The Story of “Phernando” the Bacteriophage

By Christopher Harness

Victoria TorresUniversity of Detroit Mercy 2015

Background (Bacteriophages)

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Viruses known to infect/replicate in bacteria

Caudovirales phage consist of: Siphoviruses Myovirus Podoviridae

Uses horizontal gene transfer towards hosts

Are found in almost every environment

http://www.biologyexams4u.com/

2012/11/bacteriophages.html#.VldNBvmrT

IU

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Overview

Finding the Phage

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Collected by C.H.

Date 9/6/2015 at 10:36 AM

Sample Type Soil

Depth 13.1763 cm or 5 3/16 in

Air Temp 25 Celsius

General LocationMoravian Park, Sterling Hts, MI

48312

GPS Reading 42.545778 N 82.975505 W

Description Damp, dark, shaded area Moravian Park

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Purpose: To allow the phage from the soil sample to reproduce in Mycobacterium smegmatis

Process: Preparing an enrichment culture with the

sample + various formulas Performing serial dilutions with the culture

/plating them with top agar and M. smeg Result: 8 serial dilution plates successfully

created (10 – 10 , Neg)

Soil Enrichment

0 -6

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Spot Plating

• All spot samples taken from 10 Enrichment Plate • B and C contained the most phage samples

-1

Streak Plating

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Phage chosen from Spot C were streaked

Three streaks were performed (Streak 3 not shown)

Method used to help isolate and purify plaques for titer assays

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Purpose: Further purify phage sample

First titer taken from isolated plaque (Streak 2)

Further titering taken from “10/20” plaque

Titer Assays

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Titer Assay 2 had both lysogenic (A) and lytic (B) plaque morphologies

Further Titers: A consisted of hybrid

population (lytic-lysogenic)

B had two separate as before

Problem Solving

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MTL created from flood plate of Assay 2A

Spot Plate: Series of dilutions of MTL (100 to 10-10)

Formula (MTL):

Medium Titer Lysate (MTL) Stocks

Conentration: (6 pfu on

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MTL Amount Equation:

Result:

Four, 5X flooded webplates would create the HTL

High Titer Lysate (HTL)

Plate Factor Amount of MTL ()

1/4x 17.5x10-4

1/2x 35.0x10-4

1x 7.0x10-3

2x 14.0x10-3

4x 28.0x10-3

5x 35.0x10-3

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HTL Concentration: 3.4x108 pfu/mL (17 pfu on )

HTL Plating

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DNA Restriction Analysis

Purpose: Isolate/Purify Phage DNA for restriction enzymes

DNA sample taken from 1 mL of of HTL concentration

DNA Concertation: 51.5

Restriction Enzymes Used: BamHI ClaI EcoRI HaeIII

(NOTE): All DNA was combined with ddH2O and 6X Dye

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Gel Electrophoresis Gels help identify the DNA

sequence of phage

Placements show number of fragments made by the restriction enzyme cuts

HaeIII had the highest distance from the well

The DNA may have been denatured

Ladder BamHIClaI EcoRINeg HaeIII

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TEM Imaging

• Photos taken by the curtesy of Wayne State University

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Phernando’s StoryMoravian

ParkPlate

Enrichment

Spot Plate

Spot C

Streaks 1 – 2

Assays 1 – 2(A) Assay 1 – 2

Flood

MTL

(4) 5X Plates

HTL3.4

DNA: 51.5 Gel

ElectrophoresisTEM Imaging

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Abedon, Stephen T et al. “Phage Treatment of Human Infections.” Bacteriophage 1.2 (2011): 66–85. PMC. Web. 30 Nov. 2015. <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3278644/>.

BioExams4U. “Bacteriophage Structure.” Biology Exams 4 U. 2015. Image. 26 November 2015. <http://www.biologyexams4u.com/2012/11/bacteriophages.html#.VlvicPmrTIV>.

Conant, Stephanie, and Jack Thompson. “SEA Phages.” University of Detroit Mercy. 2015. PowerPoint. 29 November 2015.

Works Cited

QUESTIONS?