1. By
Assist.Prof
Dr. Berciyal Golda. P
VICAS
Attributes required in industrial microbes to be usedas efficient
cloning and expression hosts
2. Introduction
Microbial technology refers to the use of microbes
to obtain a product or service of economic value. It is
also called as Fermentation.
The revolutionary exploitation of microbial genetic
discoveries in the 1970s, 1980s and 1990s depended
heavily upon the solid structure of industrial
microbiology, described above.
Our modern understanding of
the fermentation process comes from the work of the
French chemist Louis Pasteur.
3. The major microbial hosts for production of recombinant proteins are
E. coli, B. subtilis, S. cerevisiae, Pichia pastoris, Hansenula
polymorpha and Aspergillus niger.
The use of recombinant microorganisms provided the techniques and
experience necessary for the successful application of higher
organisms, such as mammalian and insect cell culture, and transgenic
animals and plants as hosts for the production of glycosylated
recombinant proteins.
4.
5. 13
The Production of Commercial Products by
Recombinants Microorganisms
■ Molecular biotechnology can be used to enhance the
production of many commercially important compounds
e.g. Vitamins
Amino acids
Antibiotics
■ We will be investigating the use of recombinant
organisms to improve or enhance the production of :
Restriction enzymes
Ascorbic acid
Microbial synthesis of the dye indigo
Production of xanthan gum
6. Therapeutic Agents
Before the advent of molecular biotechnology most human
proteins were available in only small (limited) quantities.
Today hundreds of genes for human proteins have been cloned,
sequenced, expressed in the host cells and are being tested as
therapeutic agents (drugs) in humans.
7. Industrial Microorganisms & Products
Properties of useful industrial microorganism:
Produces spores or can be easily inoculated
Grows rapidly on a large scale in
inexpensive medium
Produces desired product quickly
Should not be pathogenic
Amenable to genetic manipulation
10. ⦁ Microbes especially yeast have been used from time
immemorial for the production of beverages like
wine, beer, whiskey, brandy or rum.
⦁ For this purpose, the
yeast Saccharomyces cerevisiae is used for
fermenting malted cereals and fruit
juices to produce ethanol.
Wine producing bacteria Fungai
Cyberlindnera mrakii
Pichia fermentans
Acetobacter cerevisiae
Lactobacillus bucheri
actobacillus hilgardii
Lactobacillus kunkeei
11.
12. ⦁ Antibiotics produced by microbes are regarded was
one of the most significant discoveries of the
twentieth century and have made major contributions
towards the welfare of human society.
⦁ Many antibiotics are produced by microorganisms,
predominantly by Actinomycetes in the
genus Streptomycin (e.g. Tetracycline, Streptomycin,
Actinomycin D) and by filamentous
fungi (e.g. Penicillin, Cephalosporin)
13. Antibiotic Producer organism Activity Site or mode of action
Penicillin Penicillium chrysogenum Gram-positive bacteria Wall synthesis
Cephalosporin Cephalosporium acremonium Broad spectrum Wall synthesis
Griseofulvin Penicillium griseofulvum Dermatophytic fungi Microtubules
Bacitracin Bacillus subtilis Gram-positive bacteria Wall synthesis
Polymyxin B Bacillus polymyxa Gram-negative bacteria Cell membrane
Amphotericin B Streptomyces nodosus Fungi Cell membrane
Erythromycin Streptomyces erythreus Gram-positive bacteria Protein synthesis
Neomycin Streptomyces fradiae Broad spectrum Protein synthesis
Streptomycin Streptomyces griseus Gram-negative bacteria Protein synthesis
Tetracycline Streptomyces rimosus Broad spectrum Protein synthesis
Vancomycin Streptomyces orientalis Gram-positive bacteria Protein synthesis
Gentamicin
Rifamycin
Micromonospora purpurea
Streptomyces mediterranei
Broad spectrum
Tuberculosis
Protein synthesis
Protein synthesis
15. ⦁ Microbes are also used for the commercial and industrial
production of certain organic acids.
These compounds can be produced directly
from glucose (e.g. gluconic acid) or formed as end
products from pyruvate or ethanol.
Examples of acids producing microorganisms are
Aspergillus Niger (a fungus) of Citric
acid, Acetobacter acute (a bacterium) of Acetic Acid,
Lactobacillus (a bacterium) of lactic acid and many
others.
