DNA Extraction and Characterization of Salmonella‟s Lytic Bacteriophage Genome

DNA EXTRACTION AND CHARACTERIZATION OF
Salmonella’s LYTIC BACTERIOPHAGE GENOME

SALEM MOHAMED EBRAEIK

GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2013

DECLARATION

I declare that this thesis entitled “DNA Extraction and Characterization
of Salmonella’s Lytic Bacteriophage Genome” is a report of research work
carried out by me through the guidance of my academic supervisors and has not
been submitted in any form for another degree at any other university.
Information obtained from published or unpublished work of others and help
received during laboratory and field work have been acknowledged.

Bogor, June 2013

Salem Mohamed Ebraeik
G351098261

ABSTRACT

SALEM MOHAMED EBRAIEK. 2013. DNA Extraction and Characterization
of Salmonella‟s Lytic Bacteriophage Genome. Under supervision of SRI
BUDIARTI, Chairman and IMAN RUSMANA, Member of Advisory
Committee

Bacteriophages are infectious virus that can replicate only inside their
specific bacterial host. Bacteriophage can be used as a biocontrol agent for a
specific bacteria such as Salmonella and other applications. Understanding of
their molecular genetics is important due to their potential applications. The
objectives of this research was to study three isolates of Salmonella‟s phages and
to find out the best method to isolate and characterize genomic DNA of the
phages. Three different methods i.e. PEG/NaCl Method, Scraping plaque method
and Enrichment Method were used to concentrate phages for genomic DNA
extraction. Quantity and purity of extracted DNA was determine based on their
optical density ratio of 260/280 nm and agarose gel electrophoresis. Genomic

DNA size of the phages were determined using agarose gel electrophoresis with
High range ladder as a marker. The best method to prepare bacteriophage
concentrates for DNA genome extraction was enrichment method. This method
had higher concentration and purity of extracted DNA. Based on DNA genome
size, the bacteriophage genome of Salmonella sp were comparable with other
study of Salmonella phages. Phage P15 with genome size of 39,5 kb was close to
phage epsilon15. Phage P19 with genome size of 41 kb was close to phage
ST160, and phage P38 with genome size of 48,5 kb was close to phage Gifsy-1.

Keywords: Salmonella, phage, DNA, Enrichment method,

SUMMARY
SALEM MOHAMED EBRAIEK. 2013. DNA Extraction and Characterization
of Salmonella‟s Lytic Bacteriophage Genome. Under supervision of SRI
BUDIARTI, Chairman and IMAN RUSMANA, Member of Advisory
Committee

Bacteriophages are ubiquitous in nature and are known to proliferate
wherever their bacterial hosts exist. Their virion particles can exist independently
outside the host, however all phages are need their host to propagate. Several

phages are highly specific to host cell surface receptors and any slight changes in
structure results in little or no interaction between the phage and its host.
Salmonella‟s phage isolated from sewage water where their bacteria exist.
Three isolated of phages (phages 15, 19 and 38) were used in this research
that concentrated using three types of methods. PEG/ NaCl method was firstly
used to concentrate the phages based on their molecular weight to precipitate the
phage particles during centrifugation. The second method was Scraping Plaque
method. In this method, overlay plaques were scraped and collected in SM buffer
to get phage concentrates. Enrichment method was the last method used to
concentrate the phages. Phage genome isolation was done using two methods i.e.
EDTA/SDS and Proteinase K methods. The first method was done using DNase I
to eliminate bacterial DNA followed by SDS/EDTA treatment to release phages
DNA and phenol-chloroform-isoamyl alcohol to extract the DNA. DNA was
collected by isopropanol extraction. While in Proteinase K method, bacterial DNA
was eliminated by DNase I and Proteinase K/SDS was used to degrade phages
capsids and ethanol for DNA precipitation.
The result showed that PEG/NaCl method resulted the highest phage
concentration for phage 15 and phage 19, while scrapping method was the highest
for phage 38. The quantity and purity of extracted DNA were low for PEG/NaCl
and Scraping methods, while Enrichment method was higher indicated higher

purity. Run on agarose gel electrophoresis of extracted DNA confirmed that

Enrichment method results were the best while scraping and PEG/NaCl method
was appeared smear background.
Based on DNA genome size determination, the bacteriphages of
Salmonella sp were comparable with other study of Salmonella phages. Phage
P15 with genome size of 39,5 kb was close to phage epsilon15. Phage P19 with
genome size of 41 kb was close to phage ST160. And phage P38 with genome
size of 48,5 kb was close to phage Gifsy-1.

Keywords: Salmonella, phage, DNA, Enrichment method,

© Copyright by Bogor Agricultural University 2013
All rights reserved
No part or all of this thesis may be excerpted without inclusion or mentioning the
sources. Excerption only for research and education use, writing for scientific
papers, reporting, critical writing or reviewing of a problem
Excerption does not inflict a financial loss in the proper interest of Bogor
Agricultural University

DNA EXTRACTION AND CHARACTERIZATION OF
Salmonella’s LYTIC BACTERIOPHAGE GENOME

SALEM MOHAMED EBRAEIK

A thesis submitted as partial fulfillment of the requirement for the degree of
Master of Science in Microbiology

GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
2013

Thesis External Examiner in IPB: Dr. Drh. Sri Murtini, M.Si.

