SCREENING OF RECOMBINANTS - BLUE AND WHITE SCREENING (MCQS)

Introduction about genetic engineering
Steps in rDNA Technology
Screening of recombinants
Selection of recombinants
Blue and white screening
Alpha complementation
Beta galatosidase
X gal
Antibiotic resistance screening
Replica plate technique
Colony hybridization
Screening by Immunological assay
Immunological screening
Protein activity
Enzyme activity
MCQS RELATED TO SCREENING OF RECOMBINANTS

1. BHARATHIAR UNIVERSITY

srl) DEPARTMENT Of Mame BIAL ::::::::::

nn BIOTECHNOLOGY er

SUBMITTED TO: PRESENTED BY:
DR. V.S. GNANAMBAL SABARIDARAN. B
GUEST FACULTY I M.SC MICROBIOLOGY

2. Contents Runen

VVV

Vv

VVVWV

Introduction
Screening of recombinants œ—
Blue and white screening

a complementation

Antibiotic FARIAIANI screening y a
Colony hybridization

Immunological assay ——"
Screening by protein, enzyme activity
Reference —

3. INTRODUCTION

>

Poy
4

The modern biotechnology embraces all genetic manipulations,
protoplasmic fusion techniques in biotechnological process.

Genetic engineering or recombinant DNA technology or gene cloning
is a collective term includes different protocols, modifications and
transfer of DNA from one organism to another.

Vectors able to replicate and produce multiple copies of them along with
DNA insert in host. Hence they should have Origin of replication,
Selectable markers, Cloning sites.

4. @ Isolation of plasmid DNA
‘and DNA containing gene

of interest

Restriction enzymes
Endonuclease, DNA ligase
Alkaline Phosphatase
Vectors
pBR322 plasmid, Ti plasmid
Transposon as vectors
Expression vectors
Cosmid, Phagemids
Competent host for transformation with
rDNA
Methods of gene transfer
Chemical mediated, Microinjection,
Electroporation, Liposome, Biolistics

: Steps involved in r-DNA Technology

5. Screening of recombinants

contains the cloning vector with the gene of intrest refered to as recombinant cell thus it is
important to select recombinant cells from transformed cells containing the vector without
insert DNA

G After the introduction of rDNA into suitable host cells, it is essential to identify those cells which
have received the rDNA molecules. This process is called screening or selection.

(Y Generally the selection methods are based on the expression or non expression traits such

6. "1

Selection of recombinants vr:

7. The blue white screening method works by disrupting this a- P
complementation process. ESA $

The vector carries within the lacZa sequence an internal multiple
cloning site(MCS)

This MCS within the lacZa sequence can be cut by restriction enzymes
so that the foreign DNA may be inserted within the lacZa gene thereby
disrupting the gene and thus production of a peptide.

Consequently in cells containing the vector with an insert no
functional B- galactosidase may be formed

8. Recombinant

BWS & Protocol for colony
selection

@ The method is based on the principle of a complementation of the B galactosidase gene. This
phenomena of A complementation was first demonstrated by Agnes Ullman.

© The function of an inactivation mutant f3 galactosidase with deleted sequence shown to rescued
by a fragment of B galactosidase in which that same sequence that a donor peptide is still
intact

— Nonrecombinant "

@ Amore sophisticated procedure for screening of presence of recombinant plasmid which can
be carried out on a single transformed plate called blue white screening.
-@- This method also involves the insertional inactivation of a gene and uses the production of a

9. lacZAM19
Culture with X-Gal+IPTG

Competent £. coli
+
insert within lacZ

()+—

Plasmid Foreign DNA
no insert

Ligation Transformation Screening 9

10. -®- -> ==
a peptide w peptide

e

Le Active enzyme

The rescue of function of the mutant X gal \

B- galactosidase by the a- peptide is ur #
called a complementation à

10

11. /)

lacZ a and lacZ ß À »

> For example pUC18 plasmid carries the a AN i Of the 1acz Weher tng a ar is the
amino terminus of the protein and is not Iamoiional by itselt The d'flafnent is denoted by
lacZ.

> Typically the bacterivin host strain used A ‘rah Molmation is a mutant strain that has a
deletion of the alpha ffegment o£lacZ. The bacterial ¡chromosome carries only the W fragment
which is the carboxyl tefrainu-of the Protein] |

‘=> The w fragment alone is also called non functional.

