GEN 314

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GEN 314

• Gene Manipulation Lecture Two

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Prerequisites for plasmids as cloning vehicles

• Purification of plasmid DNA
• Low molecular weight
• Ability to confer readily selectable phenotypic
  traits on host cells (Table 4.1 pg 49)
• Single sites for a number of r.e.s, preferably
  in genes conferring readily scorable phenotype
• insertional inactivation

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Plasmid pBR322

• 4361 bp in length
• Three large regions (Fig. 4.7a 4.8 pg 59-60)
• the pMB1-derived region including the origin
  of replication (Ori)
• the RSF 2124-derived region including an
  ampicillin resistance (ApR) gene
• the pSC101-derived region including a
  tetracycline resistance (TcR) gene
• gt 40 restriction enzyme sites
• twelve sites fall within the TcR gene two in
  the genes promoter region
• six sites fall within the ApR gene two in
  the promoter region
• PstI site in the ApR gene is important
• 3-OH tetranucleotide extensions formed
  following digestion are substrates for terminal
  transferase
• homopolymer tailing (application in cDNA
  cloning)

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Plasmid map pBR322
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Insertional inactivation

• Suppose you insert a gene-X at the PstI site
  within the ApR gene
• Transform plasmid into E. coli
• Some cells will contain recombinant plasmid and
  others re-circularized plasmid (non-recombinants)
  Expect recombinants (ApS/TcR) and
  non-recombinants (ApR/TcR)
• Identifying the recombinants
• Grow bacteria on agar medium containing Tc
• Press on agar plate using sterile velvet cloth
  
• Replica-plate on agar medium containing Ap
• No growth on Ap-containing plate (identify ApS
  transformants on original plate)

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Replica-plating
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Promoter region

• In general, inserting a fragment into the HindIII
  site will result in loss of TcR
• However, some recombinants will retain TcR or
  even increase expression
• HindIII site lies in the promoter (not coding
  region)
• Insertional inactivation will occur if the
  inserted fragment does not carry promoter-like
  sequences that are able to initiate TcR
  transcription
• e.g. Widera et al. (1978), pg 61 in Old
  Primrose

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Transformation reactions

• Input reagents
• DNA
• plasmid
• DNA ligase
• Buffer
• Expected in the reaction mixture!
• recombinants
• non-recombinants (recircularized plasmid)
• unligated DNA
• unligated plasmid
• dimers
• bi-directional insertion (see later)
• Favouring recombinants
• High DNA concentration vs plasmid
  concentration
• Alkaline phosphatase treatment (plasmid)
  removes 5-OH terminal phosphate groups to
  minimize plasmid recircularization and plasmid
  dimer formation (Fig. 3.6 pg 39)

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pBR322-derived plasmids

• pUC vectors incorporate a DNA sequence which
  permits rapid visual detection of an insert
• smaller than pBR322 in size only 2686 bp
• lack rop gene high copy number
• contain multiple cloning site (MCS) DNA
  sequence carrying sites for many diff. r.e.s
  (inserted 5-end of lac operon)
• contains region of the lacZ operon including CAP
  protein binding site, promoter Plac, lac
  repressor binding site and the 5'-terminal part
• region encodes N-terminal fragment of
  ?-galactosidase gene
• fragment (synthesis inducible by IPTG) capable of
  complementing a defective form of gene produced
  by host bacteria
• on X-Gal rich-media, IPTG induce the synthesis of
  N-terminal complementation blue colonies
• inserting DNA in the lacZ gene insertional
  inactivation (white colonies)

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pUC18/19 map
NB Difference between pUC18 and pUC19 is
orientation of MCS
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Blue/White screening
Klug et al. (2006), Fig. 19-7, pg 461
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pBR322-derived plasmids

• pBluescriptII vectors
• 2961 bp in length
• also allow blue/white screening
• additionally, MCS is bordered by phages T7 and
  T3 promoter sequences enable expression of the
  inserted gene irrespective of its orientation
• two origins of replication
• phage f1 intergenic region, carrying the
  sequences required in cis for initiation and
  termination of phage f1 DNA synthesis and for
  packaging of DNA into bacteriophage particles
• phagemid Ori from plasmid and phage

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Plasmid map pBluescriptII
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Restriction Enzyme Mapping

• Performed to establish the number, order and
  distance (length) between restriction enzyme
  sites along a segment of DNA

Klug et al. (2006), Fig. 19-23, pg 473
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Example R. E. Mapping
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Example R. E. Mapping

• EcoRI 8.9 kb
• BamHI 6 kb 2.9 kb
• HindIII 8.9 kb
• EcoRI BamHI 6 kb 2.4 kb 0.5 kb
• EcoRI HindIII 7.4 kb 1.5kb
• BamHI HindIII 5 kb 2.9 kb 1 kb
• EcoRI BamHI HindIII 5 kb 2.4 kb 1 kb
  0.5 kb

pGEN314 8.9 kb
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Solution
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End Lecture Two