Thesis Title

: DNA Extraction and Characterization of Salmonella‟s Lytic
Bacteriophage Genome

Name

: Salem Mohamed Ebraeik

Student I.D.

: G351098261

Approved
Advisory Committee

Dr. Ir. Iman Rusmana, M.Si

Dr. dr. Sri Budiarti
Chairman

Committee Member

Agreed

Head of Study Program

Dean of Graduate School

Microbiology

Prof. Dr. Anja Meryandini, M.S.

Examination Date: 17 June 2013

Dr.Ir. Dahrul Syah, M.Sc.Agr.

Submission Date:

ACKNOWLEDGEMENT

In the name of Allah the Most Gracious, The Most Merciful. Many
individuals were responsible for the crystallization of this work, whose
associations and encouragement have contributed to the accomplishment of the
present report, and I would like to pay tribute to all of them.
I would like to share my deepest gratitude and sincere appreciation to my

advisors, Dr. dr. Sri Budiarti and Dr. Ir. Iman Rusmana for their unwavering
patience, instruction, direct guidance and valuable support during my study time. I
must also thank Bogor Agricultural University for the chance to study and
fulfillment the degree of Master of Science, also to Hibah Penelitian Tim
Pascasarjana (HPTP) 2010 which financed this research through Dr. dr. Sri
Budiarti as chairman of team Research
My sincere appreciation goes to all lab members for assistance, willingness
to share their knowledge, and friendship.
I would also like to thank Yosi Kustian, Sari Nalurita and Nani Maryani for
all the discussion time and their support during my time in Bogor. I would also
like to thank Microbiology students IPB 2009, 2010 for their support and
friendship during my time in IPB.
Finally I would like to thank my parents, my brother and my sisters for their
support and love was by far the most important element that allowed me to
continue my studies.

Bogor, June 2013

Salem Mohamed Ebraeik

AUTOBIOGRAPHY

The author was born

th

on the 17 April 1985, Misuratah, Libya. He

completed his primary education at Al-jazirah School before he joined Basic
Sciences School where he completed his high school in 2004. He later joined
Misuratah University and graduated with Bachelor of Science degree in
Microbiology in 2007.
In Augustus 2009, the author was admitted to join Bogor Agricultural
University.

TABLE OF CONTENTS
Page
LIST OF TABLES ............................................................................................. xiii
LIST OF FIGURES ............................................................................................ xiv
ABBREVIATIONS LIST ................................................................................... xv

INTRODUCTION
Background……………………………………………...…………...…… 1
Aim of this study ……………………………………………..…………... 2
LITERATURE REVIEW
Bacteriophage………………………...........................................................
Structure of Caudovirales Bacteriophage
Capsid.................................................................................................
Tail......................................................................................................
Other Structures..................................................................................
Genomic Structures............................................................................

3
4
4
5
5

MATERALS AND METHODS
Place and Duration of the Study.................................................................. 6
Bacterial Strains and Bacteriophage Isolates.............................................. 6

Preparation of Bacteriophage Concentrate.................................................. 6
PEG/ NaCl Method............................................................................ 6
Scraping Plaque Method.................................................................... 7
Enrichment Method........................................................................... 7
Quantification of Bacteriophages by Plaque Assay..................................... 8
Extraction of Bacteriophage Genomic DNA.............................................. 8
DNA Extraction Using EDTA and SDS............................................ 8
DNA Extraction Using Proteinase K................................................. 9
Measuring Concentration and Purity of Extracted DNA............................ 9
Determination of Phage Genome Size......................................................... 10
RESULTS
Concentration of Bacteriophage Concentrates and Their Plaque
Morphology………………………………………………………………. 11
Quantity and Purity of Extracted DNA...................................................... 13
Size of Phage Genome................................................................................ 14
DISCUSSION ..................................................................................................... 16
CONCLUSION ................................................................................................... 18
REFERENCES ................................................................................................... 19
APPENDIX.. ....................................................................................................... 23

LIST OF TABLES
Page
1. Plaque morphology of phage concentrates prepared using three
different methods..........................................................................................

12

2. DNA concentration and purity determined using spectrophotometer of
extracted DNA from Phage concentrates prepared using three different
methods.......................................................................................................... 13

LIST OF FIGURES
Page
1.

Concentration of phage concentrates prepared using three different
methods...........................................................................................................11

2.

Plaque morphology from plaque assay of three phage concentrates
prepared using (A) PEG/NaCl method, (B) Scraping plaque method,
and (C) Enrichment method........................................................................... 12

3.

Electrophoresis Results of phage genomic DNA extracted from phage
concentrates prepared using (A) PEG/NaCl method with EDTA/SDS
method, (B) Scraping method with Proteinase K/SDS method, and (C)
Enrichment method with Proteinase K/SDS….............................................. 14

4.

