© 2014 Pearson Education, Inc. Introduction: How to Clone a gene? Manipulation of DNA sequences in organisms –Is known as genetic engineering Techniques.

© 2014 Pearson Education, Inc.

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

© 2014 Pearson Education, Inc.

Using Plasmids in Cloning

Plasmids– 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

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What makes Plasmids good vectors?1. Contain DNA replication initiation

site (ori)

2. Contain a promoter sequence for initiating transcription of the inserted gene

3. Contain antibiotic resistance gene which allows for identification of bacteria that carry the plasmid

4. Contain restriction enzymes recognition sites for gene insertion

© 2014 Pearson Education, Inc.

ApplicationsRecombinant 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)

InsulinHepatitis B surface antigen, to

vaccinate against the Hep B virusGene for pest resistance that is

inserted into plantsGene used to alter bacteria for

cleaning up toxic waste

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Cloning Genes in Recombinant PlasmidsIn order for recombinant DNA to be useful it must be made in vast amounts.

HOW?1. Isolate 2 different DNA = bacterial plasmid

that serves as vector, and human DNA of interest

2. Treats both DNA with same RESTRICTION ENZYME

3. Sticky ends of the DNA joins by DNA ligase. Result : Recombinant DNA plasmid containing gene of interest.

4. Plasmids is inserted into bacterial cells by transformation

5. Gene Cloning – Bacteria reproduces = mass production of the recombinant DNA

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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

Origins of Restriction Enzymes

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

Restriction EndonucleasesWhy don’t bacteria destroy their own DNA with their restriction enzymes?

DNA Modification

Restriction Endonucleases

Named for bacterial genus, species, strain, and type

Example: EcoR1Genus: EscherichiaSpecies: coliStrain: ROrder discovered: 1

Restriction Endonucleases

Recognition sites have symmetry (palindromic)

“Hannah”

Bam H1 site:5’-GGATCC-3’3’-CCTAGG-5’

Restriction EndonucleasesEnzymes recognize specific 4-8 bp

sequencesSome enzymes cut in a staggered

fashion - “sticky ends” EcoRI 5’…GAATTC…3’ 3’…CTTAAG…5’

Some enzymes cut in a direct fashion – “blunt ends”

PvuII 5’…CAGCTG…3’ 3’…GTCGAC…5’

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Restriction Enzyme in Action

Sticky Ends

Human Insulin gene cut with EcoRI Plasmid cut with EcoRI

5’-C-G-G-T-A-C-T-A-G-OH3’-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’

Restriction Enzymes for Cloning

Lab 2 (Tab B): Clone That Gene

Students will prepare a recombinant plasmid by

using restriction enzymes

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

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

The New Plasmids

The Recombinant Plasmids

RFP expression

araC gene rfp genePBAD

Transcription

mRNA

Translation

araC protein

Bruce Wallace

RFP expression

rfp genePBAD

araC protein

araC gene

araC protein prevents RFP transcription by causinga loop to form in the region of the fp gener

Bruce Wallace

RFP expression

araC protein

arabinose

araC gene rfp genePBAD

arabinose – araC proteincomplex

RNA polymerase

Arabinose – araC protein complex prevents DNA loopingand helps to align RNA polymerase

on the promoter site (PBAD).

mRNA

Transcription

Translation

RFP(red fluorescent

protein)

Bruce Wallace

RFP

Bruce Wallace

Running Digested DNA Through Gel Electrophoresis

Lab 4: Verification of recombinant plasmid creation

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

Different Structural Forms

circle

“nicked-circle”

“multimer”

Different structural forms produce different bands.

Nicked Circle

SupercoiledLinear

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: 1.The restriction enzyme used.2.The plasmid’s structural shape.

Restriction analysis of pKAN-R and pARA

M K+ K- A+ A-

500

1000

1500

20003000400050008000

10000

Confirmation of restriction and ligation

L M K+ K- A+ A- L

Bruce Wallace

807 bp

4705 bp 4495 bp

377 bp

Lonza Flash Gel System

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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

preparing competent cells for transformation

Lipid bilayer(inner)

Lipid bilayer(outer)

Peptidoglycanlayer

Adhesion zone

Calcium ions

Transforming Escherichia coli with pARA-R

Recombinant Plasmids

Competent Cells

pARA-R

Bruce Wallace

Calcium ions

pARA-R

transforming Escherichia coli with pARA-R

Adhesion zone

Lipid bilayer(inner)

Lipid bilayer(outer)

Peptidoglycanlayer

Transgenic Colony Allowed to Grow in Selective Media

growth of transformed bacteria on various plates

P+ plates

P- plates

LB LB/amp LB/amp/ara

LB LB/amp

No growth