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

Bacterial DNA manipulation 1. INTRODUCTION 1 Bacterial DNA manipulation

1. INTRODUCTION 2 http://chem.agri.kagoshima-u.ac.jp/~hishamri/

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

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

Produce by LAB 1. INTRODUCTION Usually as traditional starter 5 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.

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”

2. METHOD 7 Materials

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

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

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

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