MULTIPLE CLONING SITE and BACTERIOPHAGE LAMBDA (λ - phage )

MULTIPLE CLONING SITE and BACTERIOPHAGE LAMBDA (λ - phage )

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Cloning
MULTIPLE CLONING SITE

 Many cloning vectors contain a multiple cloning site or polylinker:
- a DNA segment with several unique sites for restriction endo-
nucleases located next to each other
 Restriction sites of the polylinker are not present anywhere else in the
plasmid.
 Cutting plasmids with one of the restriction enzymes that recognize a
site in the polylinker does not disrupt any of the essential features of
the vector .
 MCSs let a molecular biologist insert a piece of DNA or several
pieces of DNA into the region of the MCS.
 This can be used to create transgenic organisms, also known
as genetically modified organisms (GMOs).

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 Gene to be cloned can be introduced into the cloning vector at one
of the restriction sites present in the polylinker

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◘ LacI
Q
: is a repressor protein to Lac Z gene , lac repressor binding to
the operator sequence causing inactivation to lac Z gene .
- In platting system I cannot detect recombination .
◘ isopropylthiogalactoside (IPTG) : Inducer protein to Lac Z
gene , which binds to the lac repressor (LacI
Q)
protein and
inactivate repressor protein , causing induction to Lac Z gene .

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BACTERIOPHAGE LAMBDA (λ - phage )


• is a bacterial virus, or bacteriophage, that infects the
bacterial species Escherichia coli (E. coli).
• The phage particle consists of a head (also known as
a capsid)(protective coat ), a tail, and tail fibers.
• The head contains the phage's double-strand
linear DNA genome.
• cloning limit : accommodate to 25 kbp of Insert .

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◘ Steps of Cloning Using λ phage :

1- Preparation :
• Lambda viral genome : linear DNA with ssDNA "sticky end" at both
ends; these ends are complementary in sequence and can hybridize to
each other (this is the cos site: cohesive ends).
• preparation The Host (E. coli)
2-Insertion :
• coupling happen in the center portion .
• Viral linear DNA have 2 arms .

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3- Packaging :

• using Capsid protein and processing Enzymes and Recombined Phage
DNA in Test Tube .
4- Transfection = Infection :-

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1- The Virus attached to The Host cell ( Attachment to the surface )
2- Inject the Viral genome ( Genatic material ) to the Host cell .
3- Replication of the viral genome ( recombinant Phage DNA ) ,
forming a new capsid and new viral genome and synthesis of new
phages ( more copies ) causing lysis of The Host cell ( E.coli ) .
5- Growing or Plating :-

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• Bacterial lawns can be produced manually by evenly spreading a
high amount of bacteria onto an agar plate .
- Bacterial lawn is a term used to describe the appearance
of bacterial colonies when all the individual colonies on a petri-
dish agar plate merge to form a field or of bacteria.
- Bacterial lawn : is a continuous Sheet of bacterial growth and a
clear area.
- clear area : clear spots in lawn called plaques .
• A clear plaque will be formed if the host is completely susceptible
to the phage ( lysis by the phage )
• After formation of the plaques , we isolate and screening the
plaques in a new agar plate .

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Comparison Between Plasmid & λ- Phage
Plasmid λ- Phage
1- Genetic
material
It is a circular double stranded
DNA
double-strand
linear DNA genome
2- Bacteria
penetration
Transformation Transfection
3- Types PBR322 , PUC 18 , PUC 19 λ- Phage
4- Protective coat Without Protective coat With Protective coat
( Capsid )
5- Size of Insert 10000 bp or 10 kb

25000 bp or 25 Kbp
6- Penetration
Steps
1- preparation
2- Insertion
3- Transformation
4- Screening
5- growing & Isolation
1- preparation
2- Insertion
3- Packaging
4- Transfection =
Infection
5- growing or Plating
7- Position Of
Insert in Vector
• insert a piece of DNA or
several pieces of DNA into
the region of the MCS.
• The Insert compliment with
the Vector at MCS .
• coupling between
insert and vector
happen in the center
portion of the vector
genome .
8- plating system • Ampicillin Agar plate
• Tetracycline Agar plate
•( X-gal ) Lac Z Agar plate

• Lawn Plate .
9- Isolation &
Growing
( Multiplication )
• The desired recombinant
colonies can be picked and
cultured , then Isolate the
Desired genome and the
vector after multiplication

• After formation of
the plaques (
multiplication ), we
isolate it and screening
the plaques in a new
agar plate , then
isolate the desired
gene from the vector .

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Transformation
◘ Definition :
• Transformation :- Insertion the Vector as recombinant DNA or Non-
recombinant DNA into a Host cell ( E.coli ) .
• Transformation :- when microorganism are able to take up and
replicate DNA from their local environment .
◘ The condition of insert the vector into the Host cell :-
1- Change the permeability of the Host cell wall by using ions like
calcium – chloride that change the structure of the cell wall and increase
permeability of the host so as to let the vector enter the cell wall of the
host.
• Or by using Electroporation :- use high electrical voltage pulses to
translocate DNA across the cell membrane .
2- Temperature of Host cell must be very low , if the temperature
increase the transformation process cannot be done .
3- Increase the number of host cell ( ex : Bacteria )
4- The Size of the vector ( plasmid ) the more smaller the easy to
enter the host cell .
5- The type of ( E. coli ) ( The Host ) :
- RecBc gene : make expression for an enzyme called exonuclease
that used to cut any linear foreign DNA .
cut only linear if the DNA is circular there is no effect on it .

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1- RecBc
+
gene : Recombinant Bacterial cell gene ( the gene is
present )
- If the bacteria have RecBC+ gene it will transform the circular
DNA 100 % but , if the DNA is Linear it will transform it only 10
% .
2- RecBc
-
gene : Recombinant Bacteria cell
- if the gene is absent , transformation of the linear and circular
DNA 100 % ( completely transformed ) .

◘ Cos – Sequence : (cohesive ends )


5՝ cos sequence Central portion 3՝ cos sequence

Order the recombinant genome to ( packaging ) and formation the capsid
around it before transformation .

The lecture is Done ♥ ☺ Q.A