1. Ch. 13.1: BIOTECHNOLOGY Objectives:
Explain how the use of bacteria has contributed to the development of DNA technology.
List some recent research trends in recombinant DNA technology.
Vocab:
Biotechnology
Recombinant DNA technology

2. Biotechnology = Use of organisms to perform practical tasks. Examples:
Use bacteria to make proteins/drug therapies
Engineer plants with natural resistance to insects, drought, fungi, etc.
Develop vaccines.

3. Recombinant DNA
= DNA from 2 different sources combined.

4. 13.2: Bioengineering Objectives: Vocab: Plasmid Restriction enzyme
1. Explain the role of plasmids in engineering bacteria.
Explain how biologist “cut and paste” DNA.
Describe the procedure used in cloning a specific gene.
Identify the usefulness of recombinant microorganisms.
Vocab:
Plasmid Restriction enzyme
Genomic library Nucleic acid probe

5. Bacteria: Work horses of Biotech.
Used to mass produce useful genes + proteins.
Simple organisms
1 chromosome.
Plasmids
Reproduce rapidly
Easy to manage in a lab.

6. Bacteria Plasmids Small, circular DNA Separate from chromosome.
Contains a few genes.
Make copies of itself
Can be shared/ transferred b/w bacteria.
Exchange of plasmids is how bacteria build genetic variation.
Genes for antibiotic resistance are shared this way :(

8. Making Recombinant DNA
Remove desired gene from donor cell.
Remove bacteria plasmid.
Insert gene into plasmid.
Return plasmid to bacteria.
Gene is transcribed and translated into protein product.
Recombinant DNA

9. Restriction Enzymes = Enzymes that CUT foreign DNA sequences.
Each enzyme is cuts a specific sequence (CCCGGG or GAGCT)
Cuts sugar-phosphate backbone of DNA
Make staggered cuts.
Leave “sticky ends” on cuts.
Evolved in bacteria to protect bacteria fr. invading viruses.

10. Restriction Enzymes: Cut DNA into fragments
BLUNT ends =no staggered cuts; not as useful
Sticky ends = made by staggered cuts; unpaired bases; useful b/c they h-bond w/ complimentary bases in other fragments. Helps to “sew” fragments together & make recomb. DNA.

11. Ligase: Glues DNA fragments together

12. Genetic Cloning = Copies of recomb
Genetic Cloning = Copies of recomb. DNA (and resulting proteins) are made by reproducing organisms

13. Genomic Library
= Complete collection of cloned DNA fragments from an organisms.
When you use restriction enzymes they cut up the donor DNA into MANY fragments.
Each fragment is incorporataed into a plasmid.
You need to figure out which bacteria has desired recomb. Plasmid!

14. Identifying Desired Recomb. DNA
Use nucleic acid probe
Radioactively labeled complimentary sequence
(TAGGCT will find and bind to ATCCGA when strands are separated).
2. Insert desired DNA into plasmid sequence for antibiotic resistance.
Recomb. plasmids will lose resistance to antibiotics and will NOT survive when exposed to antibiotic.

16. Task: Diagram Steps in Creating recombinant DNA clones
Use and illustrate the following key terms…
Host DNA
Bacteria Plasmid
Vector
Restriction Enzyme
Sticky Ends
Ligase
Bacteria reproduces/plasmid replication
Recombinant DNA
Clones

17. Lab: Recombinant Paper Plasmids
Goal: Insert human gene for insulin production into bacteria plasmid.
Construct bacteria plasmid. Color code sequences for antibiotic resistance.
Label 3’ and 5’ ends of restriction enzymes and plasmid DNA.
Identify restriction enzymes that will cut plasmid in sequence for antibiotic resistance. Mark these locations and label the enzyme used.

18. Lab: Recombinant Paper Plasmids
Goal: Insert human gene for insulin production into bacteria plasmid.
Label 3’ and 5’ of human DNA
Determine the enzyme that will allow for removal of human insulin gene AND match up with the sticky ends on the plasmid.
Create the recombinant plasmid.