18. ⦁ Amino acids such as Lysine and Glutamic acid are
used in the food industry as nutritional supplements in
bread products and as flavor enhancing compounds
such as Monosodium Glutamate (MSG).
Amino acids are generally synthesized as primary
metabolites by microbes.
However, when the rate and amount of synthesis of
some amino acids exceed the
cell’s need for protein synthesis, then cell excrete
them into the surrounding medium.
21. ⦁ Many microbes synthesize and excrete large quantities
of enzymesinto the surrounding medium.
⦁ Using this feature of these tiny organisms, many
enzymes have been produced commercially.
⦁ These include Amylase, Cellulase, Protease, Lipase,
Pectinase, Streptokinase, and many others.
⦁ Enzymes are extensively used in food
processing and preservation, washing
powders, leather industry, paper industry and in
scientific research.
25. ⦁ Vitamins are some organic compounds which are
capable of performing many life-sustaining
functions inside our body.
These compounds cannot be
synthesized by humans, and therefore they have to be
supplied in small amounts in the diet.
⦁ Microbes are capable of synthesizing the vitamins and
hence they can be successfully used for the commercial
production of many of the vitamins
e.g. thiamine, riboflavin, pyridoxine, folic acid,
pantothenic acid, biotin, vitamin b12, ascorbic
acid, beta-carotene (pro-vitaminA), ergosterol
(provitamin D)
26. ⦁ Vitamin B12 produced by Propionibacterium
freudenreichii, Pseudomonas denitrificans, Bacillus
megaterium and Streptomyces olivaceus, p.shermanii and etc
⦁ Riboflavin produced by Ashbya gossypii and
Eremothecium ashbyii,clostridum buytilcum,mycocandida
riboflavina,candida flareri and etc,.
⦁ β– Carotene is a pro vitamin produced by Blakeslea
trispora, Phycomyces blakesleeanus and Choanephora
cucurbitarum.
⦁ Blakeslea trispora commenly used for high yield production.
27.
28. ⦁ Organic solvents such as ethanol, acetone, butanol,
and glycerolare some very important chemicals that
are widely used in petrochemical industries.
⦁ These chemicals can be commercially produced by
using microbes and low-cost raw materials
⦁ (e.g. wood, cellulose, starch).
⦁ Yeast (Saccharomyces cerevisiae) is used for
commercial production of ethanol.
31. ⦁ Single Cell Protein (SCP) can serve as an alternate
source of energy when a larger portion of the world is
suffering from hunger and malnutrition.
⦁ Single cell proteins are microbial cells that are rich
in protein content and can be used as protein
supplements for humans and animals.
⦁ Microbes like Spirulina can be grown easily on materials
like waste water from potato processing plants
(containing starch), straw, molasses, animal
manure, and even sewage, to produce large quantities and
can serve as food rich in protein, minerals, fats,
carbohydrate, and vitamins.
32.
33.
34. ⦁ These are a very important group of chemicals, which
are used as anti-inflammatory drugs, and as
hormones such as estrogens and progesterone, which
are used in oral contraceptives.
⦁ Steroids are widely distributed in animals, plants, and
fungi like yeasts.
⦁ But, producing steroids from animal sources or
chemically synthesizing them is difficult, but
microorganisms can synthesize steroids from sterols
or from related, easily obtained compounds.
⦁ Mostly mycobacterium sp are usd frequently.
36. ⦁ Many pharmaceutical drugs are also produced
by microbes
e.g. Cyclosporin A, that is used as an
immunosuppressive agent in organ-transplant
patients, is produced by the fungus Trichoderma
polysporum.
⦁ immunosuppressant cyclosporin A.Statins produced
by the yeast Monascus purpureus have been
commercialized as blood-cholesterol lowering agents.
It acts by competitively inhibiting the enzyme
responsible for the synthesis of cholesterol.
37.
38. Microbes are used in dairy industry to make dairy
product such as curd, yogurt,cheese,kefir , kumies,bread
and various types of milk product.
Saccharomyces cerevisiae,
Streptococcus sp,
penicillium roqueforti,
p.camemberti,
streptococcus thermophilus,
lactobacillus bulgaricus,
Lactobacillus sp,candida sp.
