Selection and

Screening of Recombinants

D. K. Parihar
Asst. Professor
DEPARTMENT OF BIOTECHNOLOGY
Guru Ghasidas Vishwavidyalaya, Bilaspur-495009 (C.G.)
SCREENING OF RECOMBINANTS

A genetic screen or mutagenesis screen is

an experimental technique used to identify and
select for individuals who possess a phenotype
of interest in a mutagenised population.
Selection and Screening of Recombinants

Direct selection for the desired gene

 Cloning experiment is designed in such a way that the
only clones that are obtained are clones of the required
gene. Almost invariably, selection occurs at the plating-
out stage. (all ready seen in ‘Vectors’)
Identification of the clone from a gene library
 Involves an initial “shotgun” cloning experiment, to
produce a clone library representing all or most of the
genes present in the cell, followed by analysis of the
individual clones to identify the correct one.
Direct Selection of Transformants

Principle:
 Only cells transformed with the desired gene will form
colonies

Examples:
 Antibiotic resistance marker

 Auxotrophic marker after transformation of the

appropriate strain
 BLUE-WHITE SCREENING
 This uses of chromogenic substrate to
detect a particular enzymatic activity.
 The colourless compound X-gal (5-
bromo-4-chloro-3-indolyl-β-D-
galactoside) as substrate for β-
galactosidase.
 The enzyme β-galactosidase a
tetrameric protein is the product of
lacZ gene of the lac operon.
 In this system, host contains lacZ
gene without the initial region (N-
terminal) where as vector contains α-
peptide to complement the defect to
form active enzyme.
 As a result, if a vector containing α-
peptide will be transformed into the
host containing remaining lacZ, the
two fragment will reconstitute to
form active enzyme
 In addition, the α-peptide region in
vector contains MCS and insertion
of gene fragment gives inactive β-
galactosidase.
 The enzyme β-galactosidase
oxidizes x-gal to form 5-bromo-4-
chloro-indoxyl and galactose.
 The indoxyl derivative is oxidized in
air to give a blue colored
dibromodichloro derivative.
 Hence, blue colored colonies
indicate the absence of insert where
as colorless colonies indicate
presence of an insert.
Chemical conversion of X-gal
 INSERTIONAL INACTIVATION
1) Insertional Inactivation of antibiotic resistance gene-

 Bacterial plasmid PBR322 has two antibiotic resistance

gene, Apr and Tc.
 If a gene fragment will be cloned in ScaI, it will disrupt
the Apr gene. As a result, the clone will be ampicillin
sensitive and Tcr. where as the original plasmid will be
Apr and Tcr.
 INSERTIONAL INACTIVATION
2) Insertional inactivation of cI gene-
 During an infection cycle, virus undergoes a lytic and lysogenic stages. The cI gene
encodes for cI repressor which is responsible for the formation of lysogens.
 In the presence of functional cI, the plaques contains unlysed host cells and has a
turbid appearance where as in the absence it will clear.
 This feature can be use to screen the clone to detect functional cI (absence of clone)
or absence of cI (presence of insert).
ANTIBIOTIC SENSITIVITY
 In this approach, a circular plasmid containing antibiotic resistance
gene can be able to replicate into the host cell plated on a antibiotic
containing media.
 In the cloning of a fragment into this plasmid, the plasmid is cut with
restriction enzymes and a fragment in ligated to give circular plasmid
with insert.
 Cut plasmid and circularized clone into the host and plated onto the
antibiotic containing solid media.
 Only circularised clone will give colonies where as cut plasmid will
not grow as it has lost antibiotic resistance gene.
AUXOTROPHIC YEAST STRAIN

 Yeast vector has 4 different gene His3, Leu2, Trp1 and

Ura3 as selectable marker.
 Yeast host with a mutation in these gene are available
and can be use with the yeast vector to screen the
recombinant clone.
 Ura3 and Lys2 marker offer both positive and negative
selection.
Positive selection-

