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Why the improvement of microorganisms
is needed?
- 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
The major motivation for industrial strain development is economic, since:
Strain Improvement Techniques
Classical techniques
- mutagenesis (UV, mutagen)
Modern techniques
- recombinant DNA technology
Mutagenesis
A process by which the genetic information of an organism is changed in a stable manner, either in nature or experimentally by the use of chemicals or radiation.
base substitutions, insertions, and deletions.
UV light, x-rays, gamma rays, NTG (Nitrosoguanidine) , EMS (Ethyl methanesulfonate )
Ex; Hydrogen production by Bacillus pumilus Lipase production by A. japonicus
Tools
1. DNA (gene target)
2. Restriction endonucleases (molecular scissors)
3. Cloning vector/plasmid (e.g. pGEM, pBR322…)
4. Ligase enzyme (molecular glue)
Cell lysis
Removal of proteins (extraction)
DNA precipitation by ethanol
DNA dilution in water or buffer
Step 1: Isolating the gene
Crude lysate containing
nucleic acids
Mix thoroughly with
an equal volume of
organic solvent
e.g. phenol, chloroform,
or phenol:chloroform
Centrifuge
-The aqueous phase contains water-
soluble molecules, including nucleic acids.
- Proteins and lipids become trapped in
the organic phase, and are thus
separated away.
Perform additional extractions for increased purity
Collect aqueous phase
Extraction/Precipitation Method
Organic extraction
Organic
Aqueous
• Pellet down nucleic acids.
• Wash pellet with 70% ethanol to remove
residual salts and other contaminants.
• Discard ethanol and allow pellet to dry.
After
Add alcohol (100%)
and salt to precipitate
nucleic acids from the
aqueous fraction
Supernatant
Pellet
70% EtOH
Dissolve pellet
(H2O, TE, etc.)
Nucleic Acid Precipitation
Continued
Before After
Centrifuge Wash Centrifuge
DNA Purity and Concentration
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)
Step 2: Inserting gene into vector
• Vector – molecule of
DNA which is used to
carry a foreign gene into
a host cell
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.
Cloning vector
Introduction of integration vector
Principle of Electroporation Technique
Recipient cells
Plasmid
DNA
+ -
Current
Pulser
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.
kDa
97
66
45
30
14.4
Example: Expression of alanine dehydrogenase
in L. lactis
Strain Specific activity
(mmol min-1 mg protein-1)
Bacillus
sphaericus
IFO3525
0.4
Bacillus subtilis 0.5
L. Lactis
(pNZ8020)
ND
L. Lactis
(pNZ2650,+alaD)
0.39
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
Purification
Centrifugation
Ammonium sulfate precipitation
Chromatography
- Ionic exchange chromatography
- Affinity chromatography
- Hydrophobic chromatography
- Reverse phase chromatography
SDS- PAGE
M 3 3 2 2 1 1 M
Purification step 3: step 3 2: step 2 1: step 1 M: marker (molecular weight)