Post on 11-Jan-2016
Genetic EngineeringBiotechnology
The manipulation of a trait in an organism to create a
desired change
What is Genetic Engineering?
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
TACGCACATTTACGTACGCGGATGCCGCGACTATGATCACATAGACATGCTGTCAGCTCTAGTAGACTAGCTGACTCGACTAGCATGATCGATCAGCTACATGCTAGCACACYCGTACATCGATCCTGACATCGACCTGCTCGTACATGCTACTAGCTACTGACTCATGATCCAGATCACTGAAACCCTAGATCGGGTACCTATTACAGTACGATCATCCGATCAGATCATGCTAGTACATCGATCGATACTGCTACTGATCTAGCTCAATCAAACTCTTTTTGCATCATGATACTAGACTAGCTGACTGATCATGACTCTGATCCCGTAGATCGGGTACCTATTACAGTACGATCATCCGATCAGATCATGCTAGTACATCGATCGATACTGCTACTGATCTAGCTCAATCAAACTCTTTTTGCATCATGATACTAGACTAGCTGACTGATCATGACTCTGATCCCGTAGATCGGGTACCTATTACAGTACGATCATCCGATCAGATCATGCTAGTACATCGATCGATACT
human genome3.2 billion bases
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 gene2. Insert it in a host using a vector3. Produce as many copies of the
host as possible4. Separate and purify the product
of the gene
Gene Cloning Techniques
1- Grow the target microorganism
2.Extract/isolate DNA
DNA target
3- Digest fragment DNA
with restriction enzymes
4- Insert DNA fragments in a
plasmid cloning vector
Recombinant
Each bacteria will grow to form an individual colony
Continued
“Vibrio DNA library”
5- Transform E. coli with
library
Each bacteria will receive a single plasmid from the
library
Tools
1. DNA you want to clone2. Restriction endonucleases (molecular
scissors)
3. Cloning vector (e.g. pGEM, pBR322…)
4. 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
sequencesGTAACGAATTCACGCTTCATTGCTTAAGTGCGAAGTAACG|AATTCACGCTTCATTGCTTAA|GTGCGAA
Restriction enzymes Cut DNA at specific sites
____________________________
GTAACG AATTCACGCTTCATTGCTTAA GTGCGAA
GTAACGAATTCACGCTTCATTGCTTAAGTGCGAA
restriction enzyme cut site
restriction enzyme cut site
Sticky ends Cut other DNA with same enzymes
leave “sticky ends” on both can glue DNA together at “sticky ends”
GTAACG AATTCACGCTTCATTGCTTAA GTGCGAA
gene you want
GGACCTG AATTCCGGATACCTGGACTTAA GGCCTAT
chromosome want to add
gene to
GGACCTG AATTCACGCTTCCTGGACTTAA GTGCGAA
combinedDNA
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 A AGCTTTTCGAA TTCGA A
Types of Sticky Ends
5’ overhangs (HindIII)
5’AAGCTT 3’ 5’A 5’ AGCTT3’
3’TTCGAA 5’ 3’TTCGA 5’ A 5’
3’ overhangs (KpnI)
5’ GGTACC 3’ 5’ GGTAC 3’ C 3’
3’ CCATGG 5’ 3’ C 3’ CATGG 5’
Types of Overhangs
Sticky ends Examples include HindIII & KpnI
Blunt Ends Example SmaI Recognize CCCGGG Cut between C and G
CCCGGG CCC GGGGGGCCC GGG CCC
Sticky ends help glue genes togetherTTGTAACGAATTCTACGAATGGTTACATCGCCGAATTCACGCTTAACATTGCTTAAGATGCTTACCAATGTAGCGGCTTAAGTGCGAA
gene you want cut sitescut sites
AATGGTTACTTGTAACG AATTCTACGATCGCCGATTCAACGCTTTTACCAATGAACATTGCTTAA GATGCTAGCGGCTAAGTTGCGAA
chromosome want to add gene tocut sites
AATTCTACGAATGGTTACATCGCCG GATGCTTACCAATGTAGCGGCTTAA isolated gene
sticky ends
chromosome with new gene addedTAACGAATTCTACGAATGGTTACATCGCCGAATTCTACGATC
CATTGCTTAAGATGCTTACCAATGTAGCGGCTTAAGATGCTAGC
sticky ends stick together
DNA ligase joins the strands ________________ DNA molecule
Why mix genes together?
TAACGAATTCTACGAATGGTTACATCGCCGAATTCTACGATC
CATTGCTTAAGATGCTTACCAATGTAGCGGCTTAAGATGCTAGC
Gene produces protein in different organism or different individual
aa aaaa aa aa aa aa aa aa aa
“new” protein from organism ex: human insulin from bacteria
human insulin gene in bacteria
bacteria human insulin
How can bacteria read human DNA?
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)
Bacteria plus plasmid
Non-transformed bacteria
Nutrient media plus antibiotic
Overnight growth
Only coloniesfrom bacteria that
have plasmid
pBR322
• Contains: 2. Selectable Markers:
• Ampicillin Resistance (β-lactamase gene)• and Tetracycline
Resistance (tet gene)
pBR322
• Contains: 3. A few good restriction sites for
inserting foreign DNA
PstI
EcoRI Bam
HI
BamH1
BamH1
Your favorite DNA
Digest
with BamH
1
and ligate
PstI
EcoRI Bam
HI BamHI
Your favorite
DNA
pBR322
• Nice Features: √ 200 copies per E. coli cell√ Makes double stranded DNA√ All modern cloning vectors are
based on pBR322
• Advantages over pBR3221. Makes 1000’s of copies/cell
2. Small size at 2.7 kilobase pairs (kb) = easier uptake by E. coli
Next Generation: pUC Plasmids
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
+
transformedbacteriagene from
other organism
plasmid
cut DNA
recombinantplasmid
vector
glue DNA
Blue/White SelectionBacteria plus empty
plasmid Non-transformed bacteria
Only coloniesfrom bacteria that
have plasmid
Nutrient media plus antibiotic plus X-Gal
Overnight growth
Bacteria with plasmid plus insert
Colonies with insert - whiteColonies w/o insert - blue
Grow bacteria…make more
growbacteria
harvest (purify)protein
transformedbacteria
plasmid
gene fromother organism
+
recombinantplasmid
vector
Applications of biotechnology
any Questions?