genetic-engineering-vectors

Genetic engineering: vectors

• Vectors are used to transfer the desired genes into a foreign cell

• Plasmids are the most commonly used vector but viruses and liposomes (a small vesicle with a phospholipid layer) can also be used to transfer genes

Plasmids

• Plasmids are small, circular rings of double-stranded DNA

• They occur naturally in bacteria, but can also be found in archaea and eukaryotic organisms (e.g., yeast and fungi) and can contain genes for antibiotic resistance

• Plasmids are used as they can self-replicate

• A plasmid is used to transfer the desired gene to a new organism

• To insert the desired gene into the circular DNA of the plasmid it is ‘cut’ open

  • The same restriction endonuclease that was used to isolate the desired gene is used to ‘cut’ open the plasmid

  • This results in the plasmid having complementary sticky ends to the sticky ends on the desired gene fragment

• DNA ligase forms phosphodiester bonds between the sugar-phosphate backbone of the DNA fragment and the plasmid to form a recombinant plasmid (a closed circle of double-stranded DNA containing the desired gene)

• Scientists can modify bacterial plasmids or produce them artificially

  • One benefit of this is that the plasmids can have one or more marker genes so that cells that have the recombinant plasmids can be identified

• Plasmids are transferred into host cells (usually bacteria) by a process called transformation

• Only a small proportion of bacteria will become transformed and markers are used to identify transformed cells

• Transformation can occur by:

  • Bathing the plasmids and bacteria in an ice-cold calcium chloride solution and then briefly incubating at 40°C

  • This makes the bacterial membrane permeable

  • Electroporation – where the bacteria are given a small electrical shock, making the membranes very porous (this technique can be used to get DNA fragments into eukaryotic cells)