TOOLS OF BIOTECHNOLOGY SBI4U0 Ms. Manning

Introduction Biotechnology has been used for centuries in various forms i.e. Fermentation of alcohol, selective breeding of crops

More recently, major area of research has been bioremediation The use of micro-organisms to clean-up toxic chemicals in the environment

So...what is biotechnology? Biotechnology is the use of biological tools to investigate genetic information and processes

What tools are used? Restriction Enzymes Methylases DNA ligase Gel Electrophoresis Plasmids

1. Restriction Endonucleases (Enzymes) Molecular scissors that cut at a specific point (recognition site) Recognition site is usually 4-8 bp long and palindromic (same in both directions) Binds to recognition site and disrupts the phosphodiester bonds via hydrolysis

Can be used to create “sticky ends” and “blunt ends” BamH1?? Wha?? B = genus (Bacillus) am = species (amyloliquefaciens) H = strain I = order of identification

Where do restriction enzymes come from? Obtained from bacteria Bacteria use them as an immune system The enzymes break down viral DNA that are trying to invade the bacteria

Who discovered them? Restriction enzymes were first discovered in 1970 by Hamilton Smith while he was investigating bacterial resistance to viral infection He won the Nobel Prize in 1978

2. Methylases Enzymes that protect an organism from its own restriction endonucleases Found in eukaryotes and prokaryotes Adds a methyl group to the restriction site to alter chemical composition and prevent recognition Methyl group CH3 GAATTC CTTAAG

See? Its not all chopped up!

3. DNA Ligase Used to join cut strands of DNA Condensation reactions form phosphodiester bonds H-bonds will naturally form between “sticky ends”, but DNA ligase is needed to form the bonds in the backbone

4. Gel Electrophoresis Used to separate DNA fragments Like a molecular sieve, DNA fragments of different length are sorted by size A current passes through the gel and shorter DNA fragments travel faster A chemical is added that fluoresces under UV light

5. Plasmids Sequences of DNA that are small, circular, and double stranded Bacteria have 1 chromosome and 1+ plasmids Usually 1000 – 200 000 bp long

Plasmids are beneficial to bacteria! Plasmids carry genes that result in proteins for: antibiotic resistance resistance to toxic metals Breaking down herbicides Breaking down industrial chemicals

Plasmid Copy Number The number of copies of a particular plasmid found in the bacteria cell i.e. The more copies of an antibiotic-resistance gene there are, the higher the resistance

Multiple-cloning site Region in the plasmid that has been engineered to contain recognition sites of a number of restriction enzymes

So...what do we use plasmids for? We use plasmids as vectors (or vehicles) to carry a desired gene into a host cell Restriction enzymes are used to splice the DNA in specific sites that match the gene we want to insert

Match? What do you mean match? We would use the same restriction enzyme on the plasmid as we did to splice the desired gene This way they will both have sticky ends that will match up and attach The foreign gene will permanently become part of the plasmid

Recombinant DNA A plasmid carrying foreign DNA is called recombinant DNA The gene in the plasmid with be translated into protein by the bacteria i.e. The human insulin gene is inserted into a bacterial plasmid  the bacteria will produce human insulin protein

Transformation Introduction of foreign DNA, usually a plasmid or virus, into bacterial cells The bacterial cells are then allowed to reproduce and transcribe/translate multiple copies of the desired gene **The bacterial plasmid usually contains a gene for antibiotic resistance...why do you think this is?