Microbial genetics Part 3

MUTATION

Selection of mutants

Genetic recombin

ation

Gene transfer

Mutagen

Genetic EngineeringRecombinant DNA technology, gene cloning

Genetic Engineering

• Genetic engineering involves changing the genetic material in an organism to alter its traits or products

• A recombinant DNA molecule contains DNA fragments spliced together from 2 or more organisms

Outline

Stages of cloning experiment Elements:

Vectors Restriction enzymes Mechanism in joining the fragments Selection or detection of successful

cloning Gene library

Stages of Cloning

1. Joining DNA segment

2. Providing milieu that allows propagation

Clones

Vector

Recombinant Bacteria

1. Remove bacterial DNA (plasmid).

2. Cut the Bacterial DNA with “restriction enzymes”.

3. Cut the DNA from another organism with “restriction enzymes”.

4. Combine the cut pieces of DNA together with another enzyme and insert them into bacteria.

5. Reproduce the recombinant bacteria.

6. The foreign genes will be expressed in the bacteria.

Vectors : properties

1. It must be able to replicate2. There must be some way to

introduce vector DNA into the cell3. There must be some way of

detecting its presence, preferably by plating techniques

Most common vectors are: Plasmid, phage λ and viruses

Restriction Enzymes

Discovered in an experiment where bacteriophage lost its plaque formation in E.coli

Enzymes that recognize a specific base sequence in a DNA molecule

It makes two cuts, one in each strand, generating 3’-OH and 5’-P termini

Types of termini produced: Flush or blunt end Cohesive or sticky end

Examples of Restriction Enzymes

Other features of Restriction Enzymes

The number of cuts made in the DNA from specific organism is limited

A particular restriction enzyme generates a unique family fragments from a DNA molecule

•BstEII pUC19 pUC19 •HindIII •HindIII •BstEII

Restriction Mapping

Restriction maps show the relative location of a selection of restriction sites along linear or circular DNA.

HindIII BamHI PstIIPstII BamHI

HindIII

EcoRI

Simple example

Digests1: EcoRI2: HindIII3: EcoRI + HindIII Resultant Fragments: approximate sizes1: 3 kb, 5 kb2: 6 kb, 2 kb3: 2 kb, 1 kb, 5 kb,

Restriction MappingBglII BamHI PstI

BglII+BamHI

BglII+PstI

BamHI+PstI

4.2

5.2

3.6 3.53.3

2.6

1.7 1.71.4 1.41.2 1.2

1.0 1.01.2

0.7 0.9

0.5

0.3 0.30.3

BglII BamHI PstI BglII PstI

0.3 0.7 2.6 0.9 0.5 1.2

Restriction problems

A)  11, 6, 5B)  14,8C)  16,6

A x B) 8, 6, 5, 3A x C) 11, 5, 5,  1B x C) 8, 8, 6

Joining fragments

Cohesive end Blunt end

Increasing the DNA concentration and addition of ligase

Addition of homopolymers Using of linkers

Increasing DNA concentration

Homopolymers

Linkers

Selection/detection

Antibiotic resistance marker Insertional inactivation

Electrophoresis hybridization

Gene libraries

collection of all of the vector molecules, each carrying a piece of the chromosomal DNA of the organism

Applications