Arab Republic of
Egypt
Alexandria
University
Sameh E. Mohamed and Mahmoud K. Tahoun
Department of Dairy Science and Technology, Faculty of Agriculture, Alexandria University.
Aflatoon
Street, El
-
Shatby
21545
, Alexandria, Egypt
.
Propionicin
PLG
-
1
is
a
bacteriocin
produced
by
Propionibacterium
thoenii
P
127
.
Such
bacteriocin
inhibits
wide
range
of
food
-
borne
pathogens
such
as
Escherichia
coli,
Pseudomonas
aeruginosa,
Vibrio
parahaemolyticus,
Yersinia
enterocolitica
and
a
strain
of
Corynebacterium
sp
.
In
the
present
study,
plg
-
1
gene
expressing
propionicin
PLG
-
1
was
isolated
for
the
first
time
and
transferred
to
different
lactic
acid
bacterial
[LAB]
strains
using
pLEB
590
to
give
the
modified
vector
pLEBPLG
-
1
.
LAB
transformants
showed
strong
antimicrobial
activity
against
E
.
coli
DH
5
α
(most
affected
strain),
Listeria
monocytogenes
18116
,
and
Salmonella
entirica
25566
.
Such
LAB
transformants
can
be
used
in
dairy
industry
to
control
the
food
-
borne
pathogens
that
are
largely
distributed
in
worm
climates
and
to
feed
school
children
in
the
poor
countries
where
epidemic
diseases
and
diarrhea
prevails
.
DNA manipulations
Isolation
of
plasmids
from
E
.
coli
was
carried
out
using
BioBasic
EZ
-
10
plasmid
spin
column
kit,
whereas
isolation
of
plasmids
from
LAB
was
performed
using
a
mini
-
prep
isolation
method
.
Purification
of
DNA
samples
was
performed
using
Fermentas
DNA
purification
spin
column
kit
.
DNA
digestions,
DNA
ligations,
and
electroporation
of
E
.
coli
strains
were
carried
out
according
to
Sambrook
and
Russell
[
2003
],
while
electroporation
of
lactobacilli
strains
was
carried
out
according
to
Serror
et
al
[
2002
]
and
electroporation
of
lactococci
strains
was
performed
according
to
Holo
and
Nes
[
1989
]
.
Electroporation
and
stability
of
pLEBPLG
-
1
into
LAB
strains
Electroporation
efficiency
of
different
LAB
strains
harboring
pLEBPLG
-
1
was
indicated
as
4
.
2
X
10
9
cfu/µg
DNA
for
L
.
lactis
LL
108
,
while
that
for
S
.
thermophilus
was
found
to
be
4
.
1
x
10
9
cfu/µg
DNA
.
On
the
other
hand,
L
.
bulgaricus
DSM
20080
and
L
.
plantarum
TF
103
recorded
5
.
1
x
10
8
and
5
.
2
x
10
8
cfu
/µg
DNA,
respectively
.
The present work was supported using the fund of the International Center for
Genetic Engineering and Biotechnology [
ICGEB, Italy
].
plg
-
1
gene expressing propionicin by lactic
starters in dairying
Isolation
of
plg
-
1
gene
of
P
.
thoenii
P
127
using
specific
PCR
and
sequencing
of
the
PCR
product
After
PCR,
the
PCR
products
were
exposed
to
electrophoretic
migration
on
agarose
gel
[
1
%
]
in
order
to
visualize
their
bands
compared
to
the
1
Kb
DNA
ladder
.
To
our
knowledge,
this
is
the
first
time
to
isolate
such
gene
.
Furthermore,
a
sample
of
the
purified
PCR
product
is
prepared
to
be
sequenced
using
ABI
Prism
377
DNA
sequencer
[Perkin
–
Elmer,
Applied
Biosystems
,
USA]
.
Cloning
and
expression
of
plg
-
1
gene
of
P
.
thoenii
P
127
into
LAB
plg
-
1
gene
was
cloned
into
pLEB
590
and
electroporated
into
different
LAB
strains
.
pLEB
590
was
constructed
as
an
expression
vector
for
LAB
.
Such
vector
fulfilled
all
necessary
requirements
to
be
a
food
-
grade
vector
;
hence
it
has
a
small
size
[
3
.
1
Kb],
a
multiple
cloning
site
[MCS],
a
nisin
immunity
gene
[
nisI
]
as
a
dominant
food
-
grade
selection
marker,
a
high
copy
number,
and
a
high
stability
for
several
generations
.
It
has
a
potential
for
use
in
dairy
processes,
in
order
to
construct
improved
LAB
starter
strains
Screening
for
the
antimicrobial
activity
of
LAB
transformants
against
Escherichia
coli
DH
5
α,
Listeria
monocytogenes
18116
,
and
Salmonella
entirica
25566
(Table
1
)
PLG
-
1
has
strong
antimicrobial
activity
against
the
examined
pathogens
with
special
emphasis
on
E
.
coli
DH
5
α
which
was
the
most
sensitive
strain
.
Thus,
propionicin
PLG
-
1
was
expressed
using
pLEB
590
as
a
multi
-
copy
expression
vector
.
Microbial
strains,
plasmids
and
growth
conditions
All
microbial
strains
used
in
this
study
and
their
media
are
listed
in
Table
1
.
E
.
coli
DH
5
α
was
grown
in
LB
medium
at
37
°
C
.
L
.
monocytogenes
18116
and
S
.
entirica
25566
was
grown
in
BHI
at
37
°
C
.
Lactobacilli
strains
were
grown
in
MRS
medium,
whereas
lactococci
strains
were
grown
in
Elliker
medium
containing
0
.
5
%
glucose
[G
Elliker]
.
L
.
lactis
MG
1614
harboring
the
vector
pLEB
590
was
grown
in
G
Elliker
containing
nisin
(
60
IU/ml)
.
All
lactococci
and
lactobacilli
were
grown
at
30
°
C
.
All
propionibacterial
strains
were
grown
on
mNLB
.
Genomic DNA isolation, extraction from gel, primers designing, and specific PCR
Genomic
DNA
was
isolated
using
Fermentas
g
DNA
purification
kit,
whereas
DNA
bands
were
extracted
using
AxyPrep
TM
DNA
Gel
Extraction
Kit
.
Suitable
primers
[PLG
1
BAMHI
2
(G
G
A
T
C
C
A
A
T
G
T
C
G
A
T
G
C
C
A
G)
and
PLG
1
R
3
-
13
(T
G
G
G
G
T
C
G
A
G
T
T
G
C
A
G
A
C
C
C
C
A
A
T)]
were
designed
using
Geneious
software
4
.
0
.
2
to
isolate
plg
-
1
gene
of
P
.
thoenii
P
127
.
PCR
experiments
were
carried
out
with
a
thermal
cycler
[
Techne
,
UK]
;
Gold
Master
-
Mix
Beads
were
used
as
recommended
by
the
manufacturer
[
Bioron
,
Germany]
.
The
reactions
[volume,
20
µl]
were
performed
with
100
pmol
of
each
primer
.
The
PCR
conditions
used
for
amplification
of
DNA
fragments
containing
the
plg
-
1
gene
included
a
hot
start
at
94
°
C
for
3
min,
followed
by
35
cycles
of
denaturation
at
94
°
C
for
30
s,
annealing
at
59
°
C
for
30
s,
and
polymerization
at
72
°
C
for
6
min
.
Fig
1
Different
PCR
trials,
lane
5
is
the
most
perfect
trial
.
Table
1
The antimicrobial activity of different LAB transformants.
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