Biotechnology and Bioinformatics (1+1)

2.1.4. Cloning a Gene

DNA cloning or molecular cloning is the technique of producing identical copies of DNA in a larger amount with the help of host. The recombinant DNA must be introduced into a cell, within which it may replicate freely. In addition, it is required that the introduced gene also be expressed within this cell.

5.4.1. Cloning in Prokaryotes

After ligation of target into vector , the recombinant DNA is multiplied in a suitable prokaryotic host. Various bacterial host commonly employed are :

1) Gram positive bacteria E. coli: host for plasmid .

2) Gram negative bacteria, Bacillus subtilis: host for plasmid, phages and cosmid vectors.

• Chimeric DNA is inserted into bacterial cells to create transformed bacterial cells.
• Colonies of transformed cells are selected and used for multiplication in suspension cultures.
• The rDNA is retrieved and isolated from the host cell when required.

E. coli:

• Most commonly employed gram positive bacterial host which is termed as workhorse of genetic engineering .
• After suitable modifications, several commercially valuable protein s have been expressed from transformed E. coli cells.

Bacillus subtilis:

• Utilized in a large number of fermentation industries.
• Absolutely non-pathogenic.
• Most species secrete proteins which allow the desired protein to be collected exogenous ly.

• Streptomyces species:

  • Gram negative bacteria producing more than 60 percent of known antibiotic s.
  • Genetic engineering may help in improving their drug producing ability and synthesizing novel antibiotics.