1. TECHNIQUES IN BIOTECHNOLOGY

5. BIOTECHNOLOGY - the intentional manipulation of genetic material of an organism

6. https://www. youtube. com/watch

7. Common uses of biotechnology:
Making "stuff”
proteins, enzymes, medication, etc. can be produced by engineered bacteria!
Food can be altered to have new traits
Cloning (therapeutic and reproductive)
Genetic screening
crime cases, relationship, genetic screening, etc.
3. Gene Therapy

8. 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

12. Genetically modified organisms (GMOs) are organisms whose genes have been directly manipulated by scientists, often by inserting or deleting one or more genes. Inserted genes are typically from another species.

13. The only difference between living organisms is the amount and order of the four nucleotide bases of DNA.
Code is just code.

14. It can be read.
It can be analyzed.
It can be changed.
It can be compared.
It can be ignored.
It can be inserted.
It can be copied.

16. Do you agree or disagree with these statements:
I have eaten food that contains genetically modified (GM) crops.
GM foods should be available, as long as they are tested before they are sold for human consumption.
The risks of GM foods outweigh the possible benefits.
GM foods will help provide a sustainable food supply.
I am concerned about eating GM foods.
Farmers should grow corn that is genetically modified to resist insects that damage cornfields.

17. Should Canada allow farmers to grow genetically modified food plants?
Bt corn
Terminator soya
SciShow:

18. Genome: the entire sequence of DNA
Gene: the part of the code that corresponds to a protein
*genes can be transferred from one organism to another*

19. In order to do these things, we need a way to make many copies of the genes we want

20. Tools of Biotechnology
DNA Extraction

21. a) Purpose: to purify a DNA sample b) Uses
genetic testing
crime scene investigation
gene studies

22. c) Steps:
collect cells
remove DNA from cell
purify it
use it!

23. 2. PCR a) purpose: to make many copies of a specific piece of DNA
b) steps:
denature DNA using temperature
add in the ingredients (ATGC, enzymes, primers) to copy it
repeat

24. 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

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

27. c) Uses of PCR
make the desired gene/piece of DNA to use it to make proteins, study it, etc.

28. 3. Bacterial recombinant plasmids

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

30. 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

32. 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 enzymes (endonucleases)

33. 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’

35. 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

36. a) 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.

37. 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?

38. So, let’s REVIEW…

39. 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!

41. b) 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

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

43. 4. DNA SEQUENCING
purpose: find out the order of DNA, the “fingerprint”
uses: forensics, genetic testing, studying mutations

48. Steps
like PCR, put your template (DNA you want to “read”) with the nucleotides, primers, enzymes, and use heat to make “more”
some of the nucleotides have fluorescent labels and stop the chain from being made

49. results in a soup of different-length fragments
separate them by size, and “read”

50. 5. Gel Electrophoresis
used to “see” the DNA, by separating fragments using a gel
used in sequencing and to determine the origin of DNA
ex. Tsunami baby

51. Sequencing

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

53. Steps

54. 6. CLONING

55. a) Therapeutic cloning
used to produce tissue that is identical to the donor, to prevent rejection

56. b) Reproductive Cloning
creates an organism with the same genetic material (DNA) as the original organism – an EXACT COPY of the donor

58. Dolly the Sheep
the first cloned sheep

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

60. 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

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

62. 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

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

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

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

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

67. 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

68. 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

69. 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’

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

72. 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")

73. 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.

74. 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?

75. So, let’s REVIEW…

76. 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

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

78. 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

79. 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!

81. 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

82. 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

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

85. 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

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

87. Agarose Electrophoresis of DNA
used to separate fragments of DNA
DNA is negatively charged, charge is proportional to size
agarose can be used as a molecular sieve to separate the pieces of DNA by size

88. Gel electrophoresis
A current is run through the buffer surrounding the gel and it pushes the DNA away from the negative anode, towards the positive cathode

90.  A brief review of last Friday…

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

92. 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