Vector
Vector
Vector
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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
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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DNA
They supply one strand of that DNA in an
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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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phage DNA
Upto 6 times the normal length of M13 DNA
can be packaged
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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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particles in vitro
The size of the insert is dependent on the total
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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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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Note: The foreign DNA/insert can also be called as the passenger DNA
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