Genetic Engineering

Also known as recombinant DNA technology, genetic modification, genetic manipulation

-  Based to the core principle – genetic code is universal

-  The human genome project and genetically modified organisms

o Enabled identification of genes
o Enabled us to produce proteins and microorganisms such as

§ Human growth hormone

§ Insulin etc,

-  We genetically modify genes because...

o Gene products – using GMOs to produce chemicals for medical or industrial use. Mostly proteins.

§ Insulin etc

§ Modification of animals to produce the main human products o New phenotypes – to alter the characteristics of organisms

§ This can be used to make crops resistant to disease etc.
§ Bioremediation – to modify bacteria to absorb and metabolise toxins

and pollutants
o Gene therapy – altering human genes to treat disease

-  General stages (there are 4):
o The required gene is identified and obtained o A copy of the gene is inserted int o the vector o The vector carries the gene to the recipient
o The recipient expresses the novel gene

-  This seem simple but is not

-  We must get the gene

o mRNA can be extracted from the cells expressing the gene and used to synthesise cDNA (complementary DNA) the mRNA would be amplified

§ this would require the use of reverse transcriptase
o Gene can be synthesised using an automated polynucleotide synthesiser o Gene can be PCRed from genomic DNA using PCR primers if the gene

sequence is known and thus the flanking systems are known

-  After this copies must be made

-  The gene will then be isolated
o Restriction enzymes can be used to create sticky ends o DNA is used separated using electrophoresis

§ Electrophoresis separates out DNA according to size

§  Agarose is used and the DNA is loaded with a buffer and the DNA is

negatively charged so will run to the positive electrode but due to different weights it will form a ladder affect and it can then be separated by using this

§  FUNDAMENTALLY ENABLES THE SEPARATION ON THE BASIS OF THE SIZE OF DNA

-  We then need a vector: o Bacterial plasmid

o Liposomes

o Bacterial artificial chromosomes (BACs) Not used anymore though o Viruses -retroviruses
o Viruses – bacteriophages

-  The vector must be cut with the same restriction enzyme to give you complementary sticky ends

-  We can then use DNA ligase to anneal the gene and the plasmid DNA

-  Transfer into bacteria

o This can occur by electroporation – holes punched in membrane by electricity, so the plasmids taken up

o This can alternatively occur by electrofusion – Tiny currents applied to the membranes of the two cells which causes the membranes to fuse

o Lastly heat shock can be used where transfers between hot and cold are used which leads to the formation of some pores in the membrane which then allows the plasmid to be taken up

-  A gene gun could also be used which is primarily for Plants and this requires tiny gold pellets to be coated with the plasmid and fired at the plant cell (this is because the plant cell has a cell wall)

-  Isolate the transformed bacteria

o Typically an antibiotic resistant gene will be used to assure that the

transformed bacteria are easier to see and identify
o There are three types of bacteria which may then form

§ No plasmid
§ Plasmid but the plasmid reannealed so has no gene of interest § Plasmid with desired gene

o As a result, typically 2 antibiotic resistant genes will be used
o First the genes are isolated by replica plating
o We then add some antibiotic on the plate, and this leads to the death of any

that do not have the plasmid
o Then the second antibiotic can be used and we can identify that the colonies

that are not on the third plate must be the desired ones as they are not resistant to the second and this means that gene is disrupted with the inclusion of the desired gene

o These cultures can then be picked and place in liquid or batch cultures and then potentially industrial scale to extract product

- This can be tested on a small way with sequencing