Isolation and handling of

microorganism: QUESTIONS

Can the microorganism grow on simple cheap media?

Does it have better resistance to contamination?

Strain improvement, 1% increase in product output

could pay for a mutation programme
 Microorganisms: characteristics for industrial applications
i.The organism must be able to grow in a simple medium (preferably no growth factors such as pre-
formed vitamins, nucleotides, and acids)

ii.The organism should be able to grow vigorously and rapidly in the used medium and produce the
desired product

iii. Its end products should not include toxic and other undesirable materials

iv. The organism should have a reasonable genetic, and hence physiological stability.

v.The organism should lend itself to a suitable method of product harvest at the end of the
fermentation. Wherever possible, organisms which have physiological requirements which protect
them against competition from contaminants should be used.

vi. The organism should be reasonably resistant to predators

vii.Where practicable the organism should not be too highly demanding of oxygen as aeration
contributes about 20% of the cost of the finished product.

viii.The organism should be fairly easily amenable to genetic manipulation to enable the
establishment of strains with more acceptable properties.
 Limitations of naturally occurring bacteria

Bacteria are evolved for survival in competitive natural

environments, not for production of chemicals desired by
humans!
- are optimized for low nutrient levels
- have defense systems
- don’t naturally make everything we need

 -The genetic apparatus of the organism determines the organism synthetic potentialities
 What is actually synthesized depends on the available environment.
 The organism cannot ‘decide’ to manufacture and secrete certain enzymes but it can stop the
synthesis of certain compounds if they are supplied to it.
 -These sensing mechanisms for the switching on and off of the synthetic processes enable the
organism to avoid the overproduction of any particular compound.
 If these regulatory mechanisms do not work a lot of energy and resources would be wasted
(usually scarce in natural environments) in making materials that are not required.
 Manipulation of the Genome of Industrial Organisms in
Strain Improvement
The manipulation of the genome for increased productivity may be done in one of
two general procedures:
(a) manipulations not involving foreign DNA;
(b) manipulations involving foreign DNA .
 Overproduction of Metabolites by Industrial
Microorganisms /1/

- An efficient organism which does not waste its resources in producing materials will survive well in
natural environments where competition is intense, but it will NOT be of much use as an industrial
organism
- The industrial biotechnologist prefers the wasteful, inefficient and ‘relaxed’ organism with regulatory
mechanisms that overproduce the particular metabolite sought.

- Industrial microbiologist disorganize

the regulatory mechanisms to force the
organism to overproduce desired
materials
 Overproduction of Metabolites by Industrial
Microorganisms /2/

Inducible enzymes
Some enzymes are produced by microorganisms only when the substrate on which they
act is available in the medium.

Such enzymes are known as inducible enzymes.

Analogues of the substrate may act as the inducer.
When an inducer is present in the medium a number of different inducible enzymes
may sometimes be synthesized by the organism.

Feedback Regulation
Feedback or end-product regulations control exerted by the end-product of a metabolic
pathway
Two main types of feedback regulation exist: feedback inhibition and feedback
repression.
Both of them help to adjust the rate of the production of pathway end products
Feedback regulation
 Redesigning bacteria: Natural Genetic Engineering

Target: optimize industrially valuable parameters

•Redirect metabolism to specific products

• Remove unwanted products

- storage products
- excretion products
- defense systems

The newest approach for creating new metabolic

capabilities in a given microorganism is the area of
natural genetic engineering, which employs forced
evolution and adaptive mutations.
This is the process of using specific environmental
stresses to “force” microorganisms to mutate and adapt,
thus creating microorganisms with new biological
capabilities.
 Mutation
Change in the genetic material
Mutations may be neutral, beneficial, or harmful
Mutagen: Agent that causes mutations
Spontaneous mutations: Occurs in the absence of a mutagen
 Mutation