39. ❖Bacterial Transformation
•
• The ability of bacteria to
take in DNA from their
surrounding environment
Bacteria must be made
competent to take up
DNA
Microorganisms as Tools
40. Yeast are Important Too!
❖Single celled eukaryote
❖Kingdom: Fungi
❖Over 1.5 million species
❖Source of antibiotics, blood cholesterol lowering
drugs
❖Able to do post translational modifications
❖Grow anaerobic or aerobic
❖Examples: Pichia pastoris (grows to a higher
density than most laboratory strains), has a no.
of strong promoters, can be used in batch
processes
43. TYPES OF VECTORS
1) Cloning Vectors
Propagation or cloning of DNA insert inside a suitable host
cells. Examples: Plasmids, Phage or Virus
Obtaining millions of copies.
Uses :- Genomic library.
Preparing probes.
Genetic Engineering Experiments.
Selection of cloning vector depends on :-
(a)Objective of cloning experiment
(b)Ease of working.
(c) Knowledge existing about the vector.
(d)Suitability.
(e) Reliability.
44. 2) Expression Vectors
Express the DNA insert producing specific protein.
They have prokaryotic promoter.
Ribosome binding site.
Origin of replication.
Antibiotic resistance gene.
Expression vectors with strong promoters.
Inducible Expression Vectors.
Eukaryotic expression vectors.
45. VECTORS
• Plasmid
• Bacteriophages
• Cosmid
• Yeast Cloning Vectors
• Ti & Ri Plasmids
TARGET HOST CELL
Bacteria, Streptomyces Bacteria
Bacteria
Yeasts
Transformation of cloned
gene in higher plants.
46. AGENTS USED AS VECTORS
PLASMIDS
BACTERIOPHAGES
COSMID
ARTIFICIAL CHROMOSOME VECTORS
In 1973, Cohen described first successful
construction of recombinant vector.
Plasmid PSC101 - Ecoli
47. PLASMID
Extra chromosomal DNA molecules.
Self replicating.
Double stranded.
Short sequence of DNA.
Circular DNA molecules.
Found in prokaryotes.
CHARACTERISTICS
a. Minimum amount of DNA.
b. Two suitable markers for identification .
c. Single restriction site.
d. More restriction enzyme.
e. Size range 1kg – 200kg.
f. Relaxed replication control.
g. Restriction endonuclease enzyme.
48. THREE TYPES OF PLASMID
1. Fertility plasmids:- can perform conjugation.
2. Resistance plasmids:- contain genes that build a
resistance against antibiotics or poisons.
3. Col plasmids:- contain genes that code for proteins
that can kill bacteria.
49. BACTERIOPHAGE VECTORS
Cloning Vectors.
It infects bacteria.
Commonly used Ecoli phages :-
λ phage
M13 Phage
Lambda phage vector
Genome size is 48,502 bp.
High transformation efficiency.
1000 times more efficient than the plasmid vector.
Origin of replication.
Genome linear in head.
Single- stranded protruding cohesive ends of 12 bases.
Cos site – site of cleavage of phage DNA.
50. COSMIDS
Combine parts of the lambda chromosome with parts of
plasmids.
Contain the cos sites of λ and plasmid origin of
replication.
Behave both as plasmids and as phages.
Cosmids can carry up to 50 kb of inserted DNA.
Structure of Cosmid
Origin of replication (ori).
Restriction sites for cleavage and insertion of foreign
DNA.
Selectable marker from plasmid.
A cos site - a sequence yield cohesive end (12 bases).
Ampicillin resistance gene (amp).
51. ARTIFICIAL CHROMOSOME
Linear or Circular.
1 0r 2 copies per cell.
Different types –
Bacterial Artificial Chromosome(BAC)
Yeast Artificial Chromosome (YAC)
P1 derived artificial chromosome (PAC)
Mammalian Artificial Chromosome (MAC)
Human Artificial Chromosome. (HAC)
YAC – Cloning in yeast
BAC & PAC – Bacteria
MAC & HAC – Mammalian & Human cells.
52. YEAST ARTIFICIAL CHROMOSOME
Linear Plasmid Vector.
Clone large DNA segment ( 100 – 1400kb).
Occurring two forms:-
Circular – grows in bacteria. Linear –
multiplies in yeast cells.
pYAC3 - first YAC developed.
It contains :-
ARS sequence – replication
CEN4 sequence – centromeric function TRP1 &
URA3 – 2 selectable markers
Use – mapping complex eukaryotic chromosome .
