Genetic Engineering

Obtaining the Gene:

• mRNA produced from transcription can be obtained from cells where that genne is expressed. THe mRNA can be used as a template to make a copy of the gene
• The gene can be synthesised using an automated polynucleotide sequencer
• A DNA probe can be used to locate the gene on DN fragments and the gene cut from a DNA fragemtn using restriciton enzymes

Placing the Gene is a Vector

• The gene can be sealed into a bacterial plasmid using the enzyme DNA ligase
• Genes may also be sealed into virus genomes or yeast cell chromosomes
• Vectors often have to contain regularoty sequences of DNA, which ensure that the inserted gene is transcripted in the host cell

Getting the Gene into the recipient cell: - The gene once packed into a vector, can form quite a large molecule that does not easily cross the membrane to enter the recipients cell

• Electroporation - high votage pulse disrupts the membrane
• Microinjection - DNA injected with  fine micropipette
• Viral Transfer - A virus vector infects cells be inserting its DNA directly
• Liposome  - DNA is wrapped in lipid olecules. These are fat-soluble and can cross the lipid bilayer by diffusion
• Ti Plasmids - inserted int Agrobacterium tumefaciens. Plants can be infected and the DNA is inserted into the plant's genome

DNA fragment isolated using restriction enzymes

DNA frgmetns inserted to a vector (plasmid/bactriophages) - Vector DNA cut using same restriction enzymes so the sticky ends are complementary to DNA fragment. Vector DNA and Fragment mixed with ligase which seal the sugar-phosphate backbone to form recombinant DNA

The vector transfers the gene into bacteria

If a plasmid is used, the plasmids are added to the cell in ice cold CaCl2 solution so the cell walls are more permeable. The mix is then heat shocked which encourages the cells to take up the plasmid

With a Bacteriophage vector, the bacteria is infected when the bacteriophage injects its DNA which integrates into the bacterial DNA

Cells that take up the vectors are transformed

Identify the tranformed bacteria with marker genes, which are niesrted with the desired gene into the vector - only transformed bacteria contains the marker gene

• Marker gene can be antibacterial resistance so that when bacteria are grown on an antibacterial agar plate, only cells with the marker gene survive and grow
• Marker gene can be for fluorescene - when the agar plate is under UV light, only transformed cels fluorese.