Genetic Engineering
Biotechnology
What is Genetic Engineering?
The manipulation of a trait in an
organism to create a desired
change
We have been manipulating DNA
for generations!
 Artificial breeding

creating new breeds of animals & new
crop plants to improve our food
Animal breeding
Breeding food plants
 “Descendants” of the wild mustard

the “Cabbage family”
Breeding food plants
Evolution of modern corn (right) from
ancestral teosinte (left).
A Brave New World
The code is universal
 Since all living
organisms…



use the same DNA
use the same code
book
read their genes
the same way
TACGCACATTTACGTACGCGGATGCCGCGACTATGATC
ACATAGACATGCTGTCAGCTCTAGTAGACTAGCTGACT
human genome
CGACTAGCATGATCGATCAGCTACATGCTAGCACACYC
GTACATCGATCCTGACATCGACCTGCTCGTACATGCTA
3.2
billion
bases
CTAGCTACTGACTCATGATCCAGATCACTGAAACCCTA
GATCGGGTACCTATTACAGTACGATCATCCGATCAGAT
CATGCTAGTACATCGATCGATACTGCTACTGATCTAGC
TCAATCAAACTCTTTTTGCATCATGATACTAGACTAGC
TGACTGATCATGACTCTGATCCCGTAGATCGGGTACCT
ATTACAGTACGATCATCCGATCAGATCATGCTAGTACA
TCGATCGATACTGCTACTGATCTAGCTCAATCAAACTC
TTTTTGCATCATGATACTAGACTAGCTGACTGATCATG
ACTCTGATCCCGTAGATCGGGTACCTATTACAGTACGA
TCATCCGATCAGATCATGCTAGTACATCGATCGATACT
Can we mix genes from one creature
to another?
YES!
Green Fluorosceint Protein (GFP)
How do we do mix genes?
 Genetic engineering
find gene
 _______ DNA in both organisms
 _______ gene from one creature into other
creature’s DNA
 _______ new chromosome into organism
 organism _______ new gene as if it were its
own
 organism _______ gene as if it were its own
 _____________________________________:
Remember: we all use the same genetic code!

Uses of genetic engineering
 Genetically modified organisms (GMO)

enabling plants to produce new proteins
 ___________________________: BT corn
 corn produces a bacterial toxin that kills corn
borer (caterpillar pest of corn)
 ___________________________: fishberries
 strawberries with an anti-freezing gene from
flounder
 ___________________________: golden rice
 rice producing vitamin A
improves nutritional value
Basic steps in genetic engineering
1. Isolate the gene
2. Insert it in a host using a vector
3. Produce as many copies of the host as
possible
4. Separate and purify the product of
the gene
Gene Cloning Techniques
2.Extract/isolate
DNA
1- Grow the target
microorganism
4- Insert DNA
fragments in a
plasmid cloning
vector
Recombinant
DNA target
3- Digest
fragment DNA
with
restriction
enzymes
Continued
5Transform
E. coli with
library
“Vibrio DNA
library”
Each bacteria will receive
a single plasmid from the
library
Each bacteria will grow to
form an individual colony
Tools
1.
2.
3.
4.
DNA you want to clone
Restriction endonucleases (molecular scissors)
Cloning vector (e.g. pGEM, pBR322…)
Ligase enzyme (molecular glue)
Step 1: Isolating the gene
Step 1: Isolating the gene
Cutting DNA
 DNA “scissors”

____________________________

____________________________
 used by bacteria to cut up DNA of
attacking viruses
 EcoRI, HindIII, BamHI

cut DNA at specific sites
 enzymes look for specific base sequences
GTAACG|AATTCACGCTT
GTAACGAATTCACGCTT
CATTGCTTAA|GTGCGAA
CATTGCTTAAGTGCGAA
Restriction enzymes
 Cut DNA at specific sites

____________________________
restriction enzyme cut site
GTAACGAATTCACGCTT
CATTGCTTAAGTGCGAA
restriction enzyme cut site
GTAACG AATTCACGCTT
CATTGCTTAA GTGCGAA
Sticky ends
 Cut other DNA with same enzymes


