1. TOOLS OF BIOTECHNOLOGY
SBI4U0
Ms. Manning

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

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

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

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

6. 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

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

9. 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

10. 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

11. 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

12. See? Its not all chopped up!

13. 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

14. 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

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

17. 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

18. 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

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

20. 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

21. 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

23. 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

25. 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?