Electrophoresis results of three phage‟s DNA genomes. M= High
range DNA ladders; P38=Phage P38 genome; P19=Phage P19 genome.
P15=Phage P15 genome. ............................................................................... 15

LIST OF ABBREVIATIONS
°C

Degrees Celsius

μl

Microliter

μm

Micrometer

AIDS

Acquired Immunodeficiency Syndrome

bp

Base pair

Ca 2+

Calcium ions

cos

cohesive

DNA

Deoxyribonucleic acid

DNase

Deoxyribonuclease

ds

Double stranded

EDTA

Ethylene diamine tetra-acetic acid

g

gravity

h

Hours

ICTV

International Committee on Taxonomy of Viruses

kb

Kilobases

Mg2+

Magnesium ions

min

Minutes

ml

Milliliter

mm

Millimeter

mM

Millimolar

M

Molar

NaCl

Sodium Chloride

NB

Nutrient Broth

nm

Nanometer

OD

Optical Density

PEG

Polyethylene Glycol

PFU

Plaque forming unit

RNA

Ribonucleic acid

RNase

Ribonuclease

s

Second

SDS

Sodium dodecyl sulfate

SM buffer

Solely to Tris buffer with magnesium salt

ss

Single stranded

SSA

Salmonella Shigella Agar

TAE

Tris Acetate EDTA

TSB

Tryptone Soy Broth

UV

Ultraviolet

WHO

World Health Organization

INTRODUCTION
Background
Bacterial pathogens are one of the most dangerous organism infecting
human, animal and plant; the danger of these bacteria are related to their activity
causing damage and disease to human, animal and plants (Casadevall and Pirofski
2009). Salmonella is one of bacterial pathogen that causes harmful disease
(diarrhea) to human. There was estimated that 1.9-2.5 million of under 5 years old
children were died from diarrhea per year in developing countries (Nguyen et al,
2006; An 2007). In developed countries: total of 155540 confirmed cases of
salmonellosis were reported and 64.5% of the cases were caused by S. enteritidis
in 2007 in the European Union. In United States, from 1999 salmonellesis patients
caused by S. enteritidis was increased and it was more than 1400 patients in 2006
(Pan Z et al, 2009). Salmonella derby were isolated from patients with diarrhea in
Myanmar can invade Hep2 cells when tested in vitro (Budiarti et al, 1991). To
fight against these bacterial pathogens, human usually use antibiotics such as
Ciprofloxacin, Norfloxacin, Nalidixic Acid and Ampicillin taken either oral or
injection (Goodman et al, 1984).
Application of antibiotics has side effect such as the effect of antibiotics on
the body health and normal flora. Budiarti 2011, reported that E. coli isolated
from healthy human neonatus also adult were resistant to several antibiotics.
Antibiotic resistance of some pathogenic bacteria such as Salmonella spp. was
reported from Central Africa (Vlieghea et al., 2009). It was reported that
antibiotic resistance of Salmonella sp increased up to 70% in many areas of the
world, however, the resistance rate of Salmonella sp was different among
serotypes. Salmonella enteritidis is generally more susceptible to antimicrobial
agents, while S. typhimurium exhibits higher resistance (Su and Chiu 2007).
Therefore, there should be an alternative way to avoid these problems. Increasing
attention has been paid to biological control through other microorganism such as
bacteriophages. Preliminary observation showed that some bacteriophages infect
enteric bacteria with the ability to kill or lysis their bacterial hosts. The utilization
of bacteriophage to control pathogenic bacteria is not a new way but it has been

1

utilized by many researchers in Eastern Europe and Russia (Huff et al. 2005).
Bacteriophage of EPEC (enteropathogenic Escherichia coli) was reported
effective to control the EPEC growth (Budiarti et al. 2011). The bacteriophages of
Salmonella Sp ( P15, P19, P38) isolated from domestic sewage at Darmaga Bogor
Indonesia have effectivity to lysis cells of the Salmonella sp. from diarrhea
disease patient, the phage 38 was reported safe when consumed during two weeks
by rat sprague dawley (Sartika et al. 2012).
The phage can be used as a biocontrol agent for a specific bacterial disease
(Fischetti et al 2006). The specificity of the phage can also be used for rapid
detection of specific pathogenic bacteria such as Salmonella spp and
Mycobacterium tuberculosis by detecting their intracellular compounds of the
lysis bacterial cells (Goodridge and Abedon 2003). The phage can also be used for
tracing salmonellosis outbreaks and determining the sources of infected pathogens
(Ward et al, 2005). There are other applications of bacteriophages in molecular
biotechnology i.e. as delivery tools for protein and DNA vaccines, as potential
gene therapy vectors and in nanotechnology techniques (Verheust et al, 2010).
Therefore understanding of bacteriophages and their molecular genetics is
important due to their potential applications.