Ionizing radiation (X rays and gamma

rays) causes the formation of ions that
can react with nucleotides and the
deoxyribose-phosphate backbone.
Nucleotide excision repairs mutations

UV radiation causes thymine

dimers Light-repair separates
thymine dimers

Spontaneous mutation rate = 1 in

109 replicated base pairs or 1 in
106 replicated genes
Mutagens increase to 10–5 or 10–3
per replicated gene
Conventional:
auxotrophos
selection The organism (prototroph) is transferred from a
slant to a broth of the minimal medium: no
auxotroph will grow on it

The prototroph is shaken in the minimal broth

for 22–24 hours, at the end of which period it
is subjected to mutagenic treatment.
The mutagenized cells are grown on complete
medium (8 h).

The washed cells are then shaken again in minimal

medium to which penicillin is added: the antibiotic
kills only dividing cells. As only prototrophs will
grow in the minimal medium these are killed off
leaving the auxotrophs.

The washed cells are plated out on the complete

agar medium. To determine the growth factor or
compound which the auxotroph cannot synthesise
the different an agar culture is replica-plated on to
each of several plates which contain the minimal
medium and various growth factors either single or
mixed. The medium on which the auxotroph will grow
indicates the metabolite it cannot synthesize. For
example when the auxotroph requires lysine it is
designated a ‘lysineless’ mutant

Auxotrophic mutants: are those which lack the enzymes to manufacture certain required nutrients such
nutrients must therefore be added to the growth medium. Auxotrophs are largely used in biotechnology
Replica Plating
 Metabolic engineering: Deleting a gene

Gene 1 Gene 2 Gene 3

DNA

A Enzyme 1 B Enzyme 2 C Enzyme 3 D

Restriction endonucleases: Enzymes that can clip

strands of DNA crosswise at selected positions

Gene 1 Gene 2
DNA

A Enzyme 1 B Enzyme 2 C Enzyme 3

 Insertion of a gene: use of plasmide

Gene 4

1. Put the gene of interest Plasmid: a circle of DNA

into a stable carrier: a

plasmid.

Gene 4

Gene 4
Metabolic engineering: Adding a new gene

Gene 1 Gene 2 Gene 3

DNA

A Enzyme 1 B Enzyme 2 C Enzyme 3 D

Gene 1 Gene 2 Gene 4 Gene 3

DNA

A Enzyme 1 B Enzyme 2 C
Enzyme 4 Enzyme 3 D

E
 Gene 1 Gene 2 X Gene 3
DNA
X
2. Put the plasmid
into a new cell.
Gene 4

Gene 1 Gene 2 Gene 4

DNA

A Enzyme 1 B Enzyme 2 C Enzyme 4 E

Modification of Gene Expression

It also is possible to modify gene

regulation by changing gene
transcription, fusing proteins,
creating hybrid promoters, and
removing feedback regulation
controls.
These approaches, for instance,
make it possible to overproduce a
wide variety of products.
Metabolic engineering success
 Metabolic engineering mishaps

e.g. maximizing ethanol production

glucose ethanol
PFK
PFK (phosfofructokinase) was
thought to be the rate-limiting
enzyme of ethanol production, so
its levels were increased via genetic
engineering. However: rates of
ethanol production did not increase!

Why?:
Cellular metabolism is
very complicated!
 Methods in Recombinant DNA Technology

• Primary intent of recombinant DNA technology-

deliberately remove genetic material from one organism
and combine it with that of a different organism

• Form genetic clones

– Gene is selected
– Excise gene
– Isolate gene
– Insert gene into a vector
– Vector inserts DNA into a cloning host
 Genetic Recombination

Exchange of genes between

two DNA molecules

Crossing over occurs when two

chromosomes break and rejoin
 Characteristics of Cloning Vectors and cloning hosts

