Genetically modifying Bacteria

Question 1

How does gene therapy work?

Answer 1

1 The ends of the desired gene are cut using a restriction enzyme which breaks the DNA at specific base sequences to leave complementary ‘sticky ends’ on the gene. The plasmid is also cut open using the same restriction enzyme.
- The plasmid may be labelled by having marker genes (e.g., antibiotic resistance genes, an enzyme gene, or fluorescence genes) inserted into it so that it can be detected after modification.
2 Ligase enzymes ‘glue’ the sticky ends of the plasmid and the gene together with a phosphodiester bond to seal the sugar phosphate backbone, thus forming recombinant DNA.
3 Large quantities of the plasmid are incubated with the host bacteria to ensure the bacteria take up some of the recombinant DNA.

Question 2

What is Electroporation used for?

Answer 2

Electroporation (an electric shock) may be used to increase cell membrane permeability to plasmid uptake.