Cloning and Expression of Bacteriophage T4 Lysozyme Gene (Gene e) in Escherichia coli via PCR Amplification
Lysozyme (EC 3.2.1.17) is an enzyme that catalyzes the hydrolysis of b(1-->4) glycosidic bonds between N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM), which are present in peptidoglycan heteropolymers of the prokaryotic cell wall. The purpose of this study was the cloning and expression of the bacteriophage T4 lysozyme gene (gene e) via PCR amplification in Escherichia coli using the pUC18 and pRS416 vectors. PCR amplification is carried out by using the primers having Bam HI recognition sites at their 5' ends. After digesting the vectors and PCR product with Bam HI endonuclease, two recombinant plasmids were constructed by ligation reaction and named pUC18L and pRS416L respectively. Recombinant E. coli cells harboring these plasmids expressed the gene e, that is, they secreted the active T4 lysozyme (called lysozyme E) into 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 burst out easily in a hypotonic environment such as distilled water or 10 mM Tris-HCl pH 8.0. It is suggested that the cell wall of E. coli may be weakened by the action of the lysozyme E, but the osmotic pressure balanced by the NaCl concentration in the medium may protect the damaged cells from lysis. These results show that any E. coli plasmid vectors bearing gene e may facilitate the isolation of plasmid or chromosomal DNA without using cell wall and cell membrane disruptive agents such as EDTA, lysozyme and SDS. For this purpose, pUC18L (E. coli) and pRS416L (E. coli - Saccharomyces cerevisiae shuttle vector) are constructed as prototype vectors.
Cloning and Expression of Bacteriophage T4 Lysozyme Gene (Gene e) in Escherichia coli via PCR Amplification
Lysozyme (EC 3.2.1.17) is an enzyme that catalyzes the hydrolysis of b(1-->4) glycosidic bonds between N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM), which are present in peptidoglycan heteropolymers of the prokaryotic cell wall. The purpose of this study was the cloning and expression of the bacteriophage T4 lysozyme gene (gene e) via PCR amplification in Escherichia coli using the pUC18 and pRS416 vectors. PCR amplification is carried out by using the primers having Bam HI recognition sites at their 5' ends. After digesting the vectors and PCR product with Bam HI endonuclease, two recombinant plasmids were constructed by ligation reaction and named pUC18L and pRS416L respectively. Recombinant E. coli cells harboring these plasmids expressed the gene e, that is, they secreted the active T4 lysozyme (called lysozyme E) into 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 burst out easily in a hypotonic environment such as distilled water or 10 mM Tris-HCl pH 8.0. It is suggested that the cell wall of E. coli may be weakened by the action of the lysozyme E, but the osmotic pressure balanced by the NaCl concentration in the medium may protect the damaged cells from lysis. These results show that any E. coli plasmid vectors bearing gene e may facilitate the isolation of plasmid or chromosomal DNA without using cell wall and cell membrane disruptive agents such as EDTA, lysozyme and SDS. For this purpose, pUC18L (E. coli) and pRS416L (E. coli - Saccharomyces cerevisiae shuttle vector) are constructed as prototype vectors.