Vectors : plasmids and bacteriophages

• Capable of carrying a significant piece of the donor
DNA
• Readily accepted by the cloning host
• Must have a promoter in front of the cloned gene
Cloning Host:
• Rapid turnover, fast growth rate and it can be grown
in large quantities using ordinary culture methods
• Nonpathogenic
• Genome that is well delineated (mapped)
• Capable of accepting plasmid or bacteriophage
vectors
• Maintains foreign genes through multiple
generations
• Will secrete a high yield of proteins from expressed
foreign genes
Recombination
Conjugation
……..To steps 6-8
……..From steps 1-5
 Transduction

Phage protein coat

Bacterial
chromosome

Recombinant

1 A phage infects the 2 Phage DNA and proteins

donor bacterial are made, and the
cell. bacterial chromosome is
broken down into pieces.
Bacterial
DNA
Donor Recipient
Phage bacterial bacterial
DNA DNA DNA

Recipient cell Recombinant cell

3 Occasionally during phage assembly,
pieces of bacterial DNA are packaged
in a phage capsid. Then the donor cell
lyses and releases phage particles
containing bacterial DNA.
4 A phage carrying bacterial
DNA infects a new host cell,
the recipient cell.
5 Recombinant can occur,
producing a recombinant
cell with a genotype
different from both the
donor and recipient cells.
Recombinant DNA Technology
 Examples of Current Protein Products from Recombinant
DNA Technology
Immune Treatments Interferons - peptides used to treat some types of cancer, multiple sclerosis, and vira l infections such as hepatitis
and genital warts Interleukins - types of molecules that regulate the immune function of whit blood cells; used in cancer treatment

Orthoclone - an immune suppressant in transplant patients

Granulocyte - macrophage-colony-stimulating factor (GM-CSF)- used to stimulate bone marrow activity after bone marrow grafts
Tumor necrosis factor (T N F )-used to treat cancer
Granulocyte-colony- stimulating factor (Neup o g e n )- developed for treating cancer patients suffering from low neutrophil counts

Hormones Erythropoietin (EPO)- a peptide that stimulates bone marrow used to treat some forms of anemia

Tissue plasminogen activating factor (tPA ) -can dissolve potentially dangerous blood clots Hemoglobin

A form of artificial blood to be used in place of real blood for transfusions

Factor VIII- needed as replacement blood-clotting factor in type A hemophilia
Relaxin- an aid to childbirth

Human growth hormone ( H G H ) -stimulates growth in children with dwarfism; prevents wasting syndrome

Enzymes rH DNase (pulmozyme)-a treatment that can break down the thick lung secretions of cystic fibrosis

Antitrypsn - replacement therapy to benefit emphysema patients

PEG-SO D -a form of superoxide dismutase that minimizes damage to brain tissue after severe trauma

Vaccines Vaccines for hepatitis B and I-Iaemophilus influenzae Type b meningitis Experimental malaria and AIDS vaccines based on
recombinant surface antigens

Miscellaneous Bovine growth hormone or bovine somatotropin (BST ) - g iven to cows to increase milk production
Apolipoprotein- to deter the development of fatty deposits in the arteries and to prevent strokes and heart attacks
Spider s i l k -a light, tough fabric for parachutes and bulletproof vests
 Genetically Modified Organisms

• Transgenic or genetically modified organisms (GMOs): recombinant organisms

produced through the introduction of foreign genes
• These organisms can be patented

• Genetically altered strain of Pseudomonas syringae

– Can prevent ice crystals from forming

– Frostban to stop frost damage in crops

• Strain of Pseudomonas fluorescens

– Engineered with a gene from Bacillus thuringiensis

– Codes for an insecticide

• Drug therapy

• Bioremediation
Foreign genetic material for protein synthesis

Tissue plasminogen activator (abbreviated TPA or PLAT)

is a protein involved in the breakdown of blood clots.