leave “sticky ends” on both
can glue DNA together at “sticky ends”
GTAACG AATTCACGCTT
CATTGCTTAA GTGCGAA
gene
you want
GGACCTG AATTCCGGATA
CCTGGACTTAA GGCCTAT
chromosome
want to add
gene to
GGACCTG AATTCACGCTT
CCTGGACTTAA GTGCGAA
combined
DNA
Restriction Endonucleases
• Restriction endonucleases, a.k.a.
“restriction enzymes” or “enzymes” by
molecular biologists.
• Type II restriction enzymes recognize and
cut specific DNA sequences
5’-NNNAAGCTTNNN-3’
3’-NNNTTCGAANNN-5’
Example
• Hind III (Haemophilus influenza Rd)
– Recognizes: AAGCTT
– Cuts in between the two A’s
AAGCTT
TTCGAA
A
TTCGA
AGCTT
A
Types of Sticky Ends
5’ overhangs (HindIII)
5’AAGCTT 3’
5’A
3’TTCGAA 5’
3’TTCGA 5’
5’
AGCTT3’
A 5’
3’ overhangs (KpnI)
5’ GGTACC 3’
5’ GGTAC 3’
3’ CCATGG 5’
3’ C
C 3’
3’ CATGG 5’
Types of Overhangs
 Sticky ends

Examples include HindIII & KpnI
 Blunt Ends
Example SmaI
 Recognize CCCGGG
 Cut between C and G

CCCGGG
GGGCCC
CCC
GGG
GGG
CCC
Sticky ends help glue genes together
cut sites
gene you want
cut sites
TTGTAACGAATTCTACGAATGGTTACATCGCCGAATTCACGCTT
AACATTGCTTAAGATGCTTACCAATGTAGCGGCTTAAGTGCGAA
AATTCTACGAATGGTTACATCGCCG
GATGCTTACCAATGTAGCGGCTTAA
sticky ends
cut sites
isolated gene
chromosome want to add gene to
AATGGTTACTTGTAACG AATTCTACGATCGCCGATTCAACGCTT
TTACCAATGAACATTGCTTAA GATGCTAGCGGCTAAGTTGCGAA
DNA ligase joins the strands
sticky ends stick together
________________ DNA molecule
chromosome with new gene added
TAACGAATTCTACGAATGGTTACATCGCCGAATTCTACGATC
CATTGCTTAAGATGCTTACCAATGTAGCGGCTTAAGATGCTAGC
How can
bacteria read
human DNA?
Why mix genes together?
 Gene produces protein in different
organism or different individual
human insulin gene in bacteria
TAACGAATTCTACGAATGGTTACATCGCCGAATTCTACGATC
CATTGCTTAAGATGCTTACCAATGTAGCGGCTTAAGATGCTAGC
“new” protein from organism
ex: human insulin from bacteria
aa aa aa aa aa aa aa aa aa aa
bacteria
human insulin
Step 2: Inserting gene into vector
 Vector – molecule of
DNA which is used to
carry a foreign gene
into a host cell
Plasmid Vector: pBR322
 First modern cloning vector (1976)
pBR322
• Contains:
1.
colE1 origin of replication (ORI)
pBR322
• Contains:
Bacteria plus
plasmid
Non-transformed
bacteria
2. Selectable Markers:
• Ampicillin Resistance
(β-lactamase gene)
• and Tetracycline
Resistance (tet gene)
Nutrient media
plus antibiotic
Overnight
growth
Only colonies
from bacteria that
have plasmid
pBR322
• Contains:
3. A few good restriction sites for inserting
foreign DNA
BamH1
BamH1
Your favorite
DNA
PstI
Eco
RI
Bam
HI
Digest
with
BamH
1
and
ligate
PstI
Eco
RI
Your
favorite
DNA
Bam
HI Bam
HI
pBR322
• Nice Features:
√
√
√
200 copies per E. coli cell
Makes double stranded DNA
All modern cloning vectors are based on
pBR322
Next Generation: pUC Plasmids
•
Advantages over pBR322
1.
2.
Makes 1000’s of copies/cell
Small size at 2.7 kilobase pairs (kb) = easier
uptake by E. coli
Step 3: inserting vector into host
Bacteria
 Bacteria are great!
one-celled organisms
 reproduce by mitosis

 easy to grow, fast to grow
 generation every ~20 minutes
 A way to get genes into bacteria easily
insert new gene into plasmid
 insert plasmid into bacteria
 bacteria now expresses new gene

 bacteria make new protein
gene from
other organism
cut DNA
plasmid
recombinant
plasmid
+
vector
glue DNA
transformed
bacteria
Blue/White Selection
Bacteria plus empty
plasmid
Bacteria with
plasmid plus insert
Non-transformed bacteria
Nutrient media plus
antibiotic plus X-Gal
Overnight
Colonies with insert - white
Colonies w/o insert - blue
growth
Only colonies
from bacteria that
have plasmid
Grow bacteria…make more
gene from
other organism
recombinant
plasmid
+
vector
plasmid
grow
bacteria
harvest (purify)
protein
transformed
bacteria
Applications of biotechnology
any Questions?