AN INNOVATIVE LABORATORY EXERCISE: SCREENING OF LACTIC ACID BACTERIA FOR BACTERIOCINS BY MICROBIOLOGICAL AND PCR METHODS

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
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.

Example of bacteriocin application

Nisin produced by Lactococcus lactis subsp. lactis has been approved and used widely in food preservation.

Examples of Nisin and Pediocin Operons

(A) Nisin Operon
IR-mRNA processing IR rho-independent terminator

5-termini Tn5276 Tn5301 IS904

nisA

nisB
851aa

P P

nisT
600aa

nisC
418aa

nisI
245aa

nisP
682aa

nisR
229aa

(B) Pediocin (PA-1) Operon

P
pedA
62aa

kb sequenced

pedB
112aa

pedC
174aa

pedD
724aa

5.5 kb

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

weigh 1g Nham sample
UD 10-1 10-2 10-3 10-4 10-5 10-6

pipette 100 l

Serial dilution

Lactobacillus plantarum

Overlay with

30 C 24 h Colonies on plate Spread on plate

Day 2

Day 1

Method Development: Bioinformatics

Learning bioinformatics through internet
Nucleotide searching and Similarity searching

Fig. 2. Website for nucleotide searching and similarity searching.

Method Development: Bioinformatics

Learning bioinformatics through internet
Sequence alignment

Fig. 3. Website for sequence alignment.

Polymerase Chain Reaction (PCR)

Denaturation
Annealing Extension

PCR has been widely used in research. Learning PCR principles has facilitated students understanding of invitro DNA amplification.

Fig. 4. PCR diagram

Method Development: PCR

Lactic acid bacteria and bacteriocin genes
LAB Bacteriocin genes

Pediococcus pentosaceus
Enterococcus faecium Lactococcus lactis

pediocin (ped)
enterocin (ent) nisin (nis)

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

Scientific Outcome: Microbiological Results

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: 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. Students 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), 364369.

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

Questions or Comments?