53. 53
YEAST ARTIFICIAL CHROMOSOMES
•A yeast artificial chromosome (YAC) is a vector used
to clone DNA fragments larger than 100 kb and up to 3000
kb
•YACs are useful for the physical mapping of complex
genomes and cloning of large genes
•A YAC is an artificially constructed chromosome and
contains the telomeric, centromeric, and replication origin
sequences named autonomous replicating sequence
(ARS) needed for replication in yeast cells
54. Why To Use Yeast Vectors?
• Yeast are eukaryotes, contain complex internal
cell structures
• Post-translational modifications
• easy to manipulate as E. coli
• absence of pyrogenic toxins
• cell growth is faster, easier
• less expensive than other eukaryotic cells
• Higher expression levels
• a well-defined genetic system
55. • highly versatile DNA transformation system
• yeast-specific origin of replication (ORI) and a means of
selection in yeast cells, in addition to the bacterial ORI and
antibiotic selection markers
• All contain unique target sites for a number of restriction
endonucleases
• can all replicate in E. coli, often at high copy number
• The four most widely used markers are His3, Leu2, Trp1, and
Ura3.
56. Introducing DNA into fungi
Use of spheroplasts (i.e. wall-less cells) and was
first developed for S. cerevisiae (Hinnen et al., 1978)
Step 1: cell wall is removed enzymically & resulting
spheroplasts are fused with ethylene glycol in the
presence of DNA and CaCl2
Step 2: spheroplasts are then allowed to generate new cell
walls in a stabilizing medium containing 3% agar
Step 3: Electroporation provides a simpler & more convenient
Step 4: DNA can also be introduced into yeasts & filamentous
fungi by conjugation
59. Yeast Integrating plasmids (YIp):
• lack an ORI and must be integrated directly
into the host chromosome via homologous
recombination for efficient multiplication
60. Yeast Episomal plasmids (YEp):
• Beggs (1978)
• recombining an E. coli cloning vector with the naturally
occurring yeast 2 μm plasmid
• 6.3 kb in size
• High copy number of 50–100 per cell
61.
62. Yeast Replicating plasmids (YRp):
• contain anAutonomously Replicating Sequence (ARS)
derived from the yeast chromosome.
• can replicate independently of the yeast chromosome
• unstable and may be lost during budding
63. Yeast centromere plasmids
• carry an ars, most of the recombinants were unstable in yeast
• plasmid-borne centromere sequences have the same distinctive
chromatin structure that occurs in the centromere region of yeast
chromosomes (Bloom & Carbon 1982)
• Three characteristics
• Mitotically stable in the absence of selective pressure
• Segregate during meiosis in a Mendelian manner
• found at low copy number in the host cell
64. Yeast artificial chromosomes
• All autonomous plasmid vectors described above are
maintained in yeast as circular DNA molecules, even the YCp
vectors, which possess yeast centromeres
• Thus, none of these vectors resembles the normal yeast
chromosomes, which have a linear structure
65. • DNAinsert size- 500 kbp
• The ends of all yeast chromosomes, like those of all
other linear eukaryotic chromosomes, have unique
structures that are called telomeres
66. Genes for YAC selection in yeast
• URA3, a gene involved in uracil biosynthesis
• TRP1, a gene involved in tryptophan biosynthesis
• Auxotrophic method of selection
Bacterial replication origin & a bacterial selectable marker
• To propagate the YAC vector in bacterial cells, prior to insertion of
genomic
• DNA, YAC vectors usually contain the ColE1 ori and the ampicillin
• resistance gene for growth and analysis in E. coli.
67.
