1. BIOTECHNOLOGY -intentional manipulation of genetic material of an organism

2. Deoxyribose Nucleic Acid (DNA)
determines the characteristics of all living organisms.
occurs in most cells of all organisms
composed of four different nucleotides in different combinations
each cell in the human body contains more than 3 BILLION letters

3. Four bases:
Adenine
Thymine
Guanine
Cytosine
2 bonds
3 bonds
Sugar and phosphate backbone
Double helix structure
(two spirals around each other)

4. The only difference between living organisms is the amount and order of the four nucleotide bases.

5. Why would we want to do this?
BIOTECHNOLOGY The intentional manipulation of genetic material of an organism
Why would we want to do this?
To study cellular processes of an organism
E.g. Glowing gene from jellyfish to tobacco plant
To give one organism the trait(s) of another
E.g. Anti-freeze from fish blood into strawberries to survive through early frosts

7. Part 1: Manipulating Bacteria:
The Making of a Plasmid

8. bacteria exchange these plasmids to share DNA
- a small circular piece of extra-chromosomal bacterial DNA, able to replicate
bacteria exchange these plasmids to share DNA
E.g. antibiotic resistance genes

9. Since plasmid is made of DNA it can code for genes, ex
Since plasmid is made of DNA it can code for genes, ex. antibiotic resistance, and can carry specific sequences of DNA
Specific DNA sequences can be recognized by enzymes called restriction endonucleases

10. Restriction Endonucleases/Restriction Enzymes
enzymes that are able to cut double-stranded DNA into fragments at specific recognition sites in DNA sequences
Ex. EcoRI:
5’-GAATTC-3’
3’-CTTAAG-5’

12. Restriction enzymes can create “sticky ends or “blunt ends”

13. fragment end of a DNA molecule with a short single-stranded overhang
Sticky Ends
fragment end of a DNA molecule with a short single-stranded overhang
Blunt Ends
fragment end of a DNA molecule with no overhang
Once made, the ends can be re-joined together by other enzymes ("enzyme glue")

14. To Make a Recombinant Plasmid:
Insert
Cut the plasmid and the insert with the same restriction endonuclease to make complementary sticky ends.
Combine the sticky ends using ligase.
ligase: enzyme used to join DNA together
So after this step we have inserted our new DNA into the plasmid. BUT HOW DO WE GET IT INTO THE BACTERIA?
3. Introduce the recombinant plasmid into bacteria.

15. Making a Recombinant Plasmid
So after this step we have inserted our new DNA into the plasmid. BUT HOW DO WE GET IT INTO THE BACTERIA?

16. So, let’s REVIEW…

17. Bacterial Transformation
introduction of foreign DNA into a bacterial cell
plasmid is used as a vector, a vehicle by which DNA can be introduced into host cell
- - -
+ - +
phospholipid bilayer
Ca2+ ions
plasmid

18. Following transformation bacteria are grown in medium with antibiotic…
Only the bacteria that have the plasmid (and therefore the antibiotic resistance) will survive.

19. Example plasmid:
Origin of Replication:
where the plasmid starts to duplicate itself
the specific sequence MUST NOT be cut by restriction endonucleases or it won’t be able to replicate

20. Part 2: Where do we get our insert sequence?
From someone else’s DNA
ex. fish gene in strawberries,
jellyfish gene in plants
Make it!
GFP, tobacco plant
SO WE CAN HARVEST OUR THE DESIRED GENE, BUT WE ARE PROBABLY GOING TO NEED WAY MORE OF IT THAN IS FEASIBLE TO COLLECT - SO WE AMPLIFY IT!

21. Use bacterial “factories”
In order to do these things, we need a way to make many copies of the genes we want
Use bacterial “factories”
easy to grow lots
no ethical issues
small genome is easy to manipulate

22. Using Bacteria as Production Factories
easy to grow
no ethical issues
small genome
easy to manipulate

23. Step 3: Taq (heat resistant) polymerase extends sequence
Step 1: DNA becomes single stranded due to heat
Step 2: Primers anneal (attach) to desired start sites through complementarity
Step 3: Taq (heat resistant) polymerase extends sequence

24. Polymerase Chain Reaction
Making an insert:
Polymerase Chain Reaction

26. Common uses of biotechnology:
Making "stuff”
proteins, enzymes, medication, etc. can be produced by engineered bacteria!
Genetic screening
crime cases, relationship, genetic screening, etc.
3. Gene Therapy

27. Ex. RFLP: Restriction Fragment Length Polymorphism
Comparison of different lengths of DNA fragments produced by restriction enzymes to determine genetic differences between individuals

28. Gene therapy
desired gene is inserted into cell's nucleus using a retrovirus as a carrier
defective gene replaced by functional gene

29. adenosine deaminase deficiency
Ex. ADA deficiency
adenosine deaminase deficiency
little immunity with low chances of recovery
- the T-cells of a four-year-old were removed, modified and re-inserted to fix her immune system