Vector 404r29

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 30-12-2012

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• X-gal (also abbreviated BCIG for 5-bromo-4chloro-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, βgalactosidase • X-gal is one of many indoxyl glycosides and esters that yield insoluble blue compounds similar to indigo as a result of enzymecatalyzed hydrolysis 30-12-2012

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X-gal 30-12-2012

5-bromo-4-chloro-3-indolylbeta-D-galactopyranoside

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

Phage lambda plaques on a lawn of bacteria 30-12-2012

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Lysis plaques of lambda phage on E. coli bacteria 30-12-2012

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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 nonrecombinant 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 30-12-2012

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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 30-12-2012

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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 30-12-2012

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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) 30-12-2012

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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 30-12-2012

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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) 30-12-2012

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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 30-12-2012

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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 30-12-2012

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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 βgalactosidase has asparagine instead of aspartic acid in the 5th position • This change does not affect the activity of the enzyme 30-12-2012

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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 30-12-2012

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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 30-12-2012

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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 30-12-2012

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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 30-12-2012

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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 30-12-2012

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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 30-12-2012

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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 30-12-2012

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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 30-12-2012

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Ligation product is a concatemer

Note: The foreign DNA/insert can also be called as the enger DNA 30-12-2012

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