The Wild World of The Wild World of Biotechnology!!Biotechnology!!

ApplicationsApplications

Genetic Transformation

Cloning - Genes and entire organisms

Gene Therapy

Environmental Clean-Up

How do we genetically How do we genetically engineer or modify an engineer or modify an

organism?organism?

Selective breeding

Genetic transformation

Genetic Genetic Transformation 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 DNARecombinant 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 Making Recombinant DNADNA

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 Making Recombinant DNA continued...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 Polymerase Chain Reaction (PCR)Reaction (PCR)

Another DNA cloning technique

Using plasmid vectorsUsing plasmid vectorsThese 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!!!

pGLOpGLO

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

Getting bacteria to Getting bacteria to express pGLO genesexpress 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 cloningGene 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 DNAStudying DNAGel electrophoresis

A method for separating nucleic acids based on size and electrical charge

Gel ElectrophoresisGel ElectrophoresisA 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

RFLPsRFLPs

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 Draw a restriction map of the plasmid...of the plasmid...