Molecular Biology 3: Transformation AP Biology Lab 6

What you need to know ology_place/labbench/lab6/intro.ht mlhttp:// ology_place/labbench/lab6/intro.ht ml

Transgenic Procedure Locate gene of interest (chromosomes, locus) Sequence the entire region Cut out with appropriate restriction enzyme that has upstream and downstream cleavage sites Select plasmid that has a cleavage site for the same restriction enzyme Cleave both with the selected RE Bring the fragments together and add ligase (enzyme that attaches sticky ends)

Outcome of Recombination 1.Plasmid reattaches to itself or 2.Random combinations of multiple DNA fragments together or 3.Recombinant Plasmid (plasmid with inserted gene)

Production Recombinant plasmid (vector) must be inserted into bacterium –Use heat shock to insert vector Reproduction will yield multiple transgenics Feed and grow bacteria Extract protein and purify for use

Problem/Solution Problem: Bacteria do not have spliceosomes to edit out introns Solution 1.Reverse Transcriptase (retrovirus) Use reverse transcriptase on post mRNA to make DNA w/o introns 2.Recombinant Eukaryotes Use hamster ovary cells or cancer cells that know how to process pre mRNA eukaryotic genes

Recombinant Examples Glowing Tobacco: tobacco plant w/ luciferase gene from firefly (what we use) Supermouse: mouse with human growth hormone gene BT Corn: corn with bacterial (Bacillus thuringiensis) herbicide gene to fight against corn-borer Epogene: Chinese hamster ovary cells that produce human hormone erythropoietin alpha (red blood cell production stimulating hormone) Humulin: E. coli producing human insulin L-Goats: goats that produce human lysozyme and express it in their milk to treat infant diarrhea