 In the positive selection, host strain doesn't grow on

the media lacking the functional gene but the host
transformed with the recombinant clone can be able to
supply the gene product required to grow in the media.
 In yeast, an auxotrophic mutant that has non-functional
leu2 gene is used as a host. Such a mutant is able to
survive only if leucine is supplied in the growth medium.
 However, transformants are able to grow on a minimal
medium (contains no added leucine) due to presence of
leu2 gene in the vector
Negative selection-
 In the negative selection, a chemical compound is added to the
media which will be converted to the cyto-toxic agent in the
presence of gene product.
 Ura3 codes for orotine-5'-monophosphate (OMP) decarboxylase
and an active enzyme process the 5-fluoro-orotic acid to the toxic
fluorodeoxyuridine. Generation of this cyto-toxic agent kills the cells
carrying functional Ura3 gene product.
REPORTER GENE ASSAY
 In the reporter gene assay system, a chimeric construct
is produced with an enzyme gene is cloned in front of the
promoter of gene of interest.
 The general reporter gene construct contains a
eukaryotic promoter and a enzyme for easy read out.
Luciferase reporter gene system-
 Luciferase is an enzyme present in the abdomen of firefly photinus pyralis.
The enzyme utilizes D-luciferin as a substrate to form oxyluciferin.
 In the presence of ATP, Mg2+, luciferin is converted into the luciferyl
adenylate involving pyrophosphate cleavage and transfer of AMP to
luciferin.
 Luciferin adenylate undergoes oxidative decarboxylation to oxyluciferin with
simultaneous emission of light.
Chimeric Construct with green fluorescent
protein (GFP) -
 In the live cell, green fluorescent protein is a good choice as reporter
gene to screen cells containing recombinant protein fluorescently
tagged with the GFP at their c-terminus.
 The cell receiving recombinant DNA will give green fluorescence
and it can be visualized with an inverted fluorescence microscope
and it can be analyzed in flow cytometer to separate the GFP
containing cells from the untransfected cells.
 Flow cytometer analysis the cell based on its shape, size and
fluorescence level.
 A non-fluorescent cell is giving separate peak as compare to the
fluorescently labeled cells and with the help of flow cytometer, both
of these peaks can be collected in separate tubes.
 Besides, GFP, red fluorescent protein, yellow fluorescent protein,
cyan fluorescent protein are also popular to use to label the protein.
Clone identification from a gene library

Methods are based on

 Nucleic acids

 Translation products
 Nucleic acid hybridization

 (a) An unstable hybrid

molecule formed between
two non-homologous DNA
strands.
 (b) A stable hybrid formed
between two complementary
strands.
 (c) A DNA–RNA hybridmay
be formed between a gene
and its transcript.
Colony/plaque hybridization
Labeling with a radioactive marker

 Labeled by incorporating nucleotides that carry a

radioactive isotope of hosphorus, 32P

Structure of α-32P-deoxyadenosine triphosphate ([α-32P]dATP).

Nick translation
 Most purified samples of DNA contain some nicked
molecules
 DNA polymerase I is able to attach to the DNA and
catalyze a strand replacement reaction
 Supply of radioactively labeled nucleotide get incorporated
 May cause DNA damage
End filling
 Gentler method than nick translation and rarely causes
breakage of DNA
 Used to label DNA molecules that have sticky ends.
 Klenow fragment “fills in” a sticky end by synthesizing
the complementary strand.
 Filling reaction is carried out in the presence of labeled
nucleotides, the DNA becomes labeled.
Random priming
 The denatured DNA is mixed with a set of hexameric
oligonucleotides of random sequence.
 By chance, random hexamers will contain a few molecules
that will base pair with the probe and prime new DNA
synthesis.
Random priming
 The Klenow fragment only fills in the gaps between
adjacent primers
 Labeled nucleotides are incorporated into the new DNA
that is synthesized.
 Non-radioactive labeling

 dUTP nucleotides
modified by reaction
with biotin (organic
Mol.).
 Biotin has a high
affinity for a protein
called avidin.
 probe can be
determined by
washing with avidin
coupled to a
fluorescent marker
Non-radioactive labeling

 The probe DNA is

complexed with the
enzyme horseradish
peroxidase
 Detected through the
enzyme’s ability to
degrade luminol with
the emission of
chemiluminescence
 The signal can be
recorded on normal
photographic film
Identification methods based on the
translation product
Immunological screening
 (a) Antibodies in the blood
stream bind to foreign
molecules and help degrade
them.
 (b) Purified antibodies can
be obtained from a small
volume of blood taken from a
rabbit injected with the
foreign protein.
Identification methods based on the
translation product

Immunological screening
 Using a purified antibody to
detect protein in recombinant
colonies.
 Instead of labelled protein A,
the antibody itself can be
labelled, or alternatively a
second labelled antibody
which binds specifically to
the primary antibody can be
used.
Labeling of antibodies

1. Radioactively by 125 I
2. By a fluorescence marker
3. By a secondary antibody (goat, horse, etc.) with a covalently linked
enzyme (alkaline phosphatase, horseradish peroxidase)