• Prokaryotic hosts (e.g. E. coli)

• Eukaryotic genes (cDNA)
• Large amounts of protein
produced
Applications: environmental

Genetically modified organisms (GMOs)

• Genes for breakdown of

hydrocarbons already in species
of Pseudomonas

• Genes from 4 species

combined

Clean up oil spills that

endanger wildlife
 Examples BIOSENSORS

Hystory
Concept - Leland Clark (1956)
Urea Sensor - Guibault & Montalvo (1969)
Glucose Analyser - Yellow Springs Instr. Co. (1973)
Enzyme Thermistor – Mosbach (1974) Professor Leland C Clark Jnr
Microbial Electrodes – Divis (1975) 1918–2005

Fibre-Optic Oxygen Sensor – Lubbers & Opitz (1975)

Biostator – Clemens et al. (1976)
Immnosensor – Liedberg et al. (1982)
Enzyme Electrode – MediSense Inc. (1987)
BIAcore – Pharmacia, Sweden (1990)
NanoSensor – Vo-Dinh (2000)

Current definition: sensor integrating biological elements and physiochemical

transducers to produce an electronic signal proportional to a specific analyte
(which is then conveyed to detector).
 Biosensor ≠ analytical device
It is any device that uses specific biochemical reactions to detect chemical
compounds in biological samples.

FROM PAST…….

……. TO PRESENT
Biosensor: result of a multidisciplinary approach
 Why biosensor?
Biosensor generates an analogic signal proportional to a molecule concentration
(highly specifically)

CHARACTERRISTICS
Biosensor = bioreceptor +
transducer
LINEARITY Linearity of the
sensor should be high for the
detection of high substrate Tansducer
concentration.
SENSITIVITY Value of the
electrode response per substrate
concentration.
Bioreceptor: biological molecules able
SELECTIVITY Chemicals to specifically recognized target
Interference must be minimised
Trasducer: equipment able to convert
for obtaining the correct result.
RESPONSE TIME Time necessary recognition in a measurable signal
for having 95% of the response.
 Components of a Biosensor
Analyte = What has to be detect Detection/Recognition = How to specifically
Molecule - Protein, toxin, peptide, recognize the analyte
vitamin, sugar, metal ion

Detector

Sample handling = How to deliver the

analyte to the sensitive region Signal = How to know if there

(Micro) fluidics - Concentration was a detection

increase/decrease), Filtration/selection
Receptors & transducers
 Applications & Examples of biosensors
• Specific
• Biocompatible Pregnancy test:
Detects the hCG
• Small: no tissue damage
protein in urine
• Fast calibration and response
• Long lasting

APPLICATIONS (examples)
Glucose monitoring:
 Food Analysis
device for diabetes patients
 Study of biomolecules and their interaction
 Drug Development
 Crime detection
 Medical diagnosis (both clinical and laboratory use)
 Environmental field monitoring
 Quality control
 Industrial Process Control
 Detection systems for biological warfare agents
Infectous disease
 Manufacturing of pharmaceuticals and replacement organs monitoring
 Market evolution

1 Medical: glucose 90% MediSense / (Abbott), Boehringer,

Bayer, YSI, etc.
2 Medical: other 2% Lactate, urea, etc.
5 Billion US$
3 Environmental 2% Mainly BOD indicators

4 Other 6% Fermentation monitoring, food, alcohol,

etc.
 Example: Bioluminescence

luciferase
luciferine + ATP + O2 oxyiluciferin + PPi + CO2 +hν (λmax = 560 nm)
Mg2+
Bacteria luminescence

• Vibrio

• Photobacterium
Photobacterium
phosphoreum

• Xenorhabdus
Xenorhabdus
nematophilus
Genetic modification of E. coli : introduction of bioluminescence
Target; detection of organic & inorganic analytes

Hg KNO3 Ni

Phenol Benzene

Naphthalene

Octane Ethanol
 Transducer: photomultiplier

Luminometer

No luminescence Death of the receptor due to toxicity

(presence of analyte)

Yes Luminescence Survival of the receptor

(no or low presence of analyte)