Download rDNA = recombinant DNA Figure 1. Humulin®

Figure 1. Humulin®
rDNA = recombinant DNA
Figure 2. DNA Double Helix
C
5 end
G
C
Hydrogen bond
G
C
G
C
G
3 end
A
T
3.4 nm
A
T
C
G
C
G
A
T
1 nm
C
A
G
C
G
A
G
A
T
3 end
T
A
T
G
C
T
C
C
G
T
A
(a) Key features of
DNA structure
0.34 nm
5 end
(b) Partial chemical structure
(c) Space-filling
model
Figure 3. Bacterial Immunity Through DNA Methylation
Foreign DNA sequence is not chemically
modified – restriction enzyme will cut it
Harvey Lodish, et al. Molecular Cell Biology 3e, Scientific American Books
Host cell DNA sequence is chemically
modified – restriction enzyme can’t cut it
Figure 20.3-3
Restriction site
Figure 4. Using a Restriction
5
Enzyme and DNA Ligase to
DNA
Make Recombinant
3
DNA
1 Restriction enzyme
5
cuts sugar-phosphate
backbones.
5
3
5
3
GAATTC
CTTAAG
3
5
3
Sticky
STICKY
ends
3
5
5
3
ENDS
2 DNA fragment added
3
5
from another molecule
cut by same enzyme.
Base pairing occurs.
5
3 5
G AATT C
C TTAA G
3
3 DNA ligase
5 3
3 5
G AATT C
C TTAA G
5 3
3
5
One possible combination
seals strands
5
3
3
Recombinant DNA
DNAMolecule
molecule
Recombinant
5
Figure 5. Molecular Structure of Human Insulin
B Chain
http://www.biotopics.co.uk/JmolApplet/insulinjdisplay.html
A Chain
Figure 6. Cellular Mechanism of
9 Insulin Production
Step 1
Preproinsulin is synthesized
as a random coil on
membrane-associated
ribosomes
Connecting
Sequence
Leader sequence aids in
transporting the polypeptide
through the membrane
Preproinsulin
Step 2
Leader sequence is cleaved
and the resulting proinsulin
folds into a stable
conformation
Step 3
Disulfide bonds form
Proinsulin
Insulin
Copyright Peason Higher Education
Step 4
The connecting sequence is
cleaved for form the mature
insulin molecule
Figure 7. Basic Laboratory Methods to Manufacture
Human Insulin
Method 1
1. Start with gene for
entire proinsulin
polypeptide
2. Inset gene into bacteria
3. Manipulate bacteria to
make proinsulin
polypeptide
4. Process polypeptide to
make functional insulin
protein
Method 2
1. Grow bacteria to make
the two insulin
polypeptide (A and B)
chains separately
2. Mix polypeptide chains
together
3. Join chains together by
making necessary
chemical bonds resulting
in functional insulin
protein
Figure 8. Synthesis of Genetically-Engineered Human Insulin
Step 1: Synthesize Proinsulin Gene
Human
cell
Acquire source DNA and
synthesize proinsulin gene
Add appropriate
“sticky ends”
AAT T C
Proinsulin DNA with “sticky ends”
Copyright © The McGraw-Hill Companies, Inc.
Figure 9. Synthesis of Genetically-Engineered Human Insulin
Step 2: Inset Gene into Plasmid
Synthesized proinsulin DNA
Antibiotic resistance gene
Plasmid (vector)
Create recombinant DNA
AAT T C
Proinsulin DNA with “sticky ends”
Mix proinsulin DNA with
plasmid DNA to create
recombinant plasmid
Copyright © The McGraw-Hill Companies, Inc.
Join DNA molecules
together with
DNA Ligase
Restriction enzymes cut
plasmid DNA at specific
sequence to produce
same sticky ends
attached to proinsulin DNA
Figure 10. Synthesis of Genetically-Engineered Human Insulin
Step 3: Manipulate E. coli to Take Up Plasmid DNA
Culture media with
antibiotic to which cells
with plasmid will be
resistant
Insert the recombinant DNA
into a recipient cell
Recombinant
plasmid
Copyright © The McGraw-Hill Companies, Inc.
Transgenic bacterium
containing human DNA
Figure 11. Synthesis of Genetically-Engineered Human Insulin
Step 4: Culture Engineered E. coli Cells
New Brunswick Scientific
75 to 3,000 liters
1.3 to 14 liters
Collect cells
Lyse cells
Collect cellular material
New Brunswick Scientific
Figure 12. Synthesis of Genetically-Engineered Human Insulin
Step 5: Produce and Purify Insulin
Collect proinsulin polypeptide
Remove connecting sequence
Join A and B chains together
Purify Humulin®
Figure 13. Some Uses of Genetically Engineered Cells
Protein expressed from
gene of interest
Gene of
interest
Protein harvested
Basic research
and various
applications
Gene for pest
Gene used to alter Protein dissolves
Human growth
resistance inserted bacteria for cleaning blood clots in heart hormone treats
into plants
up toxic waste
attack therapy
stunted growth
Figure 14. Creating a Genetically Engineered Plant
Recombinant plasmid
Agrobacterium
When the transgenic cell divides, each
daughter cell receives the herbicide
resistance gene. The resulting tobacco
plant is transgenic.
Herbicide
resistance gene
Herbicide resistance gene
Infection
Cell
division
Chromosome
Unaltered plant
cell
Transgenic plant
cell
Copyright © The McGraw-Hill Companies, Inc.
Cell
division
Figure 15. Representative Biotechnology Products
Treatment of Humans
For
Insulin
Diabetes
Growth Hormone
Pituitary dwarfism
Tissue Plasminogen Activator
Heart Attack
Erythropoietin
Anemia
Clotting Factor VIII
Hemophilia
Human Lung Surfactant
Respiratory distress in infants
Lactoferrin (lactotransferrin)
Controls level of iron in blood
Figure 16. Bioinformatics (genomics.energy.gov)
Cloned gene
Figure 17. Gene Therapy
1 Insert RNA version of normal allele
into retrovirus.
Viral RNA
Retrovirus
capsid
2 Let retrovirus infect bone marrow cells
that have been removed from the
patient and cultured.
3 Viral DNA carrying the normal
allele inserts into chromosome.
Bone
marrow
cell from
patient
4 Inject engineered
cells into patient.
Bone
marrow