68. • Three main species of yeast
Saccharomyces cerevisiae
Pichia pastoris
Schizosaccharomyces pombe
69. Saccharomyces cerevisiae
• Baker’s yeast
• Single-celled eukaryote
• Grows rapidly (a doubling time of approximately 90 min)
• Simple, defined media
• Many, but not all, of the post-translation modifications
70. • Strong constitutive promoters
Promoters of phosphoglycerate kinase (PGK), glyceraldehyde-
3-phosphate dehydrogenase (GPD) and alcohol
dehydrogenase (ADH1)
• Suffer similar problems as constitutive E. coli expression
systems
• Inducible production
The GAL System
The CUP1 System
71. The GAL System
• Galactose is converted to glucose-6-phosphate by enzymes of
Leloir pathway
• Leloir pathway structural genes (GAL genes) are expressed at
a high level (0.5–1% of total cellular Mrna), galactose as sole
carbon source
• GAL genes promoter- sites for the transcriptional activator
Gal4p
72. Yeast autonomously replicating sequence (ARS1)
• Struhl et al. (1979)
• carry sequences that enable E. coli vectors to replicate in yeast cells
• sequences are known as ars autonomously replicating sequences
• An ars is quite different from a centromere
• ars acts as an origin of replication , Centromer is involved in chromosome
segregation
Yeast telomeres (TEL)
• Telomeres are the specific sequences (5-TGTGGGTGTGGTG-3), present at ends of
chromosomes in multiple copies, necessary for replication & chromosome
maintenance.
73. • Glucose as carbon source - less Gal4p
• Raffinose as carbon source - Gal4p is produced, binds to GAL
structure gene promoters, but a repressor, Gal80p, inhibits its
activity
• Gal80p binds to Gal4p, mask its activation domain
• Unable to recruit the transcriptional machinery
• Galactose- inhibitory effect of Gal80p
74.
75. The CUP1 System
• Copper ions (Cu2+ and Cu+) are essential, toxic at high levels
• S. cerevisiae, copper homeostasis - uptake, distribution and
detoxification mechanisms
• At high concentrations, detoxification is mediated by a copper ion
sensing metalloregulatory transcription factor-Ace1p
• Upon interaction with copper,Ace1p binds DNAupstream of the
CUP1 gene, encodes a metallothionein protein & induces its
transcription
76. • Expression vectors harbouring CUP1 promoter- induce target
gene expression in a copper-dependent fashion
• Can be grown on rich carbon sources, such as glucose, to high
cell density & protein production is initiated by the addition of
copper sulphate (0.5 mM final concentration)
• Drawback-
presence of copper ions in yeast growth media, and indeed in
water supplies
77. BACTERIAL ARTIFICIAL CHROMOSOME
BAC Vector – PBAC108L.
Cloning of large regions of eukaryotic genome.
Origin of replication from bacterium Ecoli F -factor.
BAC vectors are pBACe3.6, pBeloBAC11.
Used in analysis of genomes.
Host for BAC is mutant strain.
Streptomyces isolates have yielded the majority of human, animal,
and agricultural antibiotics, as well as a number of fundamental
chemotherapy medicines. Streptomyces is the largest antibiotic-
producing genus of actinobacteria, producing chemotherapy,
antibacterial, antifungal, antiparasitic drugs
and immunosuppressants.Streptomyces isolates are typically
initiated with the aerial hyphal formation from the mycelium
78. Why To Use Streptomyces Vectors?
One of the most effective strategies to improve the
production of a particular protein or secondary metabolite
in Streptomyces is to over-express the gene, the synthesis gene
cluster, or the positive regulatory gene for this gene/gene cluster.
The most interesting property of Streptomyces is the
ability to produce bioactive secondary metabolites such as
antifungals, antivirals, antitumoral, anti-hypertensives, and mainly
antibiotics and immunosuppressives.
79. What is the contribution of Streptomyces to the pharmaceutical
industry?
In the last 80 years, Streptomyces has made a
massive contribution to the field of medicine, not only through
antibacterial antibiotics, but also through antifungal,
antiparasitic and anticancer compounds.
What is the function of Streptomyces?
Streptomyces is the largest antibiotic-producing genus,
producing antibacterial, antifungal, and antiparasitic drugs, and
also a wide range of other bioactive compounds, such as
immunosuppressants
80. What antibiotic does Streptomyces griseus produce?
Griseus is of both historical and ecological relevance. This
species produces streptomycin, a broad‐spectrum
aminoglycoside antibiotic, that inhibits translation and was the
first clinically deployed antibiotic from Streptomyces to be
discovered.
What does Streptomyces cause?
Streptomyces spp., usually saprophytic to humans, can
cause local cutaneous fistulized nodules known as
actinomycetoma or mycetoma. Severe invasive infections have
seldom been reported, but most cases reported have occurred in
immunocompromised patients
81. How many types of Streptomyces are there?
500 species
Streptomyces, genus of filamentous bacteria of the family
Streptomycetaceae (order Actinomycetales) that includes more
than 500 species occurring in soil and water.
