MUTATION &

DNA RECOMBINANT TECHNOLOGY

AGUSTIN K. WARDANI
 Gene and Genome

• Genes and genome are made of DNA

• gene consists of enough DNA to code for one protein

• genome is a complete set of DNA

• Human genome: approximately 3 billion DNA base pairs

Bacteria genome: 130 kbp - 14 Mbp
Yeast genome: approximately 12 Mbp
Virus genome: 2 kb - 1 Mb
 Why the STRAIN IMPROVEMENT is needed?

The major motivation for industrial strain development is economic,

since:

- the metabolite concentrations produced by wild strains are too low

for economical processes.

- Optimizing the culture medium and growth conditions could not

maximize the organism ability to synthesize products.
 Strain Improvement Techniques

•Classical techniques
- mutagenesis/mutation (UV, mutagen)

•Modern techniques
- recombinant DNA technology
Genetic Mutation:
any change of DNA sequence due to some agents or
factors then the sequence of the amino acids will get
changed then change in phenotype.
MUTATION
 Chemical mutagens

• It is a mutation agent which is a chemical

substance that mimic nitrogen base in normal
DNA without coupling during DNA replication.

• Moreover, chemical mutagen has an ability to

disturb the DNA replication by inserting
themselves between nitrogen bases.

• There are various types of chemical mutagens.

 1. Base Analogs

• They are resemble purine and pyrimidine bases in

structure.
• When one of these base analogs is incorporated
at a site in DNA, replications error occur at high
frequencies at these sites.
• This result in incorporation of wrong base into the
copied strand causing mutation.
• For examples: 5-bromo uracil (analogue of
thymine), 2- aminopurine (analogue of adenine), 5-
bromodeoxyuridine, 2,6-diaminopurine, etc.
 5-bromo uracil

5-BU can cause transition mutation

A-T → G-C
2. Alkylating Agents

• They react with amino, carboxyl

and hydroxyl groups in proteins and
nucleic acid substituting them with
alkyl groups.
• They induce mutation at higher
frequencies than base analogs and
are able to introduce changes even
in non-replicating DNA.
• Both alkylating agents and base
analogs tend to induce base pair
substitutions.
• Ex: dimethyl sulpahte (DMS), ethyl
methane sulphate (EMS) and EES,
nitroso guanidine (NTG), etc.
 3. Inter-relative dyes

• The planar three ringed molecules having similar

dimensions as those of purine and pyrimidine that can be
inserted between two DNA base pairs (interaction) in aqueous
solution are called intercalative agents.
• For examples: acridine orange, proflavin, acriflavin, etc.
• They typically induce frameshift mutations.
• Ethidium bromide, which is often used to detect DNA in
electrophoresis, can also function as intercalative agent.
Mechanism of Intercalative Agents
 Radiation

• Production of highly reactive free radicals (OH

radicals).

• Interaction between the radicals and DNA,

proteins, lipids in cell membrane.

• Lead to incorrect insertion of nucleotides

opposite them in replication.

• Effects: Organelle failure, cell division blockage,

cell death
Mechanism
Genetic modification/engineering
by
Recombinant DNA Technology
What is Genetic Engineering?

The manipulation of a trait in

an organism to create a
desired change
 Tools

1. DNA (gene target)

2. Restriction endonucleases (molecular scissors)
3. Cloning vector/plasmid (e.g. pGEM, pBR322…)
4. Ligase enzyme (molecular glue)
 Step 1: Isolating the gene

• Cell lysis
• Removal of proteins (extraction)
• DNA precipitation by ethanol
• DNA dilution in water or buffer
 Extraction/Precipitation Method
Organic extraction
Mix thoroughly with Aqueous
an equal volume of
organic solvent Centrifuge Collect aqueous phase

e.g. phenol, chloroform,

or phenol:chloroform
Organic

Perform additional extractions for increased purity

Crude lysate containing -The aqueous phase contains water-

nucleic acids soluble molecules, including nucleic acids.
- Proteins and lipids become trapped in
the organic phase, and are thus
separated away.
Continued

Nucleic Acid Precipitation

Before After
70% EtOH
Supernatant
Centrifuge Wash Centrifuge

Pellet
Add alcohol (100%)
and salt to precipitate
nucleic acids from the
aqueous fraction
Dissolve pellet
(H2O, TE, etc.)
• Pellet down nucleic acids.
• Wash pellet with 70% ethanol to remove
residual salts and other contaminants.
• Discard ethanol and allow pellet to dry.
DNA Purity and Concentration

A. Spectrophotometry
Absorbance maximum
for nucleic acids 260 nm
for proteins 280 nm
• Concentration of DNA – at 260 nm
• Purity of DNA: ratio of 260/280 nm (1.8-2.0)
B. DNA confirmation by electrophoresis
 Step 2: Inserting gene into vector

• Vector – molecule of DNA

which is used to carry a
foreign gene into a host cell
 Cloning vector

• Promoter gene - A sequence of bases in a nucleic acid

strand, that serves as a signal to start transcription.
• The gene of interest.
• Antibiotic resistant gene- Are used as a marker system for
transformed cells.
Plasmid Vector: pBR322

• First modern cloning vector (1976)

pUC Plasmids
Step 3. Transformation (Electroporation)

Gene pulser
 Principle of Electroporation Technique

Introduction of integration vector

Pulser
Current Recipient cells
Plasmid
DNA
- +
 Step 4. Screening the positive clone

1. Replica plating
2. Blue/White Selection
• Modified E. coli codes for the carboxyl portion of
β-galactosidase (β-gal) enzyme. This portion alone
cannot cleave X-gal.

• Plasmid codes for amino portion (“LacZ” or α-

peptide) of β-gal.

• However, when the two peptides are expressed

together in a cell, X-gal is cleaved, and an indigo
product stains the bacterial colony blue. This
process is called α-complementation.
 Blue/White Selection
• Based on the enzymatic reaction of β-galactosidase.
 Example: Expression of alanine dehydrogenase
in L. lactis
Strain Specific activity
(mmol min-1 mg protein-1)
kDa
Bacillus 0.4
sphaericus
IFO3525
97 Bacillus subtilis 0.5
66 L. Lactis ND
(pNZ8020)
45 L. Lactis 0.39
30
(pNZ2650,+alaD)
14.4 ND: not detected
 Enzyme Gene donor Gene recipient Increase

a-amylase B. amyloliquefaciens B. subtilis 10

a-amylase Bacillus Bacillus 5

stearothermophilus stearothermophilus

a-amylase B. stearothermophilus B. brevis 100

EcoRI E. coli E. coli 50-100

DNA E. coli E. coli 100

Polymerase

Alanine B. sphaericus L. lactis 100

Multiplication of the host cells

• Small scale of fermentation (lab scale fermentation) → 500ml-

shakeflask, 2 L-fermentor
• Large scale of fermentation (industrial scale fermentation) → 10-L,
100-L fermentor