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Unit III Lecture 2
B. Tech. (Biotechnology) III Year
V th Semester
EBT-501, Genetic Engineering
Unit III
• Gene library: Construction cDNA library and genomic
library, Screening of gene libraries – screening by DNA
hybridization, immunological assay and protein activity
• Marker genes: Selectable markers and Screenable
markers, nonantibiotic markers,
• Gene expression in prokaryotes: Tissue specific
promoter, wound inducible promoters, Strong and
regulatable promoters; Increasing protein production;
• Fusion proteins; Translation expression vectors; DNA
integration into bacterial genome; Increasing secretions;
Metabolic load,
• Recombinant protein production in yeast:
Saccharomyces cerevisiae expression systems
• Mammalian cell expression vectors: Selectable markers;
Selectable and screenable
markers
A marker gene is used in molecular biology to
determine if a piece of DNA has been successfully
inserted into the host organism.
There are two types of marker genes:
• Selectable markers
• Antibiotic marker
• Non-Antibiotic marker
• Endogenous marker
• Nutritional markers
• Screenable markers.
• Flourescence marker e.g. GFP, RFP
• Colorimetric assay e.g. GUS, Lac Z
• Selectable markers are required for the
maintenance of the plasmid in the cell.
• Under the selective conditions only, the cells
that contain plasmids with the appropriate
selectable marker are able to survive.
• Commonly, genes that confer resistance to
various antibiotics (antibotics markers) are
used as selective markers in cloning vectors.
• But at times nutritional markers or
endogenous markers are used as selectable
markers.
The drawbacks of selectable markers
• loss of selective pressure as a result of
antibiotics degradation and inactivation.
• contamination of the product or biomass
by antibiotics, which may be unacceptable
from medical or regulatory considerations.
Selectable markers
A selectable marker will protect the organism
from a selective agent that would normally kill it
or prevent its growth. In most applications, only
one in a several million or billion cells will take
up DNA. Rather than checking every single cell,
scientists use a selective agent to kill all cells
that do not contain the foreign DNA, leaving only
the desired ones.
• Antibiotics are the most common selective
agents. In bacteria, antibiotics are used almost
exclusively. In plants, antibiotics that kill the
chloroplast are oftenused as well, although
tolerance to salts and growth-inhibiting
hormones is becoming more popular.
Screenable markers
• Reporter genes are also known as screenable marker
genes.
• These differ from selectable marker genes in that they
do not confer a property that allows transformed cells to
survive under selective conditions.
•
Instead, screenable markers encode a product that
can be detected using a simple and inexpensive
assay.
• When controlled by a strong constitutive promoter,
reporter genes are often used as markers to confirm
transient or stable transformation, since only cells
containing the reporter-gene construct can express the
corresponding protein
• Importantly, the assays used to detect reporter-gene
activity are quantitative, so they can also be used to
measure transformation efficiency.
• If attached to a cloned promoter, reporter genes can
therefore be used to determine transcriptional activity in
different cell types and under different conditions.
•
Transient reporter assays have been widely used to
characterize and dissect the regulatory elements driving
eukaryotic genes, as shown in the example below.
• The use of reporters is advantageous, because it
circumvents the necessity to derive different assays for
individual genes and also allows the activities of
transgenes and homologous endogenous genes to be
distinguished in the same cell.
An example of in vitro promoter analysis using
chloramphenicol acetyltransferase
• The first reporter gene to be used in animal cells
was cat, derived from E. coli transposon Tn9.
• This gene encodes the enzyme
chloramphenicol acetyltransferase (CAT),
which confers resistance to the antibiotic
chloramphenicol by transferring acetyl groups
on to the chloramphenicol molecule from
acetyl-CoA.
Commonly use Screenable markers
• A marker for screening will make cells containing the
gene look different. There are three types of screening
commonly used:
• Green fluorescent protein (GFP) makes cells glow
green under UV light. A specialized microscope is
required to see individual cells. Yellow and red versions
are also available, so scientists can look at multiple
genes at once. It is commonly used to measure gene
expression.
• GUS assay (using β-glucuronidase) is an excellent
method for detecting a single cell by staining it blue
without using any complicated equipment. The drawback
is that the cells are killed in the process. It is particularly
common in plant science.
• Blue/white screening is used in bacteria. The lacZ gene
makes cells turn blue in special media (e.g. X-gal). A
colony of cells with the gene can be seen with the naked
eye.
Markers for Screening GUS: betaglucuronidase in plants
5-bromo-4-chloro-indolyl glucuronide (
-gluc):
4-methyl-umbelliferyl-beta-D-glucuronide (
Beta-glucuronidase
37C
+ ( -gluc)
+ (
-gluc)
The GUS
histochemical
staining assay
O2
+ + + gluc
Hydrolysis of the X-Gluc
substrate by the GUS enzy
Beta-glucuronidase
-gluc)
Dimerization of the Gluc
product by reaction with O2
37C
+
+ gluc
Fluorogenic
staining
Hydrolysis of the X-Gluc
substrate by the GUS Fluorescent of the released
enzyme
fluorescent product
Endogenous Markers in Animal
cells
Selectable markers allows differential multiplication of
only transfected animal cells
are utilized to select the transfected cells
E.g. thymidine kinase TK gene whnen used with TK- (TK minus)
cells
Screenable markers or Reporters or scorable markers
produces a phenotype which helps in detection the
expression or level of transcripts of trans gene or
recombinant gene
Are used to studying promoter/enhancer activity
E.g. CAT gene from E.coli transposone Tn9
Beta galactosidase gene from E.coli
Luciferase gene from bacteria or firefly (Photinus pyralis)
de novo and salvage nucleotide synthesis pathways
De novo purine nucleotide synthesis (shown on the right of
next slide) initially involves the formation of inosine
monophosphate (IMP) which is then converted into
either adenosine monophosphate (AMP) or, via xanthine
monophosphate (XMP), guanosine monophosphate
(GMP).
The de novo synthesis of IMP requires the enzyme dihydrofolate
reductase (DHFR), whose activity can be blocked by
aminopterin or methotrexate. In the presence of such inhibitors,
cell survival depends on nucleotide salvage, as shown on the
left.
Cells lacking one of the essential salvage enzymes, such
as HPRT or APRT, therefore cannot survive in the
presence of aminopterin or methotrexate unless they are
transformed with a functional copy of the corresponding
gene.
Thus, the genes encoding salvage-pathway enzymes can be used
as selectable markers. Note that the enzyme XGPTR, which
converts xanthine to XMP, is found only in bacterial cells and not
de novo and salvage nucleotide synthesis pathways
NPT II—Kanamycin (Km)
resistance
NPT II = neomycin phosphotransferase II
• Normally, plant cells are sensitive to Km.
• Km inhibits protein synthesis and protein
translocation
across membranes.
• Expression of the NPTII in plant cells results in
synthesis of NPTII enzyme
• The enzyme detoxifies Km by phosphorylation
ATP
Km
ADP
Km-PO4
NPTII enzyme
LacZ- a screenable marker
EcoR1
EcoR1
EcoR1
Interrupted
Lac Z gene
Lac gene
pUC18
pUC18
“Recombinant
Molecules”
Beta-galactosidase
NO Beta-galactosidase
Gal + X(Blue dye)
X-gal
White colonies
blue colonies
(colorless)
Allows for easy visual “screening” of bacterial
colonies that contain recombinant DNA molecules