Genetic Engineering of INDUSTRIAL Microorganisms
Overall, genetic engineering procedures allow totally new properties to be added to the capabilities of industrial microorganisms .Microorganisms may be manipulated to synthesize and often excrete enhanced ranges of enzymes, which may facilitate the production of novel compounds or allow the utilization of cheaper complex substrates. As there is no restriction to the origins of the genes that microorganisms express, the production of plant and animal proteins is made possible.
Nevertheless, these methods have not totally replaced Industrial microorganisms traditional mutatagenesis methods and the two approaches should be viewed as complementary strategies for strain improvement.
Strategies for the genetic engineering of bacteria
Genetic engineering involves manipulation of DNA outside the cell. It necessitates the initial isolation and recovery of the gene(s) of interest from the donor organism’s genome.
Isolated DNA sequences may then be modified and the regulation of their expression altered, before insertion into host organisms via a suitable easily manipulated vector system. The first step requires total DNA extraction from the donor organism, which is then cut into smaller sequences using a specific restriction endonuclease.
Many of these restriction enzymes, found in various species of bacteria, make a staggered cut through a double-stranded DNA molecule at a specific sequence. As a result, the ends of cut molecules have complementary single-stranded sequences.
The small sections of DNA (restriction fragments) can then be joined or spliced into vector DNA molecules that have been cut with the same restriction enzyme. Splicing is performed by an enzyme, DNA ligase, and creates a synthetic DNA molecule.
Plasmids and bacteriophages have been the most useful cloning vectors. They play an important role as delivery systems to introduce the recombinant molecules into host cells via transformation or transduction. Once inside they are capable of autonomous replication, which maintains the recombinant DNA within the host cell.
Introduction of recombinant plasmids into bacterial cells can be achieved following calcium chloride treatment, which renders the cell membranes more permeable.
This generates a DNA library consisting of individual clones that contain different recombinant DNA molecules, representing all DNA sequences/genes of the donor genome. Once the library has been established, the clones are allowed to form colonies on solid selective media. At this stage, the specific clone containing the recombinant DNA molecule of interest can be identified.
22 фев 2022