How do Streptomyces grow?
Streptomyces life cycle
This is vegetative mycelia which grows into the media
following germ tube formation after spore germination. ... The
final stage of development sees a transition from aerial hyphae to
highly hydrophobic spore chains, often characterised by the
production of a spore pigment
82. Which genus of actinomycetes is most commonly used in
antibiotic production?
Streptomyces
Today, 80% of the antibiotics are sourced from
the genus Streptomyces, actinomycetes being the
most important
Is Streptomyces good or bad?
The great importance given to Streptomyces is partly
because these are among the most numerous and most versatile
soil microorganisms, given their large metabolite production rate
and their biotransformation processes, their capability of
degrading lignocellulose and chitin, and their fundamental role
in biological
83.
84. Members of the genus Streptomyces are the source for
numerous antibacterial pharmaceutical agents; among the
most important of these are:
Chloramphenicol (from S. venezuelae)
Daptomycin (from S. roseosporus)
Fosfomycin (from S. fradiae)
Lincomycin (from S. lincolnensis)
Neomycin (from S. fradiae)
Nourseothricin.
85. How many antibiotics are produced by the genus Streptomyces?
The model estimated the total number of antimicrobial
compounds that this genus is capable of producing to be of the
order of a 100,000 - a tiny fraction of which has been unearthed so
far.
Why are most antibiotics from Streptomyces?
The production of most antibiotics is species specific, and
these secondary metabolites are important so
the Streptomyces spp. can compete with other microorganisms
that may come in contact, or even within the same genus.
Which is the first antibiotic isolated from species Streptomyces?
Actinomycin
Actinomycin was the first antibiotic
isolated from Streptomyces in 1940, followed by streptomycin
three years later
86. Anticancer medicines
Doxorubicin intercalating DNA. Streptomyces, yielded the
medicines doxorubicin (Doxil), daunorubicin (DaunoXome),
and streptozotocin (Zanosar). Doxorubicin is the precursor
to valrubicin (Valstar), myocet, and pirarubicin. Daunorubicin is
the precursor to idarubicin (Idamycin), epirubicin (Ellence),
and zorubicin.
Streptomyces is the original source
of dactinomycin (Cosmegen), bleomycin (Blenoxane), pingyangm
ycin (Bleomycin A5), mitomycin
C (Mutamycin), rebeccamycin, staurosporine (precursor
to stauprimide and midostaurin), neothramycin, aclarubicin,
tomaymycin, sibiromycin, and mazethramycin.
87. Derivatives of Streptomycetes isolate migrastatin,
including isomigrastatin, dorrigocin A & B, and the synthetic
derivative macroketone, are being researched for anticancer
activity.
88. Antibiotics
Most clinical antibiotics were found during the "golden age of antibiotics"
(1940s–1960s). Actinomycin was the first antibiotic isolated from Streptomyces in
1940, followed by streptomycin three years later.
Antibiotics from Streptomyces isolates (including various aminoglycosides)
would go on to comprise over two-thirds of all marketed antibiotics.
Streptomyces-derived antibiotics include:
Chloramphenicol (Streptomyces venezuelae)
Daptomycin (Streptomyces roseosporus)
Fosfomycin (Streptomyces fradiae)
Lincomycin (Streptomyces lincolnensis)
Neomycin (Streptomyces fradiae)
Platensimycin (Streptomyces platensis)
Puromycin (Streptomyces alboniger)
Streptomycin (Streptomyces griseus)
Tetracycline (Streptomyces rimosus and Streptomyces aureofaciens)
89.
90. ⚫ Streptomyces species -source of medically useful
compounds
⚫ Many biochemical mysteries need tobe elucidated
⚫ Search for novel secondary metabolites
and their biosyntheticgene clusters
⚫ Relevant genomic databases on Streptomyces species constructed.
⚫ Systems biological approaches useful in the discoveryandengineering studiesof
Streptomyces species
91. • Factors affecting antibiotic production:
1. Medium Composition:
• Carbon source
• Nitrogen source
• Inorganic phosphates
• Inorganic salts
• Trace metals
• Precursors
• Inhibitors
• Inducers
2. Fermentation Conditions:
• pH
• Temperature
• Oxygen
• How can determine the target of inhibitor molecule which may inhibit one of
the biological pathways?
There are many different pathwaies can be applied such as reporter essay by using •
the reporter strains