Biotechnology What does it mean? Tools and Technologies Selected Applications Biotechnology 1: any method based on knowledge of biological processes that achieves a practical purpose for human civilization; 2: applied biology.

Biotechnology Past and Present: Early Civilizations: –Domesticated plants and animals –Preservation of food beer, mead, wine fermented vegetables cheese, yogurt –Bread! Emergence of Modern Biotechnology: –Mendelian genetics and hybrid organisms (e.g., crop plant advances) –Microbial fermentation; acetone and glycerol for WWI ammunitions. –Fleming’s discovery of antibiotics saved lives in WWII. –Watson and Crick described DNA (won Noble prize) Modern Genetic Engineering: (ability to create recombinant DNA in lab) –produce medications (hormones; antibiotics) –make disease / drought resistant plants –identify genetic material to help solve crimes (forensics) –clean up of toxic spills (Exxon Valdez) –whole-organism cloning of livestock species

Scope of Modern Biotechnology (Genetic Engineering)

Be able to recognize and group the examples we discuss under each of these six categories; i.e. match discussed examples with categories.

Gene Cloning Overview: Artificial transformation is used to get plasmids back into a host bacterium. How will we select for only transformed bacteria with recombinant plasmids? How do we know which is to be cloned? 1) Clone all genome fragments and find what you want from this library afterwards. 2) Find the desired DNA fragment before cloning. How is DNA cut at known sites?

Restriction endonucleases are enzymes bacteria make to cut foreign DNA (like that from an infecting virus). Each species of bacteria has a “restriction enzyme” that cuts DNA at a unique “palondromic” sequence of 4 to 8 base pairs, called recognition sites. Cutting of each strand is often unevenly, which create overhanging ends of single strand DNA, called “sticky ends”

When cloning genes, we cut the DNA of the foreign donor and that of the plasmid vector using the same restriction enzyme. This creates the same sticky ends on both vector and fragment DNA. A ligation reaction refers to the mixing, annealing of sticky ends and ligase reaction to form recombinant plasmids. In ligation reactions, not all plasmid DNA will have foreign DNA fragments inserted. Nor will all bacteria receive a plasmid after transformation.

Plasmids Designed for Cloning Genes: A plasmid vector should be designed to have genes for easily recognized phenotypes that can be used to distinguish the three different bacterial populations that result after artificially transforming a host bacterial culture. Non-transformants = host bacteria that did not receive any plasmid. Non-recombinant transformants = host bacteria with a normal plasmid vector (no inserted foreign DNA). Recombinant Transformants = host bacteria with recombinant plansmid (has inserted foreign DNA). Antibiotics, like ampicillin, in the agar media post-transformation will prevent growth of non-transformants. Genes on plasmids that can obviously change colony appearance (e.g. lacZ) are good sites for inserting foreign DNA into plasmid vector DNA. “Cloning site” Cut and insert here

Blue-White Colony Selection: 1) Cut inside lacZ with same restriction enzyme used to cut foreign donor DNA. 2) Ligation reaction. 3) Artificial transformation of host bacterium (Amp sensitive). 4) Plate to Amp+ media with the β-galactosidase substrate analog. Non-recombinants have functional enzyme; their colonies turn blue. Recombinant colonies are white.

How to clone the gene you want? Create a “Library”: Clone enough fragments of foreign donor DNA to represent the entire genome of the organism of interest. Each clone will represent a portion of the genome. Libraries may use plasmid vectors and host bacteria, or they may use a bacteriophage vector. The library can then be screened for any gene of interest, and used over and over again.

Screening a Library by Colony Hybridization Gene probes: Small DNA sequences (oligonucleotide) complementary to only the gene of interest that is “labeled” for detection, using radioactivity in this case.

Agarose Gel Electrophoresis: Separates DNA by size; many applications, including “fingerprinting” and screening fragments of foreign donor DNA with a particular gene for cloning. A fluorescent DNA stain is used to see bands under UV light.

Southern Blot Water and some DNA is blotted (or wicked) out of the gel; DNA gets trapped and binds to nitrocellulose paper. Now we can “probe”. Can’t hybridize DNA probe to sample DNA in a gel. DNA fragment detected by probe can be cut from gel and then cloned. The only thing you can see!

Polymerase Chain Reaction (PCR): Specific target sequences of DNA (e.g., gene for cloning) found in very low levels in a sample can be amplified to billions of copies to use in further manipulation (e.g., gene cloning, DNA fingerprinting, genetic screening). Repeated cycles of replication for a specific DNA sequence will exponentially increase copies on only that DNA sequence. N=2 n Each cycle has 3 major steps: 1)Denaturing DNA from helix to single strands. 2)Annealing primers; one specific to each end of the target DNA sequence. 3)Extension of new DNA strand by a heat tolerant DNA Polymerase (from a thermophilic bacterium)

Recombinant Protein Products

Transgenic Plants