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Introduction Wild-type Method s •Plaques on Plate are bull’s eye, 2-3mm after 24hrs •Electron microscopy- pure population •Size of Genome: about 70,000 bp •Gel electrophoresis: cluster assignment H •Future Directions: Kill curve determination Results Discussion Adsorption of the phagedegradative enzymes or contraction of the sheath & tail @ cell wall Enzymes degrade bacterial DNA, genetic recombination lytic or a lysogenic cycle Michaelangelo temperate phage model: Mycobacterium smegmatis (M. smeg) We hypothesize that a bacteriophage can be isolated and purified from a soil sample; that it can be characterized into a cluster; and that infectivity can be determined in a kill curve soil sample enrichment Purification: streak test, and titer assay. Acknowledgments: Sea faculty Sea Teaching Assistants, Lee Graham, The Hatfull laboratory At Pittsburgh, HHMI, Department of Biological Sciences Figure 1) Agar plate with plaques in M.smeg bacterial lawn. Shown is a Bull’s eye morphology. Note the clear sterile center and the turbid outer rings Figure 2) a typical titer assay with plaques in the M.smeg bacterial lawn. Note the density of the plaques- web pattern Figure 4) Michaelangelo bacteriophage electron microscopy image. At the tip of the tail there is a clover shaped attachment site. Figure 3) Michaelangelo electron microscopy. Note the curved tail characteristic of siphoviridae, icosahedral capsid and 2.5:1 Head:tail ratio Figure 5) Restriction digest. Bands left to right: 1-kb ladder, undigested DNA, BamH1, Cla1, EcoR1, HaeIII, HindIII. Bam H1 did not cut, while HaeII, EcoR1 and HindIII cut. In particular, HaeIII cut into such small fragments it ran off the gel. Cla1 had the clearest bands Stock solution: Medium and high titer lysates DNA extraction: alcohol precipitation Restriction digest and gel eletrophoresis M. Smeg colony formation % survival and Kill curve claI Distance traveled (cm) log fragment length Fragment length (bd) estimate of genome size (bp) 1.5 4.05 11220 79452 1.6 4.01 10232 1.6 4.01 10232 1.8 3.9 7943 2.2 3.8 6309 2.5 3.7 5011 2.7 3.7 5011 2.9 3.6 3981 3 3.6 3981 3.3 3.5 3162 3.4 3.4 2511 3.5 3.4 2511 3.6 3.4 2511 3.7 3.3 1995 4 3.2 1584 4.5 3.1 1258 Bull’s eye morphology Web pattern Gel electrophoresis Siphoviridae Attachment site

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IntroductionWild-type

Methods

•Plaques on Plate are bull’s eye, 2-3mm after 24hrs•Electron microscopy- pure population•Size of Genome: about 70,000 bp•Gel electrophoresis: cluster assignment H•Future Directions: Kill curve determination

Results

Discussion

Adsorption of the phagedegradative enzymes or contraction of the sheath & tail @ cell wall Enzymes degrade bacterial DNA, genetic recombination lytic or a lysogenic cycle

Michaelangelo temperate phage

model: Mycobacterium smegmatis (M. smeg)

We hypothesize that a bacteriophage can be isolated and purified from a soil sample; that it can be characterized into a cluster; and that infectivity can be determined in a kill curve

soil sample enrichment Purification: streak test, and titer assay.

Acknowledgments: Sea faculty Sea Teaching Assistants, Lee Graham, The Hatfull laboratory At Pittsburgh, HHMI, Department of Biological Sciences

Figure 1) Agar plate with plaques in M.smeg bacterial lawn. Shown is a Bull’s eye morphology. Note the clear sterile center and the turbid outer rings

Figure 2) a typical titer assay with plaques in the M.smeg bacterial lawn. Note the density of the plaques- web pattern

Figure 4) Michaelangelo bacteriophage electron microscopy image. At the tip of the tail there is a clover shaped attachment site.

Figure 3) Michaelangelo electron microscopy. Note the curved tail characteristic of siphoviridae, icosahedral capsid and 2.5:1 Head:tail ratio

Figure 5) Restriction digest. Bands left to right: 1-kb ladder, undigested DNA, BamH1, Cla1, EcoR1, HaeIII, HindIII. Bam H1 did not cut, while HaeII, EcoR1 and HindIII cut. In particular, HaeIII cut into such small fragments it ran off the gel. Cla1 had the clearest bands

Stock solution: Medium and high titer lysates DNA extraction: alcohol precipitation Restriction digest and gel eletrophoresis

M. Smeg colony formation% survival and Kill curve

claI Distance traveled (cm)

log fragment length

Fragment length (bd)

estimate of genome size (bp)

1.5 4.05 11220 794521.6 4.01 10232 1.6 4.01 10232 1.8 3.9 7943 2.2 3.8 6309 2.5 3.7 5011 2.7 3.7 5011 2.9 3.6 3981

3 3.6 3981 3.3 3.5 3162 3.4 3.4 2511 3.5 3.4 2511 3.6 3.4 2511 3.7 3.3 1995

4 3.2 1584 4.5 3.1 1258

Bull’s eye morphology

Web pattern Gel electrophoresis

Siphoviridae