Genetic Engineering: Vectors (Cambridge (CIE) A Level Biology)

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 (eg. 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 therefore markers are used to identify these 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)

Use of Plasmids as Vectors Diagram

A plasmid is genetically engineered with the desired gene

Viruses

• Viruses are commonly used as vectors in the process of gene therapy, which is currently used to treat genetic diseases such as cystic fibrosis

• The viruses are genetically modified to carry non-mutated genes into host cells

• Different types of viruses have been used; retroviruses, lentiviruses and adeno-associated viruses

The Use of Viruses as Vectors Diagram

An adenovirus is genetically engineered with the desired gene. Retroviruses have also been used as viral vectors however, there have been side effects from their usage

Liposomes

• Liposomes are small spherical vesicles with a phospholipid bilayer

• These vesicles can also be used in gene therapy to carry non-mutated genes into host cells

• The advantage of using liposomes as a vector is that they can fuse with the cell surface membrane

The Use of Liposomes as Vectors Diagram

A liposome is genetically engineered to carry a plasmid that contains the desired gene