> When both the a and tu. fragment

* * produced. This interaction-is called ‘complementation.

déts functionally intact B galactosidase protein a

13. on-recombinant cell

x
® -

y
8 <q

Active ß galactoside \

w peptide

When the LacZ a containing vector is transformed into the bacteria
1 then the two peptides express together and form homotetramer active

protein
This active B- galactosidase cleaves X-gal and produces blue colour

14. ecombinant cell

<------= Q

w peptide

The vector carries MCS within the lacZ a region which is cut by
restriction enzymes and foregin DNA can be inserted in this region

thereby disrupting the lacZ a gene which produce a peptides
In absence of a peptide no active B galactosidase hence colourless.

15. FB galactosidase

16. X - gal

X- Gal ( 5-Bromo -4Chloro -3indolyl B- D- Galacto pyranoside) is a
chromogenic substrate for ß galactosidase which yields a dark blue
colour after enzyme activity.

X- gal is an analog of lactose

X-gal + IPTG -- --> 5,5' -dibromo -4, 4'- dichloro- indigo

17. Antibiotic resistance screening

Recombinant
plasmid

Marker genes
(Antibiotic resistance gene)

18. 18

Usual medium

19. e,target DNA is inserted into ter ( 8) (© 8) Es)

e using Bam HI the property of No plasmid Intact plasmid Cloned plasmid
sistance to tetracycline will be lost. Ser =
Such recombinant would be tet*
| sensitive.
When such recombinantts containing
target DNA in tet* gene are grown into
medium containing tetracycline, they
will not grow because their tet* gene
has been inactivated. But they are
: resistant to ampicillin because amp*
ge functional.

1 | Plate on complete
medium with ampicillin

Only Amp’ cells
survive and grow

Replica plate on 3 | Pick, pool, and grow
complete medium cells from AmpTets
with tetracycline colonies

Only Tet cells Celis with p8R322-cloned
survive and grow DNA inserts 19

20. idizatio

Screening by Colo

y

Cells from the transformed reaction are plated onto solid agar medium under conditions
that permit transformed, but not no transformed cells to grow.

1. From each discrete colony formed on master plate and transferred to solid matrix,
nitrocellulose or nylon membrane. The pattern of the colonies on the master plate is
retained on the matrix.

2. The cells on the matrix are lysed and released DNA is denatured, deproteinized and
inversibily bound to the matrix. Labelled DNA probe is added to the matrix under
hybridization condition, after the nonhybridized probe molecule are washed away.

3. The matrix is processed by autoradiography to determine whuch cells have bound
labelled DNA. A colony on master plate that corresponds region of +ve response on Xray
film.

4. Cells from the positive colony on the master plate are subcultures because they may
carry the desired plasmid cloned DNA construct.

21. SEA, Addition of
Master plate VOB radioactively

we oO o labelled probe
Impression of
nitrocellulose
filter paper on
the master
plate

eS
AS
=
=,
‚Treatment will
Detergent
*Treatment
with NaOH

CSO «Baking at 80
Degrees

oo,
E oO = Celsius/ UV-

eos exposure No”)

DNA
hybridization
with radioactive
RNA probe

€)

Autoradiography

O

ene of
interest

22. Immunological assay

22

23. Immunological Screen

master plate

matrix cells Protein =
1 ea 2 Ges
— = ©) — (8 => &
6 transfer e © Iyse cells es e 00

cells bind protein x
À subculture ï =
cells from 3 | Add 1"Ab; HE

master plate wash

positive |
signal AS, ~~ scher EEE Ten
Mora 2 A

Ae æ/ es’ NO
Add substrate — Add2°Ab-Enzyme
wash

24. DNA hybridization and immunological assay work well for many kinds of genes and
gene products.

1. If target gene produces an enzyme that is not normally made by the host cell a
direct in situ plate assay can be identify members of a library that carry that particular

gene encoding that enzyme.

2. The genes for .a amylase, endoglucanase, B glucosidase and many other enzymes
form various organism have been isolated in this way.

3. This approach has proven effective for isolating genes encoding biotechnological
my useful enzymes from microorganisms present in environmental samples.

4. This techniques has enabled the isolation of many novel protein with interesting
properties without the need to first culture the natural host microorganism.

25. Screening by enzyme activity: : : ::::

Screening a genomic library for enzyme activity

1. Cells of a genomic library are planned are plated onto
solid medium containing the substrate for the enzyme of
intrest.

