Cloning of bacteriophage T4 lysozyme gene (Gene e)with plasmid vectors

ABSTRACT Cloning of Bacteriophage T4 Lysozyme Gene (Gene e) With Plasmid Vectors Lysozyme (EC 3.2.1.17) is an enzyme that catalyzes the hydrolysis of glycosidic bonds between N-acetyl glucosamine (NAG) and N-acetyl muramic acid (NAM) which are present in peptidoglycan heteropolymers of prokaryotic cell wall. In this study, it is aimed at the cloning of bacteriophage T4 lysozyme gene [gene e) in Escherichia coii using the pUC18 and pRS416 vectors. The primers with Bam HI recognition sites were designed for the PCR amplification of the gene e from T4 genome. After digesting the vectors and PCR product with Bam HI endonuclease, two recombinant plasmids were constructed by ligation reaction and called pUC18L and pRS416L. It is determined that, recombinant E. coli cells harboring these plasmids were expressed the gene e and secreted the active T4 lysozyme called lysozyme E in the culture medium. At the same time it was observed that, E. coli clones producing lysozyme E were grown in L and LB medium without lysis. But it is seen that cultured recombinant cells did not withstand against osmotic pressure and burst out easily in the hypotonic environment which does not contain osmotic stabilizers such as non-ionic water or 10 mM Tris-HCI pH 8.0. It is suggested that the cell wall of the E. coli may be weakened by the action of the lysozyme E, but the osmotic pressure created by the NaCI concentration in the medium may protect the damaged cells from lysis. These results show that the isolation of plasmid or chromosomal DNA may be facilitated with any E. coli plasmid vectors bearing gene e even in the absence of cell wall and cell membrane disruptive agents such as EDTA, lysozyme and SDS. For this purpose, pRS416L is constructed as a prototype shuttle vector. Key Words: Cloning, E. coii, PCR, Plasmid vectors, T4 Lysozyme gene