rDNA = recombinant DNA Figure 1. Humulin®

3.4 nm

1 nm

0.34 nm

Hydrogen bond

(a) Key features of DNA structure

Space-filling model

(c) (b) Partial chemical structure

3 end

5 end

3 end

5 end

T

T

A

A

G

G

C

C

C

C

C

C

C

C

C

C

C

G

G

G

G

G

G

G

G

G

T

T

T

T

T

T

A

A

A

A

A

A

Figure 2. DNA Double Helix

Host cell DNA sequence is chemically modified – restriction enzyme can’t cut it

Foreign DNA sequence is not chemically modified – restriction enzyme will cut it

Harvey Lodish, et al. Molecular Cell Biology 3e, Scientific American Books

Figure 3. Bacterial Immunity Through DNA Methylation

Figure 20.3-3

Recombinant DNA molecule

One possible combination DNA ligase seals strands

DNA fragment added from another molecule cut by same enzyme. Base pairing occurs.

Restriction enzyme cuts sugar-phosphate backbones.

Restriction site

DNA

5

5

5

5

5

5

5

5

5 5

5

5

5 5

5

5

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

2

3

1

Sticky ends

GAATTC CTTAAG

G G

G G

AATT C AATT C C TTAA C TTAA

STICKY ENDS

Recombinant DNA Molecule

Figure 4. Using a Restriction Enzyme and DNA Ligase to Make Recombinant DNA

B Chain A Chain

http://www.biotopics.co.uk/JmolApplet/insulinjdisplay.html

Figure 5. Molecular Structure of Human Insulin

Step 1 Preproinsulin is synthesized as a random coil on membrane-associated ribosomes

Step 2 Leader sequence is cleaved and the resulting proinsulin folds into a stable conformation Step 3 Disulfide bonds form

Step 4 The connecting sequence is cleaved for form the mature insulin molecule

Leader sequence aids in transporting the polypeptide through the membrane

Preproinsulin

Proinsulin

Insulin

Connecting Sequence

Copyright Peason Higher Education

Figure 6. Cellular Mechanism of Insulin Production 9

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 7. Basic Laboratory Methods to Manufacture Human Insulin

Copyright © The McGraw-Hill Companies, Inc.

Human cell

Acquire source DNA and synthesize proinsulin gene

C T T A A

Add appropriate “sticky ends”

Proinsulin DNA with “sticky ends”

Figure 8. Synthesis of Genetically-Engineered Human Insulin Step 1: Synthesize Proinsulin Gene

Synthesized proinsulin DNA

Restriction enzymes cut

plasmid DNA at specific

sequence to produce

same sticky ends

attached to proinsulin DNA

Plasmid (vector)

Create recombinant DNA

Mix proinsulin DNA with

plasmid DNA to create

recombinant plasmid

Join DNA molecules

together with

DNA Ligase

C T T A A

Antibiotic resistance gene

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

Insert the recombinant DNA

into a recipient cell

Recombinant

plasmid Transgenic bacterium

containing human DNA

Culture media with antibiotic to which cells with plasmid will be resistant

Copyright © The McGraw-Hill Companies, Inc.

Figure 10. Synthesis of Genetically-Engineered Human Insulin Step 3: Manipulate E. coli to Take Up Plasmid DNA

75 to 3,000 liters 1.3 to 14 liters

New Brunswick Scientific

Collect cells Lyse cells

Collect cellular material

Figure 11. Synthesis of Genetically-Engineered Human Insulin Step 4: Culture Engineered E. coli Cells

New Brunswick Scientific

Collect proinsulin polypeptide Remove connecting sequence Join A and B chains together

Purify Humulin®

Figure 12. Synthesis of Genetically-Engineered Human Insulin Step 5: Produce and Purify Insulin

Gene of interest

Protein expressed from

gene of interest

Basic research

and various

applications

Gene for pest

resistance inserted

into plants

Gene used to alter

bacteria for cleaning

up toxic waste

Protein dissolves

blood clots in heart

attack therapy

Human growth

hormone treats

stunted growth

Protein harvested

Figure 13. Some Uses of Genetically Engineered Cells

Copyright © The McGraw-Hill Companies, Inc.

Recombinant plasmid

Agrobacterium

Herbicide

resistance gene

Infection Herbicide resistance gene

Chromosome

Unaltered plant

cell

Transgenic plant

cell

Cell

division

When the transgenic cell divides, each

daughter cell receives the herbicide

resistance gene. The resulting tobacco

plant is transgenic.

Cell

division

Figure 14. Creating a Genetically Engineered Plant

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 15. Representative Biotechnology Products

Figure 16. Bioinformatics (genomics.energy.gov)

Cloned gene

2

1

3

4

Retrovirus

capsid

Bone

marrow

cell from

patient

Viral RNA

Bone

marrow

Insert RNA version of normal allele

into retrovirus.

Let retrovirus infect bone marrow cells

that have been removed from the

patient and cultured.

Viral DNA carrying the normal

allele inserts into chromosome.

Inject engineered

cells into patient.

Figure 17. Gene Therapy