Arab Republic of Egypt

1. Heterologous Expression of pctA Gene Expressing Propionicin T1 by Some Lactic Acid Bacterial Strains using pINT125 Arab Republic of Egypt Alexandria University Mahmoud K. Tahoun and Sameh E. Mohamed Department of Dairy Science and Technology, Faculty of Agriculture, Alexandria University. Aflatoon Street, El-Shatby 21545, Alexandria, Egypt. ABSTRACT pctAgene was previously transferred to some lactic acid bacterial [LAB] strains using pLEB590 as an expression vector and the modified vector pLEBA [containing pctA gene] showed moderate stability in LAB transformants. As another trial to improve pctA gene stability in LAB transformants, pctAgene was transferred into different LAB strains using another vector [pINT125] which is a multi – copy integration vector. The vector pINTB [containing pctA gene] was electroporated into different LAB strains. Such vector showed higher stability in LAB transformants than pLEBA. Fig 2. pINTB integration into chromosomal DNA of the transformants Keywords: Propionicin T1 – Propionibacteria – pINT125 – LAB– Spore formers. Total crude protein isolated from transformants media, partial purification, SDS - PAGE, and direct detection on gel RESULTS AND DISCUSSION Construction of pINTB (enclosing pctA gene of P. thoenii 419) Fig. 3.a: PAGE of LAB transformants and their parental strains Fig. 3.b: Direct detection against B. subtilis DB100 host Fig. 3.c: Direct detection against C. sporogenes DSM1446 It was noted that the inhibition zones of PT1 secreted by Lactobacillusbulgaricus DSM20080 [pINTB], Lactobacillus plantarum TF103 [pINTB], and Lactococcuslactis LL108 [pINTB] were wider than the inhibition zones of PT1 secreted by P. thoenii 419. It was also noticed that the inhibition activity of PT1 secreted by pINTB LAB transformants on C. sporogenes DSM1446 was wider than that on B. subtilis DB100 host. Finally, it can be concluded that PT1 gene (pctA) was successfully transferred via the food - grade integration vector (pINT125) to LAB. It was proved that PT1 secreted by its parental producer (Propionibacteriumthoenii 419) as well as by different LAB transformants has a bactericidal effect on B. subtilis DB100 host as well as C. sporogenes DSM1446. Fig 1. Construction of pINTB Electroporation of pINTB to LAB strains The electroporation efficiency of different LAB strains harboring pINTB was investigated and the results indicated 2.3 x 104cfu/µg DNA for Lactococcuslactis LL108, while that for Streptococcus thermophilus was found to be 1.4 x 102cfu/µg DNA. On the other hand, Lactobacillus bulgaricus DSM20080 and L. plantarum TF103 recorded 4.8 x 107 and 3.3 x 105cfu/µg DNA, respectively. Comparison between pLEBA and pINTB About the stability of pLEBA and pINTB, the data shown in the poster clearly indicated that the former is less stable than the latter. However, it is recommended to use LAB transformed with pLEBA because PT1 is more overexpressed using pLEBA than pINTB regardless of the stability from the industrial view. ACKNOWLEDGEMENT pINTB integration into the chromosomal DNA of the transformants Transformantswere grown on their appropriate media supplemented with 0.3 M sucrose (without glucose in the case of MRS). Plasmid isolation was performed using the mini - prep isolation method followed by gel electrophoresis of plasmid samples on agarose 1.0 %. It was noted that the plasmid pattern of transformants was identical to the plasmid pattern of the parental strains. Since pINTBtransformants showed strong growth on their media supplemented with 0.3 M sucrose as the sole source of carbon, pINTB was successfully electroporated and integrated into the chromosomal DNA of transformants. The present work was supported using the fund of the International Center for Genetic Engineering and Biotechnology [ICGEB, Italy]. REFERENCES 1- Takala, T M; and Saris, P E J [2002] ApplMicrobiolBiotechnol 59: 467 – 471. 2- O’Sullivan, D J; and Klaenhammer, T R [1993] Appl Environ Microbiol 59: 2730 – 2733. 3- Ben – Shushan, G; Zakin, V; and Gollop, N [2003] Peptides 24: 1733 – 1740.