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08/04/2023 1
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