selection ,screening & expression

1. BY- SUNANDA ARYA
MSc . Microbiology

2. • INTRODUCTION FOR SELECTION
• DIRECT SELECTION OF RECOMBINANTS
• INSERTIONAL INACTIVATION
• BLUE- WHITE SCREENING
• COLONY HYBRIDISATION
• IMMUNOLOGICAL TESTS
• PROTEIN EXPRESSION INTRODUCTION
• MAMMALIAN PROTEIN EXPRESSION
• INSECT EXPRESSION
• EXPRESSION IN BACTERIA
• CONCLUSION
• REFERENCE

3. • After the introduction of recombinant DNA into the host
cells, it is essential to identify those cells which received
rDNA molecule - screening (or) selection.
• The vector or foreign DNA present in the recombinant
cells expresses certain characters or traits, while non-
recombinants do not express the traits.
• Transformed cells (or recombinant cells) are those host
cells which have taken up the recombinant DNA
molecule. In this step the transformed cells are separated
from the non-transformed cells by using various methods
making use of marker genes.

5. Methods
1- Direct selection of Recombinants
2- Insertional inactivation
3- Blue - White screening
4- Colony Hybridization
5 -Immunological Tests

6. • Vector designed to contain an antibiotic resistance genes.
• After transformation , cells are plated on medium containing
antibiotic .
• Ultimately the transformed cell will grow i.e recombinants
showing antibiotic resistance.
Example- to clone the gene for kanamycin resistance from
plasmid R6-5.
• This plasmid carries genes for resistances to four antibiotics:
Kanamycin, chloramphenicol, streptomycin and
sulphonamide.

7. • In this gene the EcoRI fragments of R6-5 would be inserted into
the EcoRI site of a vector such as PBR322.
• The ligated mix will comprise many copies of 13 different
recombinant DNA molecules, one set of which carries the gene
for kanamycin resistance.
• Insertional inactivation cannot be used to select recombinants
when the EcoRI site of PBR322 is used. This is because this site
does not lie in either the ampicillin or the tetracycline resistance
of this plasmid. But this is immaterial for cloning the kanamycin
resistance gene because in this case the coloned gene can be
used as the selectable marker.
• Transformed are plated onto kanamycin agar, on which the only
cells able to survive and produce colonies are those
recombinants that contain the cloned kanamycin resistance
gene(c).

10. • Insertional inactivation is a technique used in
recombinant DNA technology. In this procedure, a
bacteria carrying recombinant plasmids or a fragment of
foreign DNA is made to insert into a restriction site inside
a gene to resist antibiotics, hence causing the gene to
turn non-functional or in an inactivated state.

11. Three essential features for a
vector est
1)An origin of replication
2)Many Unique restriction sites
within selectable markers
3) At least 2 selectable
markers
• Upon insertion of desired
gene on Tetracycline region,
the gene is no more
functional and the process is
called insertional
inactivation.

13. • Blue-white screening is a rapid and efficient technique for the
identification of recombinant bacteria. It relies on the activity of β-
galactosidase, an enzyme occurring in E. coli, which cleaves
lactose into glucose and galactose.
• The presence of lactose in the surrounding environment triggers
the lacZ operon in E. coli. The operon activity results in the
production of β-galactoisdase enzyme that metabolizes the
lactose.
• β-galactosidase is a protein encoded by the lacZ gene of the lac
operon, and it exists as a homotetramer in its active state.
However, a mutant β-galactosidase derived from the M15 strain
of E. coli has its N-terminal residues 11—41 deleted and this
mutant, the ω-peptide, is unable to form a tetramer and is
inactive.

14. • This mutant form of protein however may return fully to its
active tetrameric state in the presence of an N-terminal
fragment of the protein, the α-peptide. The rescue of
function of the mutant β-galactosidase by the α-peptide is
called α-complementation.
• The blue/white screening method works by disrupting this
α-complementation process.