2. If a functional enzyme is produced by a colony that
carries a cloned gene encoding the enzyme the substrate is
converted to a coloured product that can be easily detected.
.3.. No coloured colonies on the medium also contain

: gene for the enzyme of intrest.

26. Reference

27. . Which of the following techniques is used in recombinant
identification?
A. Ligation. B. Isolation
C. Replica plating. D. Restriction digestion

2. lacZ’ gene present in a Puc series plasmid codes for which of

: : the enzyme?

- -A. Lactase. B. B glactosidase

“ °C. Amylase. D. Nuclease

28. 3. In screening for Puc8 recombinants, which color colonies will be - . -

the desired recombinants?
A. Blue. B. Colorless

y C. White. D. Yello wish

4. What is the compound X-gal?
A. Plasmid. B. Lactose analog
C. Antibiotic. D. Inducer

mes C. Antibiotic. D. Inducer

29. 6. In molecular cloning, Blue White screening is used
r 2 :

A. To screen for recombinant vectors
B. To detect gene mutations

C. To identify desired chromosomal DNA insert in plasmid vectors
D. To detect host DNA in with

7. Which of the following characteristic is not present in a plasmid on
a general basis?

30. 8. Which of the characteristics is present in lacZ gene?
. It encodes for beta galactosidase enzyme
B. Beta galactosidase enzyme is responsible for
cleaving monosaccharides into the constituent elements
C. It doesn’t cleaves a substrate called as X-gal
D. But if X-gal is cleaved, it liberates pink coloured dye.

9. Molecules having new combination of sequences that were not
present before are called as

: + : A. Intermolecular ligants. B. Recombinants

6; Couple. D. Intramolecular ligants

31. 10. After carrying out the cloning experiment, the cells are plated. on.

se Along with agar, it also contains antibiotic resistant genes, X-:
9

al and an inducer of lacZ gene. Which of the following would'grow?: :

A. Cells that have taken up plasmid DNA

B. Cells that have taken up genomic DNA

C. Cells having no insert

D. Cells either having no insert or having genomic DNA.

-11. The phenomenon of alpha complementation is

ii A.a+ß=w. B.a=B+w

- C.B=a+0. D. His eithera+fB=worfB=a+w

32. 12. If genomic DNA has been inserted instead of the plasmid, what
will happen?
y: It would lead to inactivation of lacZ gene
ie B. The X-gal substrate would be broken down
C. The colonies formed are blue in colour
D. The lacZ gene would be intact.

13. Which of the following gene helps in identifying transformed
cells?
A. Plasmid

33. Which statement is false regarding a-complementation?

that the recipient strain of bacteria (the one receiving DNA) has a
deletion in its lacZ gene such that it can only produces the w
fragment of B-galactosidase.

B. a-complementation involves the lacZ ’ gene.

C. a-complementation is successful when a cell is bioluminescent
(glows in the dark).

‘D. a-complementation involves the a and w fragments of B-

- ‘galactosidase.

34. eee recombinant DNA can be formed by joining both by: : :

A. Polymerase Ill. B.EcoRl oO
C. Ligase. D. Taq polymerase

16. Find the incorrect statement about plasmids

A. They are circular. B. They replicate independently

C. They are transferrable. D. They are single stranded
:17.:The DNA molecule used for integrating foreign gene for cloning is
- ++ -ealled

"A. Vector. B. Carrier. C. Template. D. Transformer

35. Antibiotics are used in genetic engineering. They are useful : : : : : - -

A. To keep culture free of microbialinfections = = (°°: °°:
B. To select healthy vectors = = | °°
C. To identify replication start sites

D. As selectable markers

19. The vector that can clone only a small DNA fragment is
A. Cosmid
B. Plasmid

36. In blue-white screening ........ Is used in medium. ae op
A. Chromogenic substrate B.Ampicillin = ....---
Ñ C. Tetracycline D. Lactose

21. Phenomena of a- complementation was demonstrated by
A. Agnes Ullman. B. Tatum
C. Edinberg. D. Koch

: : À. a complementation B. Gradient plate

: €. Replica plate. D. Colony hybridization

37. 23. LacZ gene produces ß galactosidase which converts lactose into-

glucose & ____ HÉRESE
A. Maltose. B. Sucrose RER

” C. Fructose. D. Galactose

24. B galactosidase is a polypeptide of aminoacids
A. 1029. B. 1039
C. 1049. D. 1059

... À. Protein assay. B. Immunological assay

GC. Enzyme activity D. Colony hybridization