Bacterial Transformation Genetic Engineering – scientists put new genes into cells to develop organisms that are beneficial to people – uses include: 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 Bacteria that can produce hormones such as human growth hormone and insulin Making plants frost and pest resistant Making plants frost and pest resistant Developing plants that do not need fertilizer Developing plants that do not need fertilizer Bacteria that eat oil slicks Bacteria that eat oil slicks

Escherichia coli Often used for genetic engineering Often used for genetic engineering Common inhabitant of human colon – easy to get Common inhabitant of human colon – easy to get Can be easily grown in suspension culture in a nutrient such as Luria broth Can be easily grown in suspension culture in a nutrient such as Luria broth Has a simple circular chromosome with about 1/600 th the haploid amount of DNA in a human cell Has a simple circular chromosome with about 1/600 th 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 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 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 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 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: 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 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 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) 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!) 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) 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 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 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 CaCL 2 E. coli cell membrane is weakened using ice cold CaCL 2 E. coli cells are then “heat shocked” to induce them to take up the plasmid E. coli cells are then “heat shocked” to induce them to take up the plasmid Sterile technique must be used Sterile technique must be used Transformation Lab – we will transform bacteria by introducing a plasmid that will convey resistance to the antibiotic, ampicillin 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 Ampicillin kills bacteria by interfering with their ability to make cell walls