vector

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VECTORS

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• The artificial plasmid pUC18 has been genetically engineered to include a gene for antibiotic resistance to Ampicillin (ampR), and a gene (and its promoter) for the enzyme beta-galactosidase (lacZ)

• The lacZ gene contains a polylinker region, with a series of unique restriction sites found nowhere else in the plasmid

• Digestion with any one of these endonucleases will make a single cut that linearizes the circular plasmid DNA, and allow it to recombine with foreign DNA that has been cut with the same endonuclease

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• X-gal (also abbreviated BCIG for 5-bromo-4-chloro-indolyl-β-D-galactopyranoside) is an organic compound consisting of galactose linked to a substituted idole

• X-gal is much used in molecular biology to test for the presence of an enzyme, β-galactosidase

• X-gal is one of many indoxyl glycosides and esters that yield insoluble blue compounds similar to indigo as a result of enzyme-catalyzed hydrolysis

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X-gal5-bromo-4-chloro-3-indolyl- beta-D-galactopyranoside

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08/04/2023 9Concatemer: Multiple copies of the same  sequence joined tandemly end to end

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Phage lambda plaques on a lawn of bacteria

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Lysis plaques of lambda phage on E. coli bacteria

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• Commonly used λ insertion vector – λgt10• In λgt10 the EcoRII cloning site is in a gene

which is deleterious to phage replication in certain host strains

• This property allows selection against non-recombinant phage

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• The major advantage of the λ phage vector is

its high transformation efficiency, about 1000

times more efficient than the plasmid vector

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The vectors lambda gt10 (insertion vector) and 

Charon 16A (replacement vector)

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Filamentous phages as cloning vectors

• Includes Ff class of filamentous phages

• Strains include f1, fd, and M13

• Infects E. coli cells

• Ff virions are long and thin, they contain

closed loop of ssDNA

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• These phages readily accept inserts of foreign

DNA

• They supply one strand of that DNA in an

easily isolated form

• For these reasons the vectors based on Ff

phages became a standard choice

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M13 bacteriophage

• Filamentous bacteriophage of E.coli• 870nm long• 6nm wide• Protein coat called capsid, made up of 3 kinds

of capsomeres• Infect cells by adsorbing to and entering through F pili – they only infect F+ or Hfr E.coli cells, they do not infect F- cells

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• These phages do not lyse the host cells like phage λ during the lytic cells

• Instead the progeny viruses are extruded through the layers of the cell membrane and cell wall without major interference with cell growth

• Infected cells continue to grow and extrude thousands of progeny virus particles into the medium

• Each of these particles consists of a ss genome

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• Virus particles are very small compared to host – low speed centrifugation is used to remove host cells and separate the virus particles

• Virus particles are further collected by high speed centrifugation of the supernatant

• ssDNA molecules can be isolated by simple phenol-chloroform extraction

• The “+” strand of the virus is packaged (the same DNA strand of the virus is always packaged, that is called “+” strand. Strand complementary to the “+” strand is called the “-” strand)

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• The “+” has the same “sense” as mRNA

• Its nucleotide triplets correspond to the mRNA

codons, but with “T” in place of “U”

• Packaging of ss of phage DNA in progeny

provides a neat biological purification of

ssDNA

• This property is exploited for its use as vectors

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Genetic Organization of Wild type BACTERIOPHAGE M13

• ss and circular DNA• 6407 bases • 10 genes that are closely packed – all are

essential for phage replication• A segment of 507 nucleotide intergenic sequence (IS) contains the origin of replication (ori) 

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• The IS can be manipulated for cloning 

without disrupting the ori

• Therefore, wild type M13 has limited use in 

gene cloning experiments

• Size of phage particle is determined by size of

phage DNA

• Upto 6 times the normal length of M13 DNA 

can be packaged

08/04/2023 25M13 Cloning Vectors

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Construction of M13 based vectors

• IS region is the only region that can be manipulated for cloning

• This region has only two restriction sites AsaI and AvaII

• Wild type M13 phage is not an efficient vector• But the IS can be modified to introduce

additional restriction sites

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M13mp vectors (mp1&2)

• lac Z gene is introduced into the wildtype IS to get the M13 mp1 phages

• These phages produce blue plaques on X-gal agar plates

• The lacZ does not consist of any restriction site• It has a hexanucleotide sequence, GGATTC 

near the start

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A LB agar plate showing the result of a blue white screen

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• If the second G residue (GGATTC) is substituted by an A residue, the sequence becomes an Eco RI site i.e., GAATTC

• Such a vector is called as the M13 mp2 vector• The conversion from G to A is achieved by invitro

mutagenesis• Due to this change the lacZ enzyme β-galactosidase has

asparagine instead of aspartic acid in the 5th position• This change does not affect the activity of the enzyme

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• M13 mp2 is the simplest cloning vector derived from M13 phage

• The unique Eco RI site of M13 mp2 is cleaved to insert foreign DNA

• The insertion leads to the inactivation of lacZ (insertional inactivation)

• Recombinants fail to produce blue plaques on X-gal agar, instead they produce clear plaques

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M13 mp7

• It is a more complex vector• Derivative of M13 mp2• A polylinker is inserted into the Eco RI site of

lacZ gene• Polylinker is designed in such a way that it

does not inactivate the lacZ gene• The polylinker consists of restriction sites for

Bam H1, Sal I, Pst I

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• Therefore it consists of 4 possible insertional sites

• Foreign DNA corresponding to these restriction sites can be inserted in their respective sites

• Recombinant M13 mp7 phage cannot produce blue plaques on X-gal agar because of insertional inactivation of the lacZ gene

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Cosmids 

• Hybrid (plasmid + λ phage) DNA molecules• Can live dual lives• Plasmid part enables them to replicate as it contains the ori

• Plasmid part also helps in selection due to the presence of selectable markers 

• Cleavage site is located in the marker gene

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• The lambda phage part (cos sequences)

allows them to be packaged in a phage coat 

• It also enables them to be transduced into a

recipient by the lambda infection machinery

• It consists no genes for viral proteins,

therefore viral particles are not formed within

the host cells

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• Hence host cell lysis is absent

• The vector cosmids are then ligated with

foreign DNA and then packaged into viral

particles in vitro

• The size of the insert is dependent on the total

size of the recombinant DNA molecule

required for stable packaging into viral

particles

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Cosmid pHC79

• Suitable for cloning DNA fragments (upto 40kb)• In vitro packaging systems are used• Vector is 6.5kb in size• Contains a part of the pBR322 and a fragment of λ

phage DNA containing the cos region• Cos region is required for packaging into phage

heads• The pBR322 region consists of genes for Ampicillin

and Tetracyclin resistance

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Cosmid pJB8• Cosmid is 5.4kb in size• Consists of amp resistance genes of pBR322 

and cos site of λ phage DNA• The enzymes that package the DNA molecules

into a viral coat require only the cos sites for functioning

• The in vitro packaging can accommodate 37 – 52kb of DNA

• The foreign DNA has to lie between 2 cos sites to enable packaging

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Cloning using cosmid vector

• Cosmid vectors are used along with the in

vitro packaging system

• Cosmid is cut at the restriction site with an

appropriate RE (Bam H1) and is made linear

• Foreign DNA are prepared using the same RE

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• The Linear vector and the foreign DNA are ligated

• Ligation occurs in such a way that concatamers of recombinant cosmids are formed

• These concatamers are packaged in vitro into λ phage heads

• Recombinant cosmids can be transduced into E. coli host by infection

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 Ligation product is a concatemer

Note: The foreign DNA/insert can also be called as the passenger DNA