Aim of this study
The objectives of this research were to study three isolates of Salmonella‟s
phages and to find out the best and easiest method to isolate and characterize
genomic DNA of the phages.

2

LITERARY REVIEW
Bacteriophages
Bacteriophages are ubiquitous in nature and are known to proliferate
wherever their bacterial hosts exist (Hendrix et al. 1999). Virion particles can
exist independently outside the host, however all phages are obligate intracellular
parasites and need their host to propagate (Jensen et al. 1998). Several phages are
highly specific to host cell surface receptors and any slight changes in structure
results in little or no interaction between the phage and its host. Therefore, many
phage typing schemes for the identification of bacterial species or subspecies are
based on this specificity (Welkos et al. 1974).
Bacteriophages are found in almost all environments on Earth, from the
depths of the ocean to hot springs, and can be isolated from almost any material
that will support bacteria (Dabrowska et al. 2005). There is evidence that the
diversity of bacteriophage is about an order of magnitude higher than that of
bacteria (Weinbauer and Rassoulzadegan 2004) which has implications in the
classification of bacteriophage.
Bacteriophages discovered early by Frederick W. Twort and Felix
d„Herelle each one independent. However there has been considerable
controversy with regards to who actually discovered the bacterial viruses first. In
1896, British bacteriologist Ernest Hankin described his observations with regards
to the presence of antibacterial activity against Vibrio cholerae in the Jumna and
Ganges rivers of India. He proposed that an unidentified chemical substance was
responsible for the decline in the spread of cholera. A few years later, other
researchers made similar observations although they did not investigate their
findings further (Sulakvelidze et al. 2001). Nearly 20 years after Hankin„s report,
Frederick W. Twort reported on a phenomenon referred to as the „glassy
transformation‟ while working with Vaccinia virus which had been contaminated
by micrococci. He speculated on the possibility that he had come across an
ultramicroscopic virus and concluded that the glassy transformation was caused
by an infectious agent that killed bacteria and multiplied itself in the process
(Duckworth 1976). In 1917, Felix d„Herelle independently discovered „ultra

3

viruses‟ that resulted in the death of bacteria (Summers 2001). He proposed the
name „bacteriophage‟ from „bacteria‟ and „phagein‟ (Greek word for to eat or
devour) therefore implying that bacteriophages „eat‟ bacteria. D„Herelle believed
that a phage was an obligate parasite which is particulate, invisible, filterable, and
self-reproducing in nature (Stent 1963).

Structure of Salmonella’s Bacteriophage

Capsid
The capsid is icosahedral in shape, with rare elongated variations.

It

appears smooth under electron microscopy and ranges in diameter from 34 to
160nm with a majority at 60nm. Capsomers (the morphological subunits) are also
present (Ackermann 2003; Bradley, 1967). The family Myoviridae are generally
larger than Podoviridae and Siphoviridae families (Ackermann 1998).

Tail
The tail is structured of a hollow tube of fixed length and width, built of
stacked rows of subunits and generally has a six-fold symmetry (Ackermann
1998). Members of the family Siphoviridae have long non-contractile, flexible
tails, the family Podoviridae have short variants on this, while the family
Myoviridae have long, rigid, contractile tails (Ackermann et al. 1992).

The

family Myoviridae tails consist of a tail tube surrounded by a sheath, separated
from the head by a neck. They are of sixfold symmetry, with subunits arranged in
helix format. On contraction, these subunits slide over each other, forming a short
cylinder (Ackermann 1998). Tail lengths can vary widely, but are typically
conserved within a species. A ruler protein has been identified in some
bacteriophage and this acts as a tape-measure around which tail tube monomers
polymerize. Alterations to the ruler protein will alter the length of the tail tube
(Katsura and Hendrix 1984).

4

Other Structures
There is a small disk located inside the head at the site of tail attachment
which is known as the connector. The connector holds the head and tail together
and has functions in head assembly and DNA encapsidation. Tailed bacteriophage
can also have base plates, tail spikes and tail fibres, though the number and shape
of these can vary (Ackermann 1998).

Genomic Structure
Genomes of Caudovirales consist of linear double stranded (ds) DNA.
They range from 17kb to in excess of 700kb in length. Some genomes contain
cohesive (cos) sites near end allowing circularisation of the genome after
infection. Packaging of DNA can be either of a single genome, or by a headful
mechanism, where the genome is continually copied into the capsid until it is full
(Streisinger et al, 1967). DNA may be concatemeric (head-to-tail repeats of a
sequence) or unit length prior to packaging with concatemeric DNA formed from
recombination between linear DNAs or rolling circle replication. Cleavage of
concatemeric DNA for packaging can occur at; a) unique sites resulting in blunt
termini or cos ends with packaging starting and finishing at a cos site, b) pac sites
(sequences recognised by terminase complex) to produce DNA molecules with
limited circular permutation and terminal redundancy (excess coding DNA at the
terminal end) and where packaging starts at the pac site and continues until the
head is full or c) random sites to produce circularly permuted, terminally
redundant DNA (Ackermann, 1998; Ackermann 2003; Maniloff and Ackermann
1998).
From comparisons of bacteriophage genomes, it is apparent that
bacteriophage genomes have a mosaic nature, with gene order not conserved
between species (Chopin et al. 2001; Hertveldt et al. 2005). The following
observations of gene order can be made. Genes with related function generally
cluster together, though non-structural gene order does not follow any general
pattern. Structural genes are generally separate from other genes and, of these
head genes precede tail genes (Casjens 2003).

