Download Recombinant DNA

Recombinant DNA
Genetics
Bio 36404
Recombinant DNA
• aka Genetic Engineering
• Combining DNA from 2 sources
• e.g. putting gene into bacteria to produce
product
• e.g. putting genes into plants to improve
crops
Bacteria
main
DNA
ring
Plasmid
Plasmid Map
replication
origin
amphicillin
resistance
gene
Antibiotic
• Drug that kills bacteria
• Examples:
– Penicillin
– Amphicillin
– Tetracyclin
Antibiotic Resistance
• Ability of bacteria to withstand antibiotic
• Have gene to make enzyme to inactivate
antibiotic
Plasmid Map
replication
origin
amphicillin
resistance
gene
Restriction Enzymes
• Enzymes that cut DNA in specific region
• Derived from bacteria
• Named after bacteria of origin
EcoRI
CG
TA
TA
AT
AT
GC
Restriction Enzymes
EcoRI
Escherichia coli R
CG
TA
TA
AT
AT
GC
T
T
A
A
GC
CG
A
A
T
T
C
HindIII
Haemophilus
influenzae Rd
AT
AT
GC
CG
TA
TA
sticky ends
BamHI
Bacillus
amyloliquefaciens H
GC
GC
AT
TA
CG
CG
Plasmid Map
Eco RI
HinDIII
replication
origin
HinDIII
amphicillin
resistance
gene
Step 1 - Get and Cut Plasmid
Remove plasmid
from bacteria
cut with
restriction
enzyme
Step 2 - Get and Cut Other DNA
Remove DNA
from other
cut with same
restriction
enzyme
Step 3 - Combine DNA
DNA Ligase enzyme that joins
DNA fragments
Step 4 - Insert into Live Bacteria
Disrupt Membrane
•heat shock
•CaCl2
Plate with Antibiotic
e.g. Amphicillin
Transformation
take up plasmid
untransformed E. Coli
not resistant to amphicillin
+
transformed E. Coli
Fluorescent Green
Amphicillin Resistant
pFG plasmid
(has genes for amp resistance and green
fluorescent protein)
Products from Recombinant Bacteria
Insulin
Diabetes
Human Growth Hormone Dwarfism
Interleukin 2
Cancer
Erythropoietin
Hemophilia
Hepatitis B Vaccine
Hepatitis
GM Crops
Bt Corn (+ others)
Pest Resistant
Round-up Ready
Soybeans
Resistant to herbicides
(weed killers)
Golden Rice
Carotene Gene
Drought Resistance
Toxin Removal