The Wild World of Biotechnology!!

Applications Genetic Transformation Cloning - Genes and entire organisms Gene Therapy Environmental Clean-Up

How do we genetically engineer or modify an organism? Selective breeding Genetic transformation

Genetic Transformation This is the process by which we get an organism to express foreign DNA e.g. making a tomato synthesize antifreeze proteins that are commonly found in fish e.g. making a bacterial cell synthesize human insulin

Recombinant DNA Recombinant DNA = a molecule of DNA that contains DNA from two or more organisms Making recombinant DNA is the first step in producing genetically engineered (GE) or genetically modified (GM) organisms

Making Recombinant DNA Isolate the gene of interest (e.g. the gene for insulin) using restriction enzymes This is a hit or miss process that requires a great deal of luck to be successful Restriction enzymes generally cut at palindromic DNA sequences

Making Recombinant DNA continued... Some method is needed to get the gene of interest into the cells of the organism we wish to transform e.g. plasmid vectors and gold particles or electroportation

Polymerase Chain Reaction (PCR) Another DNA cloning technique

Using plasmid vectors These are small circular bits of DNA that are separate from the main prokaryote chromosome. They are easily transformed because of there chemistry Bacterial cells will absorb foreign plasmids and express their genes. You and I cannot do this!!!

pGLO pGLO are plasmids that carry the genes for fluorescent proteins from jellyfish

Getting bacteria to express pGLO genes The trick is getting the recombinant plasmid past the cell membrane and into the cell and then getting the cell to express the genes. We use chemicals (CaCl2) and heat shock to get recombinant plasmids into the cell. We include antibiotic resistance genes in the recombinant plasmid so that only the successfully transformed bacteria live. We make sure the gene of interest is near a known operon and we intentionally turn that operon on (e.g. arabinose, tryptophan, lactose)

Gene cloning Once the bacteria take up the recombinant plasmid they will go through DNA replication and, in effect, clone the gene of interest many times

Studying DNA Gel electrophoresis A method for separating nucleic acids based on size and electrical charge

Gel Electrophoresis A DNA sample is cut with restriction enzymes. The cut up DNA is placed in one end of a gel and electricity is passed through the gel Because DNA carries a negative charge the electric current is able to carry the DNA through the gel The smallest pieces of DNA move the furthest distance

RFLPs Restriction fragment length polymorphisms This term refers to variation in a gene sequence in a population RFLPs can be used to determine paternity, in forensics, to map genes, and to detect alleles

Draw a restriction map of the plasmid...