15. • Based on concept of Lac- operon

16. • If LACTOSE is there in environemnt, bacteria use this lactose &
activate the lac z,y,a genes.
• Lac z gene produced enzyme β-galactoisdase, which
hydrolyses…
LACTOSE GLUCOSE + GALACTOSE
- β-galactoisdase – HOMOTETRAMER (active state)
- β-galactoisdase (mutant form) –unable to form tetramer
(inactive state)
- MUTANT FORM NORMAL FORM
- BY getting α-peptide.
- The rescue of function of the mutant β-galactosidase by the α-
peptide is called α-complementation

17. The blue/white screening method works by disrupting
this α-complementation process.
• The presence of an active β-galactosidase can be detected by
X-gal, a colourless analog of lactose that may be cleaved by β-
galactosidase to form 5-bromo-4-chloro-indoxyl, which then
spontaneously dimerizes and oxidizes to form a bright blue
insoluble pigment 5,5'-dibromo-4,4'-dichloro-indigo.
• This results in a characteristic blue colour in cells containing a
functional β-galactosidase. Blue colonies therefore show that
they may contain a vector with an uninterrupted lacZα (therefore
no insert), while white colonies, where X-gal is not hydrolyzed,
indicate the presence of an insert in lacZα which disrupts the
formation of an active β-galactosidase.

20. • The colony hybridization technique is based on the
availability of radioactively labeled DNA probe.
• A probe is radioactively labeled p32 nucleic acid (20-40
nucleotide long) with a sequence complementary to at
least one part of the desired DNA.
• The probe may be partially pure mRNA, a chemically
synthesized oligonucleotide or a related gene, which
identifies the corresponding recombinant DNA.
• DNA probes have commercial significance. They
diagnose specific DNA sequences (genes) and are useful
in the diagnosis of diseases, microbiological tests and in
research as well.

21. • In this technique master plates are prepared as described
earlier. Replica plating of colonies is done onto a nitrocellulose
filter disc which is then placed on the surface of a gelled nutrient
medium and both master plate and disc are incubated to develop
colonies.
• Cells growing on nitrocellulose filter disc are nourished through
the diffusion of nutrients from gelled nutrient medium.
• The filter disc is removed and put on blotting paper soaked with
0.5 N NaOH solution. The alkali diffuses into nitrocellulose, lyses
bacterial cells and denatures their DNA. Thereafter, the filter disc
is neutralized by tris (hydroxymethyl) aminomethane-HCl buffer
by keeping high salt concentrations. This results in binding the
DNA with nitrocellulose disc in the same pattern as the bacterial
colonies; to fix the cDNA properly the filter disc is baked at 80°C
.

22. • Then it is incubated with a solution containing radioactive
chemical labeled probe (p32 DNA) at suitable conditions. The
probe will hybridize any bound DNA which contains sequences
complementary to the probe .
• By thorough washing unhybridized (unbound) probes are
removed from the hybridized probe (colonies containing
sequences complementary to probe) and is identified by
autoradiography of the nitrocellulose filter disc .
• Colonies which develop positive X-ray image are compared
with master plate and picked up, and multiplied on the nutrient
medium.

24. • The immunological techniques are the final test analogous to colony
hybridization technique as described earlier. It is an alternative
screening procedure which relies on expression, and generally
applicable approach to identify a clone synthesizing a particular
polypeptide.
• This is potentially a very powerful method since the only absolute
requirement is that the required mRNA encodes a protein for which a
suitable antibody is available.
• In the immunological test, instead of radio-labeling of DNA molecules,
antibodies (immunoglobulins) are used to identify the colonies or
plaques developed on master plates that synthesize antigens encoded
by the foreign DNA present in plasmids of the bacterial clones.
• For this purpose a special vector, known as expression vector, is
designed where the foreign DNA is transcribed and translated within
the bacterial cell

25. The growth medium
containing specific anti
serum may help in
detection of viable
immunoprecipitate
(precipitin) around the
colonies or plaques. The
method follows :

26. • Broome and Gilbert (1978) purified immunoglobulin G (IgG)
and bound it to an agar plate so that the antigens released
through in situ lysis of bacterial colonies can bound to the
fixed antibody .
• The antigen is then detected by using the same IgG
preparation which is radioactively labeled. This recognizes
determinants on the bound antigen.

29. • Protein expression refers to the way in which proteins are
synthesized, modified and regulated in living organisms.
In protein research, the term can apply to either the
object of study or the laboratory techniques required to
manufacture proteins. This article focuses on the latter
meaning of protein expression. However, in practical
terms, recombinant protein production depends on using
cellular machinery.