5

MATERIALS AND METHODS
Time and Place of Study
This research was carried out from September 2011 to December 2012 at
Biotechnology of Animal and Biomedical Laboratory, Study Center for Life
Sciences and Biotechnology and Microbiology Laboratory, Biology Department,
Faculty of Science and Mathematics, Bogor Agricultural University. While past of
this research was carried out at Indonesian Institut of Sciences, Cibinong.

Bacterial Strains and Bacteriophage Isolates
Salmonella spp and their bacteriophages used in this study were
collections of Dr. Sri Budiarti, Department of Biology Bogor Agricultural
University. Three isolates of Salmonella spp were used and maintained on SSA
media. Culturing Salmonella spp were on TSB or NB and incubated at 37C° for
16-24h until their concentration reached to OD600nm=1 that (1x109 CFU/ml).
The bacteriophages were stored in SM buffer. The bacteriophage stocks can be
stored up to 5 months.

Preparation of Bacteriophage Concentrate
Three different types of methods were tried for preparing bacteriophage
concentrates. The methods were method using Polyethilene glycol PEG/NaCl
(PEG/NaCl Method), scraping of plaque on soft agar (Plaque Scraping Method),
and enrichment in broth media (Enrichment Method).

PEG/ NaCl Method
Single colony of Salmonella was cultured in 40 ml of TSB media overnight
at 37C°. Centrifugation was done at 4000g for 10 minutes at 4C°. The supernatant
was discarded and the pellets were resuspended in 1 ml of TSB. Salmonella
suspension was mixed with 200 μl of bacteriophage suspension, and incubated for
20min at 37C°. Sterile TSB media was Added up to 500 ml and incubated at 37C°
for 16-24h. The suspension was treated with 100µl chloroform followed by
centrifuging at 4000g. PEG 8000 and NaCl were added at final concentrations of

6

10% and 1 M respectively. The mixture was stored at 4 C° overnight. The phage
particles were centrifuged at 10,000 g for 20 min and the pellet was resuspended
in 5 ml TM buffer. The remaining of PEG 8000 and cell debris was extracted
from bacteriophage suspension by adding an equal volume of chloroform
followed by gently vortex for 30 seconds. The organic and aqueous phases were
separated by centrifugation at 3000g for 15 minutes at 4C°. Then concentration of
bacteriophage particles was checked using Double Layers Agar Plaque Assay
(Seyediras et al 1991; Sambrook and Russell 2001).

Scraping Plaque Method
Six petri dishes of plaque assay were prepared for each bacteriophage with
suspension dilution of 10 -3 and two petri dishes of each bacteriophage were
scraped their overlay plaques and diluted in 3 ml of SM buffer. The suspension
was mixed thoroughly using a Vortex for 30s and it was incubated at room
temperature for 1-2 h to allow bacteriophage to diffuse from agar. Centrifugation
was done at 4000g and 4 C° for 10 minutes and the supernatant was transferred to
sterile tubes. The bacteriophage concentration was checked using Double Layers
Agar Plaque Assay (Sambrook and Russell 2001).

Enrichment Method
One Petri dish of each bacteriophage isolates was prepared with low
bacteriophage dilution. The plaque formation was checked after 7-9 h to affirm
that the plaques were already formed then they were incubated overnight at 37 C°.
Four ml of SM buffer was added on the soft top agar followed by incubation
overnight at 4 C°. The SM buffer was transferred and the bacterial debris was
removed by centrifugation at 4000g for 10 minutes at 4 C°. Supernatant was
transferred to sterile tubes as a stock of bacteriophages.
Single colony of Salmonella was transferred to 25 ml of TSB followed by
incubation overnight at 37 C°. Salmonella suspension was centrifuged at 4000g
and 4 C° for 10 minutes and the supernatant was discarded. Pellet was
resuspended with 200 µl of TSB media followed by mixing with 200 µl of the
bacteriophage stock. Incubation was done at 37C° for 20 min. As much as 3.5 ml

7

of TSB was added and incubated at 37 C° for 16-24 h. The suspension was
centrifuged at 4000 g and 4 C° for 10 minutes. 200 µl of supernatant was taken
and incubated with 200 µl of concentrated Salmonella culture. Bacteriophage
suspension was incubated at 37 C° for 20 minutes. And 2 ml of TSB media was
added followed by overnight incubation. Concentration of the bacteriophage was
checked by plaque assay (Sambrook and Russell 2001).

