AN INNOVATIVE LABORATORY EXERCISE:
SCREENING OF LACTIC ACID BACTERIA
FOR BACTERIOCINS BY
MICROBIOLOGICAL AND PCR METHODS
Duongdearn Suwanjinda, Mahidol University
Chris Eames, University of Waikato
Watanalai Panbangred, Mahidol University
Outline
Introduction
Rationales of the Study
Laboratory Unit Development
Lactic Acid Bacteria (LAB)
Bacteriocins
Polymerase Chain Reaction (PCR)
Science and Education Outcomes
Rationales of the Study
A
need for the integration of current molecular
biology together with conventional methods for
undergraduate laboratory courses
Positive laboratory experiences can encourage
students to continue their education in molecular
biology or provide them an appreciation for how
technology applies to their daily lives.
Rationales of the Study
Topic of selected experiment
Concern for food safety: Lactic acid bacteria (LAB)
are recognized as GRAS. LAB and their products
(Bacteriocins) are used in many foods.
There are many kinds of fermented foods in
Thailand in which LAB can be found.
Experiment on LAB and bacteriocins
Lactic Acid Bacteria (LAB)
LAB are widely used in food fermentation.
LABs produce bacteriocins.
Bacteriocins can inhibit the growth of
undesirable bacteria.
Bacteriocins
Example of bacteriocin application
Nisin produced by
Lactococcus lactis
subsp.
lactis
has been
approved and used widely in food preservation.
Substances
used
in
defense
systems
to
compete
for
growth
and
survival
of
bacteria
against
other
genetically
related
species
Bacteriocin is a protein by nature and there are
many types of bacteriocins.
724aa
Examples of Nisin and Pediocin Operons
IS904
nisA
nisB
P
P
nisT
nisC
nisP
nisR
229aa
682aa
245aa
418aa
600aa
851aa
IR rho
-
independent
terminator
IR
-
mRNA
processing
5’
-
termini
Tn5276
Tn5301
(A) Nisin Operon
174aa
62aa
(B) Pediocin (PA
-
1) Operon
112aa
kb sequenced
5.5 kb
P
nisI
pedA
pedB
pedC
pedD
Fig. 1. Organization of nisin A (A), and pediocin PA
-
1 (B) operons.
Method Development: Microbiology
Isolation of LAB on selective media (MRS).
An overlay method was chosen for testing
bacteriocin activity.
Lactobacillus plantarum
was used as a tester
strain.
Method Development: Microbiology
Nham sample
Serial dilution
Spread on plate
Colonies on plate
Overlay with
Lactobacillus plantarum
Day 1
Day 2
UD
10
-
1
10
-
2
10
-
3
10
-
4
10
-
5
10
-
6
pipette
100
µl
weigh
1 g
30
˚
C
24 h
Method Development:
Bioinformatics
Nucleotide searching and Similarity searching
Fig. 2. Website for nucleotide searching and similarity searching.
Learning bioinformatics through internet
Method Development:
Bioinformatics
Sequence alignment
Fig. 3. Website for sequence alignment.
Learning bioinformatics through internet
Polymerase Chain Reaction (PCR)
Denaturation
Annealing
Extension
Fig. 4. PCR diagram
PCR has been
widely used in
research.
Le
arning PCR principles
has
facilitated students’
understanding of invitro DNA
amplification.
Method Development
: PCR
LAB
Bacteriocin genes
Pediococcus pentosaceus
pediocin
(
ped
)
Enterococcus faecium
enterocin
(
ent
)
Lactococcus lactis
nisin
(
nis
)
Lactic acid bacteria and bacteriocin genes
Three pairs of three bacteriocin gene
-
specific primers were designed and
tested.
Method Development:
PCR
DNA template preparation by simple cell boiling method
TE buffer
Boil
Laboratory Unit
Teaching unit development: Students activities
chose their own food samples (Day1).
screened for bacteriocin
-
producing bacteria (Day1
-
3).
selected bacteria to perform the PCR procedures (Day 3).
learned bioinformatics (Day 3).
performed PCR (Day 4).
PCR analysis by gel electrophoresis (Day 5).
Laboratory Unit
Teaching unit trial
trialed in February 2006 and 2007
students worked in groups of five
Scientific Outcome: Microbiological Results
Fig. 5. MRS agar plate with lactic acid bacterial colonies overlaid with soft agar containing
L. plantarum
. The black
arrow indicates one of seven positive colonies with a clear halo zone around it.
The white arrow indicates a negative
colony with no clear halo zone around it. The photograph was taken from above with the cover of Petri dish removed
Conclusion: Each student group
Several LAB colonies were obtained on
MRS agar.
Students can observe inhibition zone.
Three colonies were picked for PCR.
Scientific Outcome: Microbiological Results
Scientific Outcome: Bioinformatics Experience
Students have an experience on using
free online software to design primers
for the detection of pediocin, enterocin,
and nisin genes.
Students know how to do DNA
sequence alignment.
Scientific Outcome: PCR Results
Fig. 6. Sample agarose gel of PCR products from amplification of pediocin (
ped
), enterocin (
ent
), and nisin (
nis
)
genes.
Lane 1
,
100
-
bp DNA ladder;
Lane 2
, amplicons from DNA isolated from
Pediococcus pentosaceus
,
Enterococcus faecium
and
Lactococcus lactis
;
Lanes 3
-
5
, amplicons from DNA from boiled cells of
Pediococcus
pentosaceus
,
Enterococcus faecium
and
Lactococcus lactis
, respectively;
Lane 6
, amplicons from DNA from a nham
sample spiked with
Pediococcus pentosaceus, Enterococcus faecium
and
Lactococcus lactis
;
Lanes 7
-
8
, amplicons
from DNA from food samples;
Lanes 9
-
11,
amplicons from DNA from colonies producing clear halos in a
Lactobacillus
plantarum
overlay.
Scientific Outcome: PCR Results
Conclusion: Each student group can
detect 608, 412, and 332 bp PCR
products.
Educational Outcome
Questionnaires and Interviews
Students were asked with series of questions to
monitor their perceptions of technical skills,
scientific knowledge, inquiry skills, and
enjoyment of science gained through the lab
unit.
Student’s responses were positive.
Observation
Observation results were corresponded with
the questionnaires and interviews.
Educational Outcome
Findings from the study
Students had gained
•
Technical
skills
•
Scientific knowledge
•
Scientific inquiry skills
•
Enjoyment of science
D
.
Suwanjinda, C
.
Eames, W
.
Panbangred
(
2007
)
Screening of Lactic Acid Bacteria for Bacteriocins by Microbiological
and PCR Methods
,
Biochem. Mol. Biol. Educ.
35
(5),
364
–
369
.
Acknowledgement
Prof. Dr.
Watanalai Panbangred
The institute for Innovation and Development of Learning
Process
The Institute for the Promotion of Teaching Science and
Technology (IPST), Thailand
Promotion of Science and Mathematics Teachers project
A research
grant from Mahidol University
Third year undergraduate biotechnology students
Members of K535
-
539
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