plg-1 gene expressing propionicin by lactic starters in dairying Alexandria University Arab Republic of Egypt 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. Table 1 The antimicrobial activity of different LAB transformants. ABSTRACT Propionicin PLG-1 is a bacteriocin produced by Propionibacterium thoenii P127. 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 pLEB590 to give the modified vector pLEBPLG-1. LAB transformants showed strong antimicrobial activity against E. coli DH5α (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. Fig 1 Different PCR trials, lane 5 is the most perfect trial. Cloning and expression of plg-1 gene of P. thoenii P127 into LAB plg-1 gene was cloned into pLEB590 and electroporated into different LAB strains. pLEB590 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 MATERIALS AND METHODS Microbial strains, plasmids and growth conditions All microbial strains used in this study and their media are listed in Table 1. E. coli DH5α 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 MG1614 harboring the vector pLEB590 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. Electroporation and stability of pLEBPLG-1 into LAB strains Electroporation efficiency of different LAB strains harboring pLEBPLG-1 was indicated as 4.2 X 109 cfu/µg DNA for L. lactis LL108, while that for S. thermophilus was found to be 4.1 x 109 cfu/µg DNA. On the other hand, L. bulgaricus DSM20080 and L. plantarum TF103 recorded 5.1 x 108 and 5.2 x 108 cfu/µg DNA, respectively. 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 AxyPrepTM DNA Gel Extraction Kit. Suitable primers [PLG1BAMHI2 (G G A T C C A A T G T C G A T G C C A G) and PLG1R3-13 (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 P127. 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. Screening for the antimicrobial activity of LAB transformants against Escherichia coli DH5α, Listeria monocytogenes 18116, and Salmonella entirica 25566 (Table 1) 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]. RESULTS AND DISCUSSION Isolation of plg-1 gene of P. thoenii P127 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]. PLG-1 has strong antimicrobial activity against the examined pathogens with special emphasis on E. coli DH5α which was the most sensitive strain. Thus, propionicin PLG-1 was expressed using pLEB590 as a multi-copy expression vector. ACKNOWLEDGEMENT The present work was supported using the fund of the International Center for Genetic Engineering and Biotechnology [ICGEB, Italy].