recombinant dna. restriction enzymes enzymes that break down dna by cutting it at a specific site...
TRANSCRIPT
![Page 1: Recombinant DNA. Restriction Enzymes Enzymes that break down DNA by cutting it at a specific site (restriction site) Originally used as cellular defense](https://reader031.vdocuments.net/reader031/viewer/2022032006/56649e365503460f94b2512b/html5/thumbnails/1.jpg)
Recombinant DNA
![Page 2: Recombinant DNA. Restriction Enzymes Enzymes that break down DNA by cutting it at a specific site (restriction site) Originally used as cellular defense](https://reader031.vdocuments.net/reader031/viewer/2022032006/56649e365503460f94b2512b/html5/thumbnails/2.jpg)
Restriction Enzymes• Enzymes that break down DNA by cutting it at
a specific site (restriction site)• Originally used as cellular defense against
invaders• Used in Biotech to create recombinant DNA or
for DNA fingerprinting• Each enzyme has a unique restriction site that
it recognizes
![Page 3: Recombinant DNA. Restriction Enzymes Enzymes that break down DNA by cutting it at a specific site (restriction site) Originally used as cellular defense](https://reader031.vdocuments.net/reader031/viewer/2022032006/56649e365503460f94b2512b/html5/thumbnails/3.jpg)
Example of Restriction Sites
• EcoRI5’ GAATTC 3’3’ CTTAAG 5’
5’ G AATC 3’3’ CTTA G 5’
“Sticky ends” (sections of single stranded DNA) are created.
![Page 4: Recombinant DNA. Restriction Enzymes Enzymes that break down DNA by cutting it at a specific site (restriction site) Originally used as cellular defense](https://reader031.vdocuments.net/reader031/viewer/2022032006/56649e365503460f94b2512b/html5/thumbnails/4.jpg)
Time to Practice
• DNA Scissors Activity will enable you to visualize how restriction enzymes work!
![Page 5: Recombinant DNA. Restriction Enzymes Enzymes that break down DNA by cutting it at a specific site (restriction site) Originally used as cellular defense](https://reader031.vdocuments.net/reader031/viewer/2022032006/56649e365503460f94b2512b/html5/thumbnails/5.jpg)
What is Recombinant DNA?
• DNA that contains a gene or genes from more than one organism
![Page 6: Recombinant DNA. Restriction Enzymes Enzymes that break down DNA by cutting it at a specific site (restriction site) Originally used as cellular defense](https://reader031.vdocuments.net/reader031/viewer/2022032006/56649e365503460f94b2512b/html5/thumbnails/6.jpg)
What are some uses for recombinant DNA?
• Create GMO’s– Protein production– Organisms with specific traits
![Page 7: Recombinant DNA. Restriction Enzymes Enzymes that break down DNA by cutting it at a specific site (restriction site) Originally used as cellular defense](https://reader031.vdocuments.net/reader031/viewer/2022032006/56649e365503460f94b2512b/html5/thumbnails/7.jpg)
How do you create recombinant DNA?
• Identify desired gene and cut it out with restriction enzymes. (sticky ends)
• Cut a plasmid with same restriction enzyme.
![Page 8: Recombinant DNA. Restriction Enzymes Enzymes that break down DNA by cutting it at a specific site (restriction site) Originally used as cellular defense](https://reader031.vdocuments.net/reader031/viewer/2022032006/56649e365503460f94b2512b/html5/thumbnails/8.jpg)
• Join your gene to plasmid and seal with ligase to form a recombinant plasmid
![Page 9: Recombinant DNA. Restriction Enzymes Enzymes that break down DNA by cutting it at a specific site (restriction site) Originally used as cellular defense](https://reader031.vdocuments.net/reader031/viewer/2022032006/56649e365503460f94b2512b/html5/thumbnails/9.jpg)
• Put recombinant DNA into a host cell to replicate.
• Detect cells that have recombinant DNA and can make the desired product.– Example – antibiotic resistance or ability to glow
![Page 10: Recombinant DNA. Restriction Enzymes Enzymes that break down DNA by cutting it at a specific site (restriction site) Originally used as cellular defense](https://reader031.vdocuments.net/reader031/viewer/2022032006/56649e365503460f94b2512b/html5/thumbnails/10.jpg)
How does this relate to class?• Using recombinant plasmid pGLO in transformation
lab• Contains Green Fluorescent Protein gene (GFP)
that glows green in UV light and is ampicillin resistant
![Page 11: Recombinant DNA. Restriction Enzymes Enzymes that break down DNA by cutting it at a specific site (restriction site) Originally used as cellular defense](https://reader031.vdocuments.net/reader031/viewer/2022032006/56649e365503460f94b2512b/html5/thumbnails/11.jpg)
Why is this important in the real world? (not just in science class)
• Real uses of recombinant DNA?• Ethical issues?
![Page 12: Recombinant DNA. Restriction Enzymes Enzymes that break down DNA by cutting it at a specific site (restriction site) Originally used as cellular defense](https://reader031.vdocuments.net/reader031/viewer/2022032006/56649e365503460f94b2512b/html5/thumbnails/12.jpg)
Time to Practice!
• We are going to practice creating recombinant DNA using paper plasmids. If you master this, you will get to make real recombinant plasmids during our transformation lab.
![Page 13: Recombinant DNA. Restriction Enzymes Enzymes that break down DNA by cutting it at a specific site (restriction site) Originally used as cellular defense](https://reader031.vdocuments.net/reader031/viewer/2022032006/56649e365503460f94b2512b/html5/thumbnails/13.jpg)
Transformation Lab Vocab• Sterile Technique– Proper way of handling bacteria to avoid
contamination.
• Streaking– Process of spreading bacteria on an agar plate to
separate the cells
![Page 14: Recombinant DNA. Restriction Enzymes Enzymes that break down DNA by cutting it at a specific site (restriction site) Originally used as cellular defense](https://reader031.vdocuments.net/reader031/viewer/2022032006/56649e365503460f94b2512b/html5/thumbnails/14.jpg)
• Agar– Substance on plate that provides support and food
for bacterial growth
• Arabinose– Added to Agar plates, sugar that’s food for bacteria
• Ampicillin– Added to Agar plates, Antibiotic that kills bacteria
![Page 15: Recombinant DNA. Restriction Enzymes Enzymes that break down DNA by cutting it at a specific site (restriction site) Originally used as cellular defense](https://reader031.vdocuments.net/reader031/viewer/2022032006/56649e365503460f94b2512b/html5/thumbnails/15.jpg)
• Bacterial Library – A collection of bacterial cells that contain recombinant
plasmids with random DNA inserts from a single species
• Colony – A clump of genetically identical cells growing on an agar
plate (clones)
• Cloning – When a population of cells is prepared by growth from a
single cell (entire population is genetically identical)