2 Genetic Engineering learning outcomes Be able to explain that genetic engineering involves the extraction of genes from one organism and placing them into another organism.Be able to describe how sections of DNA containing a desired gene can be extracted from a donor organism using restriction enzymes.Be able to explain how isolated DNA fragments can be placed in plasmids, with reference to the role of ligase.
3 Restriction enzymes(a) Cutting DNA using a restriction enzyme produces a staggered cut and leaves sticky ends. They cut at specific sequences in the DNA called restriction sites (or recognition sites)(b) Joining DNA from two sources cut with the same restriction enzyme; complementary sticky ends allow the base pairs to anneal or match up and form H bondsMake sure you can explain the naming of restriction enzymes
7 Transformation in bacterial cells Bacteria are injected into miceS strain of bacterium contains a gene to make a toxic protein but R strain of bacterium does not.Heat killed S strain bacteria can’t make the protein so the mouse remains healthy.R strain mixed with killed S strain kills the mouse because the live bacteria can take up the DNA from the dead bacteria and use its “instructions” to make the toxic protein.This is called TRANSFORMATION7
8 State other vectors into which fragments of DNA may be incorporated. Eg: virus genomes or yeast cell chromosomes
11 Identifying Transformed Bacteria By using plasmids that carry genes for resistance to ampicillin and tetracycline it is possible to identify those bacteria that have taken up plasmids that contain the desired gene.The desired gene is inserted part way along the tetracycline resistance gene.Bacteria are grown on ampicillin agar so all bacteria that have plasmids in them growReplica plating is carried out on tetracycline agar – only those bacteria that HAVEN’T taken up the insulin gene will grow on this.So it is possible to identify those colonies on the ampicillin plates that DO have the insulin gene.11
12 Golden Rice 500,000 affected by Vit A blindness annually 1-2 million deathsVit A found only in animal sourcesBeta carotene is a plant-made precursor to Vit A converted to vit A in gutNeeded for making rhodopsin, glycoproteins, epithelial cells, bone growthAlso need sufficient fat in diet as it is lipid soluble
13 Golden RiceRice plants have the gene for beta carotene (photosynthetic pigment) but in the endosperm of rice it is not expressedNeed to add genes for “Phytoene synthetase” from daffodils and “Crt enzyme” from soil bacteria Erwinia uredovora for beta carotene to be made in endospermThese 2 genes were inserted close to the promoter site that initiates endosperm development so that they are switched on at the same time.Cross breeding of Golden Rice varieties has increased Beta carotene production in endosperm by 20 foldNeed to eat about grams of rice per day to get sufficient betacarotene