1. Introduction: How to Clone a gene?
Manipulation of DNA sequences in organisms
Is known as genetic engineering
Techniques used to engineer genes
Are called recombinant DNA technology

2. Using Plasmids in Cloning
Are small, circular DNA molecules
Are often found in bacteria
Replicate independently of the chromosome
Plasmids can serve as a vector
A vehicle for transferring recombinant genes to a new host

3. What makes Plasmids good vectors?
Contain DNA replication initiation site (ori)
Contain a promoter sequence for initiating transcription of the inserted gene
Contain antibiotic resistance gene which allows for identification of bacteria that carry the plasmid
Contain restriction enzymes recognition sites for gene insertion

4. Applications
Recombinant Plasmids are extremely useful because it allows manufacturing mass quantity of biological molecules. For example:
Protein used to dissolve blood clots in heart attack therapy (tissue plasminogen activator)
Insulin
Hepatitis B surface antigen, to vaccinate against the Hep B virus
Gene for pest resistance that is inserted into plants
Gene used to alter bacteria for cleaning up toxic waste

5. Cloning Genes in Recombinant Plasmids
In order for recombinant DNA to be useful it must be made in vast amounts.
HOW?
Isolate 2 different DNA = bacterial plasmid that serves as vector, and human DNA of interest
Treats both DNA with same RESTRICTION ENZYME
Sticky ends of the DNA joins by DNA ligase. Result : Recombinant DNA plasmid containing gene of interest.
Plasmids is inserted into bacterial cells by transformation
Gene Cloning – Bacteria reproduces = mass production of the recombinant DNA

6. Restriction Endonucleases
1962: “molecular scissors” discovered in bacteria. Also called restriction enzymes
E. coli bacteria have an enzymatic immune system that recognizes and destroys foreign DNA
3,000 enzymes have been identified, around 200 have unique properties, many are purified and available commercially

7. Origins of Restriction Enzymes
Discovered in bacteria. They restrict the growth of bacteriophage by cutting the viral DNA and rendering them harmless.

8. Restriction Endonucleases
Why don’t bacteria destroy their own DNA with their restriction enzymes?
DNA Modification

9. Restriction Endonucleases
Named for bacterial genus, species, strain, and type
Example: EcoR1
Genus: Escherichia Species: coli Strain: R Order discovered: 1

10. Restriction Endonucleases
Recognition sites have symmetry (palindromic)
“Hannah”
Bam H1 site:
5’-GGATCC-3’
3’-CCTAGG-5’

11. Restriction Endonucleases
Enzymes recognize specific 4-8 bp sequences
Some enzymes cut in a staggered fashion - “sticky ends”
EcoRI ’…GAATTC…3’
3’…CTTAAG…5’
Some enzymes cut in a direct fashion – “blunt ends”
PvuII ’…CAGCTG…3’
3’…GTCGAC…5’

12. Restriction Enzyme in Action
This slide shows a restriction enzyme cutting DNA. The enzyme used here is EcoRI, which cuts at any site where the base sequence is GAATTC.
Sticky Ends

13. Restriction Enzymes for Cloning
Human Insulin gene cut with EcoRI Plasmid cut with EcoRI +
5’-C-G-G-T-A-C-T-A-G-OH
3’-G-C-C-A-T-G-A-T-C-T-T-A-A-PO4
PO4-A-A-T-T-C-A-G-C-T-A-C-G-3’
HO-G-T-C-G-A-T-G-C-5’
5’-A-C-G-G-T-A-C-T-A-G A-A-T-T-C-A-G-C-T-A-C-G-3’
3’-T-G-C-C-A-T-G-A-T-C-T-T-A-A G-T-C-G-A-T-G-C-5’
Complementary base pairing
+ DNA Ligase, + rATP
recombinant DNA molecule
5’-A-C-G-G-T-A-C-T-A-G-A-A-T-T-C-A-G-C-T-A-C-G-3’
3’-T-G-C-C-A-T-G-A-T-C-T-T-A-A-G-T-C-G-A-T-G-C-5’

14. Lab 2 (Tab B): Clone That Gene
Students will prepare a recombinant plasmid by using restriction enzymes

15. Goals of Activity Know the characteristics of plasmids
Explain how plasmids are used in cloning a gene
Understand the function of restriction enzymes and how they are used in creating a recombinant plasmid

16. Students will …..
Lab 2: use restriction enzymes to digest the 2 plasmids OR
Lab 2a: digest a plasmid with 2 restriction enzymes.
Good to do a visual to help students see what they are doing: paper plasmids

17. The New Plasmids See ordering information in meeting binder appendix

18. The Recombinant Plasmids

19. RFP expression Bruce Wallace araC gene PBAD rfp gene Transcription
mRNA
Translation
araC protein 19

20. RFP expression araC protein prevents RFP transcription by causing
Bruce Wallace
araC protein prevents RFP transcription by causing
a loop to form in the region of the fp gene r
araC gene
araC protein
PBAD
rfp gene 20

21. arabinose – araC protein
RFP expression
Bruce Wallace
RFP
(red fluorescent
protein)
Arabinose – araC protein complex prevents DNA looping
and helps to align RNA polymerase
on the promoter site (PBAD).
arabinose
Translation
RNA polymerase
arabinose – araC protein
complex
araC protein
mRNA
Transcription
araC gene
rfp gene
PBAD 21

22. Bruce Wallace
RFP

23. Running Digested DNA Through Gel Electrophoresis
Lab 4: Verification of recombinant plasmid creation
Running Digested DNA Through Gel Electrophoresis

24. How does it work? DNA is cut into smaller fragments.
Loading dye is used to sink the DNA into the wells in the gel and to track the movements of DNA
The negative DNA molecule is attracted to the positive (RED) electrode.
DNA moves based on size and structure; with the smallest fragments move fastest

25. Different Structural Forms
circle
“multimer”
Nicked Circle
Linear
Supercoiled
“nicked-circle”
Different structural forms produce different bands.

26. Goals of this Hands-On Lab
Compare cut vs uncut plasmid DNA by running them through a gel.
Look for different banding patterns and understand how to read them.
Predict what kind of banding pattern a plasmid will make based on:
The restriction enzyme used.
The plasmid’s structural shape.

27. Confirmation of restriction and ligation
Restriction analysis of pKAN-R and pARA
Bruce Wallace
Confirmation of restriction and ligation M K+ K- A+ A- L M K+ K- A+ A- L
500
1000
1500
2000
3000
4000
5000
8000
10000
4705 bp
4495 bp
807 bp
377 bp

28. Lonza Flash Gel System

29. Why? Lab 5: Transformation
Plasmid vectors can be introduced into bacteria by transformation: the process of taking up DNA from the environment
Why?
If a recombinant plasmid can be inserted into a bacterial or yeast cell
The foreign DNA will be copied and transmitted to new cells as the host cell multiplies
Produces millions of identical copies of specific genes

30. preparing competent cells for transformation
Lipid bilayer
(inner)
Adhesion zone
Peptidoglycan
layer
Lipid bilayer
(outer)
Calcium ions

31. Transforming Escherichia coli with pARA-R
Bruce Wallace
Competent Cells
pARA-R
Recombinant Plasmids

32. transforming Escherichia coli with pARA-R
Lipid bilayer
(inner)
Adhesion zone
Peptidoglycan
layer
Lipid bilayer
(outer)
Calcium ions
pARA-R

33. Transgenic Colony Allowed to Grow in Selective Media

34. growth of transformed bacteria on various plates
P+ plates LB
LB/amp
LB/amp/ara
P- plates
No growth LB
LB/amp