Control of Gene Expression

Recombinant Gene Technologies

In Vivo gene cloning (vectors)

Preparing to insert into a vector:
  1. Promoter regions for RNA polymerase to bind to are added to the start of the sections of DNA
  2. Terminator regions, which stop transcription, are added at the relevant points
Inserting into a vector:
  1. After adding the relevant sections to the target gene, it can be added to a carrying unit
  2. The carrying unit, also called a vector, is used to transport the target gene into a host cell
  3. Usually, a plasmid is used to insert the vector into bacteria, and there is usually a gene for antibiotic resistance or fluorescence to allow separation later
  4. Ligase, an enzyme, is used to permanently bind the DNA together in the plasmid
Introducing the DNA to a host cell:
  1. The bacterial hosts are mixed together with the plasmid containing the target DNA
  2. Only a small number of bacteria will take up the plasmid, and an even smaller number will take up the plasmid containing the target gene
  3. An antibiotic, typically ampicillin, is used to kill the bacteria not containing the target gene
  4. The surviving bacteria are then replicated in a fermenter, producing many copies of the target gene
Transformation – the process of plasmids and bacterial cells being mixed together in a growth medium containing calcium ions