Quantification of Bacteriophages by Plaque Assay
Bacteriophage suspensions were diluted serially in NB media. And 100 μl of
desired dilution of phages was incubated with 100 µl of specific strain of
Salmonella at 37 C° for 20 min to allow the bacteriophage particles to attach
bacterial cells of Salmonella. Suspension was added to a tube of overlay medium
and mixed throughly. The mixture was poured over underlay plate. The plates
were incubated at 37C° for 18–24 h. The plaques were counted on plates. The
result was accepted from plates with 30-300 plaques. The titer of the original
phage preparation was determined using the following calculation (Clokie and
Kropinski 2009):

Phage Concentration (pfu/ml) = Number of plaques × 10 × reciprocal of counted
dilution

Extraction of Bacteriophage Genomic DNA
Two different methods for DNA isolation of phage genomes were used.
The methods were DNA extraction using EDTA and SDS and DNA extraction
using Proteinase K.

DNA Extraction Using EDTA and SDS
DNAase I was added at concentration of 1 unit for 10 ml of bacteriophage
concentrate and incubated at 37 C° for 30 minutes. Phage DNA was extracted
using 0,5 ml SDS and EDTA at final concentrations of 1% and 10 mM
respectively, followed by two phenol-chloroform -isoamyl alcohol extractions.
Centrifugation was done at 4000g for 10 minutes at room temperature and the

8

upper aqueous phase was taken. DNA was precipitated using isopropanol by
centrifugation at 10,000g. The pellet was washed by 1 ml of ethanol 70% at room
temperature and then centrifuged at 10,000g for 5 minutes. The pellet was dried
and added 20 µl TE buffer. 2 µl of RNase was added and incubated for 30min at
37C°. DNA was stored at 4C° (Oakey and Owens 2000; Sambrook and Russell
2001).

DNA Extraction Using Proteinase K
Bacteriophage DNA was extracted by adding 5 µl of DNase I to 0,5 ml of
bacteriophage suspension, and incubated at 37C° for 30 minutes. Proteinase K (1
mg/ml) was added to a final concentration of 50 µg/ml and SDS to a final
concentration of 0.5%. The solution was mixed by vortex for a few seconds.
Incubation was done at 56 C° for an hour. One volume of equilibrated phenol was
added and mixed well by gently inverting. The phases of complex were separated
by centrifugation at 4000g for 10 minutes at room temperature. Aqueous phage
was transferred to sterile tube and one volume of 1:1 mixture of equilibrated
phenol and chloroform was added and centrifugation was repeated at same
condition. Extraction with an equal volume of chloroform was also done.
Precipitation of DNA was done with ethanol absolute followed by washing with
ethanol 70%. And 20 µl of TE buffer was added. 2µl of RNase was added and
incubated for 30 min at 37C°. Then the DNA was stored at 4 C° (Sambrook and
Russell 2001).

Measuring Concentration and Purity of Extracted DNA
Concentration and purity of extracted DNA was determined using a
spectrophotometer at 260/280nm. The DNA concentration was calculated from
acceptable value of OD (OD 1.0 = 50 µg/ml of DNA). Spectrophotometer
measurement was between 0.1–1.0. Optical density ratio of 260/280 nm were used
to estimate DNA purity. Good DNA purity is at the ratio of 1.8 to 2.0 (Fong
2008).
Extrated

DNA

quality

was

also

determined

using

agarose

gel

electrophoresis. DNA samples were run on gel electrophoresis with 1% agarose in

9

1× TAE. DNA ladder was One Kb DNA ladders were used as DNA size marker.
Electrophoresis was set up at 60 volt for 50 minutes. DNA staining used 1 mg/mL
ethidium bromide. DNA gels were visualized and photographed under a UV
Transilluminator (Fong 2008).

Determination of Phage Genome Size
DNA genomes of three phage isolates were run on low agarose gel‟s
concentration 4% electrophoresis for 8 hours at 30V. Size of DNA genomes bands
were estimated by comparing the band with High Range DNA ladder. Excel
program was used for more accurate calculation based on their migration distance.

10

RESULTS
Concentration of Bacteriophage Concentrates and Their Plaque Morphology
Concentration of bacteriophage concentrates from three different methods
showed that PEG/NaCl method was the highest concentration for concentrate of
phage 15 and phage 19 while scrapping method was the highest concentration for
phage 38 (Figure 1). 15.8X109

Phage concentration (PFU/ml)

35
30
25
20

P15

15

P19
P38

10
5
0

PEG/NaCl Method

Scraping Plaque Method

Enricment Method

Figure 1. Concentration of phage concentrates (phage P15, P18 and P38) prepared
using three different methods
As shown on Figure 2 and Table 1, plaque morphology of bacteriophages
prepared using three different methods showed different plaque morphology.
Diameter and morphology of phage plaques prepared using PEG/NaCl method
were fuzzy with smaller diameter than the other methods (Table 2).

