Genetic Engineering learning outcomes Define the term recombinant DNA. A section of DNA (often in the form of a plasmid) which is formed by joining DNA sections from 2 different sources © Pearson Education Ltd 2009 This document may have been altered from the original

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.

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 bonds Make sure you can explain the naming of restriction enzymes

Explain how engineered plasmids may be taken up by bacterial cells in order to produce a transgenic microorganism that can express a desired gene product. Describe the advantage to microorganisms of the capacity to take up plasmid DNA from the environment. © Pearson Education Ltd 2009 This document may have been altered from the original

Engineering plasmids Bacterial cells can be encouraged to take up plasmids (circular pieces of DNA found in bacteria) containing foreign genes by treating them with calcium salts. The cells receiving the plasmids are transgenic. Transgenic organisms contain additional DNA which has come from another organism The transgenic bacteria can be cultured and will express the inserted genes as if they were their own. © Pearson Education Ltd 2009 This document may have been altered from the original

Bacterial conjugation Bacteria often transfer plasmids from one to another. Because the plasmids often carry genes for antibiotic resistance this speeds up the spread of this characteristic in bacterial populations, leading to strains such as MRSA. This is an advantage to the bacteria but creates problems for the medical profession. © Pearson Education Ltd 2009 This document may have been altered from the original 6

Transformation in bacterial cells Bacteria are injected into mice S 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 TRANSFORMATION 7

State other vectors into which fragments of DNA may be incorporated. Eg: virus genomes or yeast cell chromosomes

LO’s for Insulin and Golden Rice Outline the process involved in the genetic engineering of bacteria to produce human insulin. Outline how genetic markers in plasmids can be used to identify the bacteria that have taken up a recombinant plasmid Outline the process involved in genetic engineering Golden Rice™. © Pearson Education Ltd 2009 This document may have been altered from the original

Production of bacteria containing the human insulin gene mRNA for insulin extracted from pancreatic cells by differential centrifugation, 153 base pairs corresponds to 51 amino acids. Use Reverse Transcriptase enzyme to produce single strand of DNA Then use DNA polymerase and free nucleotides to produce complementary strand. Double strand cDNA (copy DNA) is the gene for insulin Sticky ends added, sometime promoter site is added Cut plasmid with restriction enzyme to give complementary sticky ends Add cDNA and plasmid together and use ligase to join the sugar phosphate backbones. Add the recombinant plasmids to bacteria with calcium salts Some bacteria will take up plasmids, some plasmids will have taken up the insulin gene. © Pearson Education Ltd 2009 This document may have been altered from the original

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 grow Replica 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

Golden Rice 500,000 affected by Vit A blindness annually 1-2 million deaths Vit A found only in animal sources Beta carotene is a plant-made precursor to Vit A converted to vit A in gut Needed for making rhodopsin, glycoproteins, epithelial cells, bone growth Also need sufficient fat in diet as it is lipid soluble

Golden Rice Rice plants have the gene for beta carotene (photosynthetic pigment) but in the endosperm of rice it is not expressed Need to add genes for “Phytoene synthetase” from daffodils and “Crt enzyme” from soil bacteria Erwinia uredovora for beta carotene to be made in endosperm These 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 fold Need to eat about 200-300 grams of rice per day to get sufficient betacarotene

Golden Rice 14 © Pearson Education Ltd 2009 This document may have been altered from the original 14