Lecture 8 Genetic Engineering

Medically important substances produced by genetic engineering Human Insulin- used to treat diabetes Past: extracted insulin from pancreas of cattle and pigs Today: Human insulin produced from bacteria

Medically important substances produced by genetic engineering Vaccines: genetically engineered microorganisms used to make vaccines Hepatitis B vaccine: gene coding for viral protein in Hepatitis B cloned into bacteria Bacteria make viral protein Protein used to make vaccine

Importance of Genetic Engineering Genetic traits transferred to plants Pest resistant plants: engineered to resist natural pests –Examples: corn, cotton, potatoes Bt-toxin: made in B. thuringiensis –Toxic to insects- dissolves their gut –Insert gene that makes Bt-toxin into plants’ –Plants now make Bt-toxin Transgenic: plants and animals into which new DNA has been introduced

Figure 9.5

Table 9.2

Genetic Engineering Transferring genes from one organism to another organism –Can be prokaryote to prokaryote –Prokaryote to eukaryote –Or eukaryote to prokaryote Requires several things: –DNA of interest (must be isolated) –Various enzymes (restriction enzymes) –Suitable vector –Method for introducing DNA into new host

Isolating DNA of interest 1.Cells are lysed (burst open) 2.As cells burst DNA is sheared into many pieces of varying lengths 3.Cut purified DNA into smaller fragments using restriction enzymes

Restriction Enzymes When DNA is cut with restriction enzymes the DNA pieces generated either have sticky (cohesive ends) or blunt ends The cohesive ends will adhere to any piece of DNA with complementary sequence

Table 9.3

Vectors After isolating DNA, need to insert into vector Vector- modified plasmid Contains: –Origin of replication –Selectable marker, example: antibiotic resistance –At least one restriction enzyme recognition site

Figure 9.15

Generation of Recombinant Vector Recombinant vector: vector containing the piece of DNA you isolated Put piece of DNA you isolated with sticky ends together with vector that is cut with same restriction enzyme Sticky ends of isolated DNA will join to sticky ends of vector Add ligase to glue isolated DNA to vector

Same restriction enzyme used to isolate DNA of interest

Introducing recombinant vector into new organism Three steps: 1.Select a suitable host 2.Insert DNA into cells 3.Select for transformants

Select a suitable host Usually use E.coli E.coli is easy to grow and much is known about it’s biochemistry and genetics Also have known phenotypes such as sensitivity to certain antibiotics

Inserting the recombinant vector into bacterial cells Two methods: 1.DNA-mediated transformation –In this process competent cells take up DNA from surrounding environment 2.Electroporation - Process involves treating cells with electric current that puts pores in the membrane so that plasmid can enter

Selecting for bacteria that contain vector First, grow bacteria on selective media- one that contains antibiotic Remember, vectors contain genes for certain antibiotic resistance

Introducing vectors into eukaryotic cells If introducing to plant cells, can use the T i plasmid –Advantage of this is that T-DNA and any genes you have inserted is naturally transferred from Agrobacterium to host cell Other methods: –Electroporation: same as with bacterial cells –Gene gun: device that uses a gas pulse or other mechanism to propel DNA-coated gold particles into cell