11

Table 1. Plaque morphology of phage concentrates prepared using three different
methods
PEG/NaCl method
Phage

Diameter
(mm)

Morphology

Scraping plaque method
Diameter
(mm)

Morphology

Enrichment method
Diameter
(mm)

Morphology

P15

1

Fuzzy

2

clear-cut edge

2

clear-cut edge

P19

1

Fuzzy

2

clear-cut edge

2

clear-cut edge

P38

2

Fuzzy

2.5

clear-cut edge

2.5

clear-cut edge

(A)

(B)

(C)
Figure 2. Plaque morphology from plaque assay of three phage concentrates
prepared using (A) PEG/NaCl method, (B) Scraping plaque method,
and (C) Enrichment method.

12

Quantity and Purity of Extracted DNA
Results of determining quantity and purity of extracted DNA were shown on
Table 3. Optical density (OD) measurement of extracted DNA for three different
methods using spectrophotometer showed that PEG/NaCl and Scraping methods
were low. However, OD value ratio of 260/280nm from Enrichment method was
higher indicated higher purity. While the highest DNA concentration was from
phage P19 prepared using Enrichment method (Table 3).
Based on agarose gel electrophoresis results (Figure 2), it was shown that
effect of PEG 8000 used in precipitation of phage on agarose gel. There was
appeared smear background even using chloroform to wash it, but its impact was
still appeared. The same problem was appeared when using the scraping method,
dusky smear was appeared because of the high concentration of agar whose had
been dissolved in SM buffer. Even after washing out using phenol and chloroform
three times, but its effect on electrophoresis results was still appeared. However
the extracted DNA from enrichment method showed a clear DNA band indicated
a good DNA purity.

Table 2.

DNA concentration and purity determined using spectrophotometer of
extracted DNA from Phage concentrates prepared using three different
methods
Method

Bacteriophage
Concentrate
PEG/NaCl

Scraping

Enrichment

DNA Extraction
EDTA /SDS

Proteinase K /SDS

Proteinase K /SDS

DNA concentration
Phage

Purity

(µg/ml)

P15

1.04

6.6

P19

1.07

10.6

P38

1.20

6.6

P15

1.13

6.8

P19

1.12

3.9

P38

1.26

9.9

P15

1.70

8.4

P19

1.81

13.5

P38

1.78

11.3

isolate

13

(A)

(B)

(C)
Figure 3. Electrophoresis Results of phage genomic DNA extracted from phage
concentrates prepared using (A) PEG/NaCl method with EDTA/SDS
method, (B) Scraping method with Proteinase K/SDS method, and (C)
Enrichment method with Proteinase K/SDS.
Size of Phage Genome
Based on electrophoresis analysis on agarose gel of extracted DNA from
phage concentrates, it was shown that the size of phage P15 was approximately

14

39,5 kb (Figure 3). While DNA genom of phage P19 was approximately 41 kb.
And DNA genom of phage P38 was approximately 48,5 kb.

Figure 3. Electrophoresis results of three phage‟s DNA genomes. M= High range
DNA ladders; P38=Phage P38 genome; P19=Phage P19 genome.
P15=Phage P15 genome utilizing Enrichment Method and Proteinase K
/SDS.
.

15

DISCUSSION
In this study, three methods of isolation phages were used and compared to
find that Enrichment method is the best with regard to results that obtained, and
the advantages of this method are its simplicity, rapidity of isolation and reduction
in the cost, In addition all chemicals used are equipments and available in the
laboratories. The method is best suited for isolating DNA from a small scale.
Although using 500ml of media in PEG/NaCl method to propagate phage
but concentrated phage suspension using centrifuge were low concentration
comparing with large amplification occurred, that is because phage particles were
often broken into head and tail components and would obviously not be viable
(Oakey and Owens 2000). In contrast, the concentrates obtained through other
methods contained a larger number of intact bacteriophage particles comparing
with small amplification.
A few modifications were done in concentration of phages and extraction of
phage DNA methods which DNase I addition was delayed until concentration of
phage particles was done in PEG/NaCl method to avoid any DNA contamination
maybe happen, while RNase A was added after DNA precipitation by ethanol
70% at all of methods. Enrichment method also was modified by adding an extra
time of incubation phage with bacteria, and the second incubation was added to
avoid any agar transferred with first phage suspension. In addition scarping soft
agar in SM buffer can be very useful which concentration of phage particulates
could be increased by adding more times of soft agar from Plaque Assay to the
same volume of SM buffer, that is very sample and possible but not for isolation
DNA because of the difficulties to remove the high concentration of agar.
Two methods for DNA isolation of phage genomes were utilized. The
methods were DNA extraction using EDTA and SDS and DNA extraction using
Proteinase K. The first method was utilized for phage samples that precipitated
using PEG/ NaCl Method. Few different were between two methods which used
EDTA instead of Proteinase K due to PEG affection on Proteinase action, also
utilizing isopropanol instead of ethanol absolute due to ethanol need be chilled.

