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Bacterial Transformation. Genetic Engineering – scientists put new genes into cells to develop organisms that are beneficial to people – uses include: Bacteria that can produce hormones such as human growth hormone and insulin Making plants frost and pest resistant
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Bacterial Transformation Genetic Engineering – scientists put new genes into cells to develop organisms that are beneficial to people – uses include: Bacteria that can produce hormones such as human growth hormone and insulin Making plants frost and pest resistant Developing plants that do not need fertilizer Bacteria that eat oil slicks
Escherichia coli • Often used for genetic engineering • Common inhabitant of human colon – easy to get • Can be easily grown in suspension culture in a nutrient such as Luria broth • Has a simple circular chromosome with about 1/600th the haploid amount of DNA in a human cell • E. coli often contain small circular DNA molecules called plasmids (extrachromosomal) – confer a particular trait such as resistance to antibiotics • So we can easily introduce our own plasmids to produce desired products
Plasmids are produced by cutting desired DNA (using restriction enzymes) and inserting a gene into a plasmid to act as a carrier • The gene is often inserted into a plasmid with genes for antibiotic resistance so that the transformed bacteria can be easily selected from other cells that did not pick up the plasmid
In nature, genes can be transferred between bacteria in three ways: • Conjugation – mating process during which genetic material is transferred from one bacterium to another of a different mating type • Transduction – a virus acts as a vector (carrier) to transfer small pieces of DNA from one bacterium to another • Bacterial Transformation – involves the transfer of genetic information into a cell by direct uptake of the DNA (occurs only rarely in nature)
Transformation in the Laboratory • Transformation was first performed in the laboratory by Griffith and later by Avery, MacLeod, and McCarty (experiment using mice and pneumococcus bacteria – please review!) • Bacteria can take up DNA only during the period a the end of logarithmic growth – cells are said to be competent (can accept DNA that is introduced from another source)
E. coli competence can be induced under carefully controlled chemical growth conditions • Plasmids can transfer genes and act as carriers for introducing DNA from other bacteria or from eukaryotic cells • E. coli cell membrane is weakened using ice cold CaCL2 • E. coli cells are then “heat shocked” to induce them to take up the plasmid • Sterile technique must be used • Transformation Lab – we will transform bacteria by introducing a plasmid that will convey resistance to the antibiotic, ampicillin • Ampicillin kills bacteria by interfering with their ability to make cell walls
