1. Recombinant Production of Plantaricin W from Lactobacillus plantarum S34 :
Cloning and expression in Lactobacillus acidophilus
Aksar Chair Lages1, Ike Rahmawati2, Apon Zaenal Mustopa2, Suharsono1
Affiliations :
Department of Biotechnology, Graduate School of Bogor Agricultural University
Research Center for Biotechnology, Indonesian Institute of Science
Presented at:
International Conference for Pre-breeding and Gene Discovery
Bogor, 13-14th August 2014

2. Bacterial DNA manipulation
1. INTRODUCTION 1
Bacterial DNA manipulation

3. 1. INTRODUCTION 2

4. Produce by E.coli 1. INTRODUCTION 3 Escherichia coli
Short generation time
Broad usage for development many tools such as expression system
Possibilities for high yield when used for recombinant protein production, but
Some recombinant protein able to form inclusion bodies
For the “food grade” purpose, use this bacteria is not recommended

5. Lactic Acid Bacteria (LAB)
1. INTRODUCTION 4
Lactic Acid Bacteria (LAB)

6. Produce by LAB 1. INTRODUCTION Usually as traditional starter5
Produce by LAB
Usually as traditional starter
GRAS organism
Safe and sustainable use as overall safety food-grade expression system
Several recombinant peptide in LAB :
β-Galactosidase
Aminopeptidase
Enterocin
Sakacin, etc
Lactobacillus sp.

7. 1. INTRODUCTION 6
“Aim of this study is to obtain crude extract of recombinant plantaricin W of Lactobacillus plantarum S34 that produce by Lactobacillus acidophilus”

8. 2. METHOD 7
Materials

9. Research Scheme 1 2 3 4 2. METHOD 8 flow of process Preparation
Gene isolation (PCR)
Plasmid extraction
Construction
Cutting and ligation DNA
Introduction to E.coli
Transformation
Introduction to Lactobacilli host by electroporation
Expression
Growth trans-gene species
Harvest & disrupt cell
Analyze crude extract by SDS-PAGE 1 2 3 4

10. 3. RESULT & DISCUSSION 9
(a)
(b)
(c)
Figure 1. Construction of pSIP409-WS34 as expression vector in LAB. (a) Colony PCR of transformants, (b) recombinant plasmid, (d) map of recombinant plasmid
Recombinant plasmid has been constructed successfully , based on sequencing result (data not shown)
Both of genes sppK and sppR are regulatory system that encode Histidine Kinase and Response Regulator
Rep-region contains pUC(pGEM)ori and 256rep
Further, antibiotic resistance marker will be exchanged by food-grade selection marker

11. 3. RESULT & DISCUSSION
(c) 10
(a)
(b)
700 bp
Figure 2. Cloning and expression in LAB. (a) The transformants, (b) colony PCR, (c) SDS-PAGE of Plantaricin WS34 expression. Red arrow indicates Plantaricin WS34 (~ 15 kDa). P mean pellet or insoluble fraction, and SN mean supernatan or soluble fraction.
Recombinant Plantaricin WS34 has been over-expressed intracellularly in Lactobacillus acidophilus C1-9
The cultures were induced with 50 ng ml-1 SppIP at OD600 0,2-0,3 and grown to OD600 1,8-2
Level of expression may be increased by optimizing induction or exchanging the replicon with replication determinant that gives rise to a higher copy number

12. ACKNOWLEDGMENT
This study financially supported by Competitive Program 2014 from Indonesian Institute of Science. The authors also wish to thank Dr. Lars Axelsson from Matforsk Skining Institute for providing plasmid pSIP409