16

The chilling leads to assemble PEG molecules which precipitate by
centrifugation.
This study results on the Phage DNA genome size have similarity with other
published results. Phage 15 that was determined its genome size about 39,5 kb is
close to phage epsilon15 (ε15) genome size that is 39671bp (Kropinskia et al
2007). Phage P19 with genome size about 41 kp is approximately similar to
genome size of phage ST160. This phage is Salmonella enterica phage with
genome size of 40986 bp (Price-Carter et al 2011). McClelland et al (2001)
published Salmonella phage Gifsy-1 that has genome size 48491 bp, this phage
has genome size a few bases different with genome size of phage P38 that was
48.5 kb.

17

CONCLUSION
Isolation of DNA genome of Salmonella‟s phages were successfully
performed for all of the three phages. The best method to prepare bacteriophage
concentrates for DNA genome extraction was enrichment method. Bacteriophage
concentrate from enrichment method resulted in extracted DNA of bacteriophage
genome with higher purity.
Based on DNA genome size determination, the bacteriphages of Salmonella
sp were comparable with other study of Salmonella phages. Phage 15 with
genome size of 39,5 kb was close to phage epsilon 15. Phage 19 with genome
size of 41 kb was close to phage ST160. And phage 38 with genome size of 48,5
kb was close to phage Gifsy-1.

18

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22

APPENDIX

23

Appendix 1- Media Composition

1) Tryptone Soy Broth (TSB)
Sodium chloride

5g

Glucose

2.5g

Tryptone (Pancreatic digest of casein)

17g

Soytone (Peptic digest of soybean meal)

3g

Di-Potassium hydrogen phosphate

2.5g

Media components were dissolved in 1000 ml distilled water, pH was adjusted to
7.3 and broth was autoclaved.

2) Nutrient broth (NB)
Peptone

10g

Sodium chloride

5g

Beef Extract

10g

Media components were dissolved in 1000 ml distilled water, pH was adjusted to
7.3 and broth was autoclaved

3) Salmonella Shigella Agar (SSA)
Peptic digest of animal tissue

5g

Beef extract

5g

Lactose

10g

Bile salts

8.5g

Sodium citrate

10g

Sodium thiosulfate

8.5 g

Ferric ammonium citrate

1g

Neutral red

25mg

Brilliant green

0.33 mg

Bacteriological agar

15.0 g

Media components were dissolved in 1000 ml distilled water, pH was adjusted to
7 and media was not autoclaved.

24

Appendix 2- Buffers

1) TE Buffer
1M Tris-HCl (pH8.0)

10mM

EDTA

1mM

2) 50X TAE Buffer
EDTA (0.5 M)

100ml

Glacial acetic acid

57.1ml

Tris Base

242g

Buffer components were dissolved in 1000 ml distilled water

3) SM Buffer
Sodium chloride (NaCl)

5.8g

1M Tris-HCl, pH7.5

50ml

Hydrated magnesium sulfate (MgSO4-7H2O)

2g

Buffer components were dissolved in 1000 ml distilled water

25

ABSTRACT

SALEM MOHAMED EBRAIEK. 2013. DNA Extraction and Characterization
of Salmonella‟s Lytic Bacteriophage Genome. Under supervision of SRI
BUDIARTI, Chairman and IMAN RUSMANA, Member of Advisory
Committee

Bacteriophages are infectious virus that can replicate only inside their
specific bacterial host. Bacteriophage can be used as a biocontrol agent for a
specific bacteria such as Salmonella and other applications. Understanding of
their molecular genetics is important due to their potential applications. The
objectives of this research was to study three isolates of Salmonella‟s phages and
to find out the best method to isolate and characterize genomic DNA of the
phages. Three different methods i.e. PEG/NaCl Method, Scraping plaque method
and Enrichment Method were used to concentrate phages for genomic DNA
extraction. Quantity and purity of extracted DNA was determine based on their
optical density ratio of 260/280 nm and agarose gel electrophoresis. Genomic
DNA size of the phages were determined using agarose gel electrophoresis with
High range ladder as a marker. The best method to prepare bacteriophage
concentrates for DNA genome extraction was enrichment method. This method
had higher concentration and purity of extracted DNA. Based on DNA genome
size, the bacteriophage genome of Salmonella sp were comparable with other
study of Salmonella phages. Phage P15 with genome size of 39,5 kb was close to
phage epsilon15. Phage P19 with genome size of 41 kb was close to phage
ST160, and phage P38 with genome size of 48,5 kb was close to phage Gifsy-1.

Keywords: Salmonella, phage, DNA, Enrichment method,

SUMMARY
SALEM MOHAMED EBRAIEK. 2013. DNA Extraction and Characterization
of Salmonella‟s Lytic Bacteriophage Genome. Under supervision of SRI
BUDIARTI, Chairman and IMAN RUSMANA, Member of Advisory
Committee

Bacteriophages are ubiquitous in nature and are known to proliferate
wherever their bacterial hosts exist. Their virion particles can exist independently
outside the host, however all phages are need their host to propagate. Several
phages are highly specific to host cell surface receptors and any slight changes in
structure results in little or no interaction between the phage and its host.
Salmonella‟s phage isolated from sewage water where their bacteria exist.
Three isolated of phages