Bacterial Transformation

Introduction

  • Cell culture: the process by which cells can be gathered from their natural locations and grown inside laboratory containers under controlled conditions.
  • Appropriate food and environment must be provided for the cells to grow.
  • Bacteria and yeast are very easy to grow in culture.
  • After cell growth is complete, cells in culture can be harvested and studied.
  • A fundamental assumption of recombinant DNA technology is that permanent and desirable changes in the functioning of living cells can be accomplished by changing the linear sequence of their DNA.
  • Genetic transformation: involves the insertion of some new DNA into E. coli cells, or a number of other organisms, predominantly bacteria.
  • In addition to one large chromosome, bacteria often contain one or more small circular pieces of DNA called plasmids.
  • Plasmid DNA usually contains genes for more than one trait.
  • Genetic engineering: a process used to insert genes coding for new traits into a plasmid.
  • Restriction enzymes: proteins made by bacteria as a defense against foreign, invading DNA.
    • Each restriction enzyme recognizes a unique sequence of typically 4-6 base pairs, and will cut any DNA whenever that sequence occurs.
    • Restriction enzymes will cut DNA from any source, provided the recognition sequence is present.
  • DNA ligase: an enzyme that glues pieces of DNA together, provided the ends are compatible.
    • This allows the creation of recombinant DNAs, or hybrids, created by joining pieces of DNA from two different sources.
  • Genes can be cut out of human DNA or plant DNA, and placed inside bacteria.
  • Because of their small size, plasmid DNAs is easy to extract and purify from bacterial cells.
    • When cut with a restriction enzyme, they can be joined to foreign DNAs – from any source – which has been cut with the same enzyme.
    • The resulting hybrid DNAs can be reintroduced into bacterial cells by a procedure called transformation.
    • Now the hybrid plasmids can perpetuate themselves in the bacteria just as before, except that the foreign DNA which was joined to it is also being perpetuated.
    • The foreign DNA is called a cloning vehicle or vector.

DNA Libraries

  • When DNA is extracted form a given cell type, it can be cut into pieces and the pieces can be cloned in masses into a population of plasmids. This process produces a population of hybrid (recombinant) DNAs.
  • After introducing these hybrids back into cells, each transformed cell will have received and propagated on unique hybrid.
  • Every hybrid will contain the same vector DNA but a different insert DNA.
  • DNA library: a collection of DNA fragments that have been cloned into vectors
  • If the original extract came from human cells, the library is a human library.