screening method in recombinant clone

1. JIWAJI UNIVERSITY,GWALIOR
TOPIC- SCREENING METHOD FOR RECOMBINANT CLONES
SUBMITTED TO- SUBMITTED BY-
head of department Manisha Singhai
SOS CENTRE FOR GENOMICS MSc M.H.G
3RD SEM
DATE -18-12-2020
1

2. Contents
 Introduction
 Screening of recombinant clone
 Methods for screening:
1.Blue- white screening
2.Insertional inactivation of cI gene
3.Insertional inactivation of lac Z gene
4.Antibiotic resistant gene
5.Antibiotic sensitivity
6.Complimentation of mutation
 Reference
2

3. Introduction
 After the introduction of recombinant
DNA into the host cells, it is essential to
identify those cells which receiced rDNA
molecule is known as screening or
selection.
 The vector or foreign DNA present in
the recombinant cells expresses certain
characters or traits,while non-
recombinant do not express traits.
3

4. 4

5. 1. Blue white screening
 The use of chromogenic substrate to detect a particular enzymatic activity is the basis to screen the
desired clone.
 The colourless compound X- gal is 5 bromo- 4 chloro-3indolyl beta-D galactosidase.
 The enzyme beta galactosidase is the product of lac Z gene of the lac operon.
 Beta galactosidase is a tetrameric protein and initial N terminal region of the protein is important for
activity of the protein.
 In this system host contain lac Z gene without the initial region whereas vector contain alpha peptide
to compliment the defect to form active enzyme. As a result if a vector containing alpha peptide will
be transformed into the host containing remaining lac Z the two fragment will reconstitute to form
active enzyme.
 The alpha peptide region in vector contains multiple cloning site and as a result of insertion of gene
fragment consequently alpha peptide will not be synthesize to give fully active beta galactosidase.
 The enzyme beta galactosidase oxidizes X gal to form 5 bromo -4 chloro – indoxyl and galactose. The
indoxyl derivative is oxidized in air to give a blue colour dibromo-dichloro derivative.
 Hence blue coloured colonies indicate the absence of insert whereas colourless colonies indicates
presence of an insert.
5

6. 6

7. 2. Insertional inactivation of cI gene
 During an infection cycle virus undergoes lytic
and lysogenic stages.
 The lytic phase is responsible for lysis of host
to release the virus particle whereas a lysogenic
stage allow the replication of virus without lysis
of host.
 The cI gene encodes for cI repressor and which
is responsible for the formation of lysogens.
 In the presence of functional cI the plaques
unlysed host cells and turbid appearance
whereas in the absence it will be clear.
 This feature can be use to screen the clone to
detect functional cI (absence of clone)
or absence of cI (presence of insert).
7

8. 3.Insertional inactivation of lac Z gene
 Lac Z is a part of lac operon and responsible
for synthesis of beta galactosidase.
 X gal system can be used to detect the
insertional inactivation of lac Z gene to
screen the cloned fragment .
 If the gene is inserted into the lac Z,
the clone will be able to produce a
functional beta galactosidase .
 Hence blue coloured colonies indicate
the presence of an active enzyme or
absence of insert whereas colourless
colonies indicate presence of an insert.
8

9. 4.Antibiotic resistance gene
9

10. 5.Antibiotic sensitivity
 Vector carries a functional selection marker such
as antibiotic resistance gene to be use to select
the clone.
 The antibiotic resistance gene product has multiple
mechanism to provide resistance in host cell.
 A circular plasmid containing antibiotic resistance
can be able to replicate into the host cell plated
on a antibiotic containing media.
 In the cloning of a fragment into this plasmid
is cut with restriction enzyme and a fragment is
ligated to give circular plasmid to insert.
 The transformation of both DNA speices cut
plasmid and circularized clone into the host and
plated onto the antibiotic containing solid media.
 Only circularized clone will give colonies whereas
cut plasmid will not grow as it has lost antibiotic
resistance gene.
10

11. 6. Complimentation of mutation
 In this approach, a mutant host strain can be used to screen the plasmid containing the missing gene and
the transformant will grow only if the gene product from clone will complement the function.
 In general genes taking part in metabolic pathway or biosynthetic pathway are routinely been used for this
purpose.
 3 important requirements:
1. Host strain deficient in a particular gene, if the gene belongs to the biosynthetic pathway, the mutant host
in this case are called auxotroph as host dependent on the gene product or final product of the
biosynthetic pathway as a supplement in media for growth.
2. A defined media with missing nutrient.
3. A vector containing the gene to supply the gene product to compliment.
 Yeast has 4 different gene :His3, Leu2, Trp1, Ura3 as selectable marker.
11

12.  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 media.
 Negative selection- In the negative selection the chemical compound is added to the media which
will be converted to the cytotoxic agent in the presence of gene product, and as a result it doesn’t
allow the growth of wild type cells, but host strain transformed with the recombinant clone has non
functional gene product and grows in the presence of the compound in the media.
 Ura3 codes for oritine 5 monophosphate (omp) decarboxylase and an active enzyme process the 5
fluro oritic acid to the toxic florodeoxyuridine. Generation of this cytotoxic agent kills the cells
carrying functional Ura3 gene product.
12

13. Reference
 AUTHOR: T.A. BROWN: 2016 PUBLISHED: SEVENTH EDITION: GENE CLONING & DNA ANALYSIS:
PAGE NO-78-88.
 AUTHOR: PRANAV KUMAR & USHA MINA: 2019 PUBLISHED: SEVENTH EDITION: PATHFINDER:
PAGE NO-284-287.
13

14. 14