Biochemical and Biotechnology - Method for Specific integration of T7 RNA Polymerase gene into the Chromosome of Corynebacteria - T7 promoter based shuttle vector and shuttle vector system

Introduction

E.coli has been the host of choice for the production of recombinant proteins since the last many years. However being a gram negative bacteria, the secretion of proteins is limited in E.coli. It forms inclusion bodies from which the recombinant protein has to be isolated by using harsh chemical treatment. Besides, not only is the reducing environment in the cytoplasm not conducive for several proteins, the periplasmic space of this bacteria has got several proteases, which degrade the expressed proteins reducing their yield considerably. For these reasons, a method for effectuating a specific integration of T7 RNA polymerase gene into the chromosomal DNA of Corynebacteria to obtain high success rates of getting the desired strain .has been developed. This enable to construct E.coli Cornebacteria shuttle vector as well as Cornebacteria - T7 shuttle vector system.

The method involve integration of T7 RNA polymerase gene into the chromosome of corynebacteria (Cacetoacidophilum). The said method for specific integration uses an E.coli plasmid pGP1-2 carrying the gene for kanamycin resistance, the genes for T7 RNA Polymerase and the cl repressor. This plasmid is digested with restriction enzyme Bam H1 and ligated to the Sau3A1dugested genomic DNA of C, acetoacidophilum.. Thereafter a ligation mixture is used to transform C, acetoacidophilum.. protoplasts. Transformants are then screened for Kanamycin resistance and seniitivity to aminoglycoside such as streptomycin, to ensure targeting of plasmid vector pGP1-2 into the chromosome of C, acetoacidophilum. The process will lead to the modification of the chromosomal DNA of C, acetoacidophilum, which now enables the said bacterium designated B-30T7R to express T7 RNA polymerase. These cells are then processed for isolation of pGP1-2 to detect plasmid DNA. The absence of plasmid DNA establishes that the plasmid has integrated into the chromosome of the cell. It was also noticed that this chromosomal integration did not affect the growth of this strain. Figure 1 outlines the strategy adopted for the integration of T7 RNA polymerase gene of plasmid pGP1-2 into the genomic DNA of C, acetoacidophilum.

Special Features:

Screening of the clones is simple and cost effective No expensive chemical is required for regulation No adverse effect is noticed on cell growth There is no protease induction when the temperature is raised to 400 C Expensive chemicals is not required Provide a novel shuttle vector pBKET29aS and a novel vector system, Provides a method for obtaining optimum expressed proteins in a transformed gram positive bacteria.

Prospective Users:

Pharmaceutical industries

Type of Technology:

Process

Status of IPR Protection:

Indian Patent Application No 96/DEL/2002 dated 5.2.2002 with title "Method for Specific integration of T7 RNA Polymerase gene into the Chromosome of Corynebacteria and resultant novel Corynebacteria -T7 promoter based shuttle vector and shuttle vector system "

Contact for more information

The Director
Foundation for Innovation and Technology Transfer
Indian Institute of Technology Delhi
Hauz Khas, New Delhi-110016
Tel : 91-011-26597167, 26857762, 26581013
Fax : 91-011-26851169
E-mail : mdfitt@gmail.com

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