10^3 and 1 in 10^-4)

Spot test to determine web plateCountable plate to determine titer

Titer: 2.8x10^-4 pfu/mL

Web PlatePlate webbed very wellSmall morphology appeared againGoing to set up 10 web platesHarvest HTL and TiterAll 10 plates completely plaqued outTiter came out to be 2.75x10^-9 pfu/mL

DNA QuantificationSpectrophotometer gave me .119 mg/mL

Restrict and analyze phage DNASolution123456Reaction buffer2 L2 L2 L2 L2L2 LPhage DNA4.2 L4.2 L4.2 L4.2 L4.2 L4.2L10x BSA2 L2 L2 L2 L2 L2 LBamHI1.5 LClaI2.5 LEcoRI1.5 LHaeIII1.5 LHindIII1.5 LH2O11.8 L10.3 L9.3 L10.3 L10.3 L10.3 LRestrict and analyze phage DNA

Hind IIIHaeIIIEcoRIClaIBam HIDNALadderRestrict and analyze phage DNA (again)Solution1234Reaction Buffer2 L2 L2 L2 LPhage DNA3.3 L3.3 L3.3 L3.3 L10x BSA2 L2 L2 L2 lPstI1.5 LBclI1.5 LNcoI1.5 LEcoRV1.5 LH2O11.2 L11.2 L11.2 L11.2 L

EcoRINcoIBclIPstILadderEM Picture

The tails are approximately 70 nmThe heads are approximately 30 nm longWhy choose my phage?I dont think we should use my phage. My phage was my own sample, but has a surprising resemblance to a Hudson phage.

Results - NoahNames: Pyero because it is a pretty cool namePhage Titer: The first time I did phage titer I got 2.72 x 104 pfu/ml, the second time I got 1.06 x 105 pfu/ml, the third time was 5.00 x 103 pfu/ml, the fourth time was 1.91 x 108 pfu/ml, and the final time was 6.52 x 1010 pfu/mlI got 1.41 x 10-1 ug/ul for my DNA quantification Photos of this Process

This picture is a spot test to help determine which plates would be webbed platesThis picture is my countable plate, which can be used for determining titerPhotos of this Process

This is my closest example of a web plate. For creating the web plates, I used 30 ul of the 10-3 My DNA sample is the sixth well from the bottom of the gel, and is being compared to a DNA ladderPhotos of this Process

This is a picture of the gel, being cut with various enzymes. From top to bottom, it goes Hind III, Hae III, Eco R I, Cla I, Bam H I, undigested DNA, and the DNA ladderThis is a picture of the gel, being cut with various enzymes. From top to bottom, it goes Eco R V, Neo I, Bcl I, Pst I, and the DNA ladderEM Pictures

The tails are approximately 60 nm longThe heads are approximately 30 nm longPhage Most Similar To

I believe that my phage is most similar to Jawanski however, there arent any real distinctive cuts in mine that I can compare using the program because of the lack of enzymesWhy Should We Choose my Phage? I dont believe that we should choose my phage It is one of the Hudson phage, which is common in this classroomIt seems remarkably similar in the gel electrophoresis results

ConclusionNeither of us think our phage should be selected. There is a large possibility we somehow ended up with the same phage.

Tail: 60 nmHead: 30 nmTail: 70nmHead: 30 nm