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 betagalactosidase (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-4chloro-indolyl--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, galactosidase X-gal is one of many indoxyl glycosides and esters that yield insoluble blue compounds similar to indigo as a result of enzymecatalyzed hydrolysis
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X-gal
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5-bromo-4-chloro-3-indolylbeta-D-galactopyranoside

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Concatemer: Multiple copies of the same sequence joined tandemly end to end 30-12-2012 9

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 insertion vector gt10 In gt10 the EcoRII cloning site is in a gene which is deleterious to phage replication in certain host strains This property allows selection against nonrecombinant phage

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 The major advantage of the 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 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
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M13 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 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 + 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 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 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 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
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