30. • Traditional strategies for recombinant protein expression involve
transfecting cells with a DNA vector that contains the template and then
culturing the cells so that they transcribe and translate the desired
protein. Typically, the cells are then lysed to extract the expressed
protein for subsequent purification. Both prokaryotic and eukaryotic in
vivo protein expression systems are widely used. The selection of the
system depends on the type of protein, the requirements for functional
activity and the desired yield.
• These expression systems are summarized in the table, include
mammalian, insect, yeast, bacterial, algal and cell-free. Each system
has advantages and challenges, and choosing the right system for the
specific application is important for successful recombinant protein
expression.

33. • Mammalian expression systems can be used to produce mammalian
proteins that have the most native structure and activity due to its
physiologically relevant environment. This results in high levels of post-
translational processing and functional activity. Mammalian expression
systems are the preferred system for the expression of mammalian
proteins and can be used for the production of antibodies, complex
proteins and proteins for use in functional cell-based assays. However,
these benefits are coupled with more demanding culture conditions.
• Mammalian expression systems can be used to produce proteins
transiently or through stable cell lines, where the expression construct is
integrated into the host genome. While stable cell lines can be used
over several experiments, transient production can generate large
amounts of protein in one to two weeks. These transient, high-yield
mammalian expression systems utilize suspension cultures and can
produce gram-per-liter yields. Furthermore, these proteins have more
native folding and post-translational modifications, such as
glycosylation, as compared to other expression systems.

34. • Insect cells can be used for high level protein expression
with modifications similar to mammalian systems. There
are several systems that can be used to produce
recombinant baculovirus, which can then be utilized to
express the protein of interest in insect cells. These
systems can be easily scaled up and adapted to high-
density suspension culture for large-scale expression of
protein that is more functionally similar to native
mammalian protein. Though yields can be up to 500
mg/L, recombinant baculovirus production can be time
consuming and culture conditions more challenging than
prokaryotic systems.

35. Baculovirus Expression System
–
The Invitrogen BaculoDirect
Baculovirus Expression System
utilizes Invitrogen Gateway
technology for cloning. After a
1-hour recombinase reaction
and transfection in insect cells,
baculovirus containing the
gene of interest is produced. A
quick expression test can then
be performed before amplifying
the viral stock and scaling up
expression. Use of this system
allows for baculovirus
expression in insect cells.

36. • Bacterial protein expression systems are popular
because bacteria are easy to culture, grow fast and
produce high yields of recombinant protein. However,
multi-domain eukaryotic proteins expressed in bacteria
often are non-functional because the cells are not
equipped to accomplish the required post-translational
modifications or molecular folding. Also, many proteins
become insoluble as inclusion bodies that are very
difficult to recover without harsh denaturants and
subsequent protein-refolding procedures.

37. • Although eukaryotic cells can be used to express
eukaryotic proteins, bacteria are simpler to grow and
manipulate genetically. Therefore, it is often desirable to
express eukaryotic proteins in bacteria.
• Eukaryotic genes must be adapted for expression in
bacteria. First, the mRNA from the gene of interest is
converted to cDNA to provide uninterrupted coding DNA.
The cDNA is cloned between a bacterial promoter and a
bacterial terminator so the bacterial transcription and
translation machinery express the coding sequence.

39. • In production of recombinant protein, the gene for the protein of
interest is cloned into a vector and expressed into protein in a model
organism.
• The plasmids of the copy of the interest gene, or the expression
vectors, are often used to enhance gene expression. These vectors
provide a strong promoter to drive expression of the cloned gene.
Expression vectors also contain genes for antibiotic resistance to
allow selection of the vector and the recombinant protein.
• Expression vectors are used to make eukaryotic proteins in bacteria.
The vector has the ribosome-binding site, terminator sequences, and
a strong regulated promoter. The eukaryotic gene is a cDNA copy of
the mRNA. Once cloning is completed, plasmids are taken up into
competent cells (chemically competent or electro competent E. coli)
for propagation and storage, by a process called transformation.

40. • Because bacteria cannot process introns so it is standard
procedure to clone the cDNA version of eukaryotic
genes, which lacks the introns and consists of
uninterrupted coding sequence. And the cDNA version of
eukaryotic genes is generally used, even for expression
in eukaryotic cells.

41. • Prescott's Microbiology, 10th Edition by Joanne Willey
and Linda Sherwood and Christopher J. Woolverton.
• Overview of Protein Expression.
Thermofisher.scientific.com
• Expression of recombinant proteins in insect and
mammalian cells. Article .sciencedirect.com
• www.ncbi.com
• Slideshare. Selection and screening of recombinant
clones.neeru02.