L4-6 quiz

• yeast artificial chromosomes can be used to clone fragments up to 1000 kbp
• bacterial plasmid vector can be used for fragments up to 20kbp
• lambda vectors an clone up to 25 kbp
• useful plasmid cloning vector contains

  • origin of replication
  • antibiotic resistance marker
  • polylinker (multiple cloning site)
  • method of identifying recombinants
• in plasmid only need small number of restriction sites, otherwise:


  make it difficult to find single site to insert DNA into and would complicate analysis
• an origin of replication is an essential feature of an expression vector
• vector needs an origin of replication to multiply in cells
• bacterial promoter is needed to drive expression of the foreign DNA
• bacterial RNA polymerase (a bacterial promoter) is used to initiate transcription of foreign gene in bacteria
• subtilisin is a bacterial protease which has been engineered for improved characteristics, is a key constituent of

  washing powder
• subtilisin is present as a stain remover in biological washing powder
• Arabidopsis' genome is 100MB long, and has 25,000 genes encoded
• the strategy of fusing a eukaryotic coding sequence to a poly-histidine (6) N-terminal tag aids recombinant protein production primarily because:

  it allows for rapid and easy purification of the fusion protein
• screening recombinant with specific insert done by:

  • colony hybridisation (specific labelled probe will hybridise only with the gene of interest
  • antibody screening of an expression library (as antibodies are highly specific and will bind to the protein encoded by the gene of interest)
  • complementation cloning (a mutation in a gene can be rescued only if the correct version of that same gene is expressed)
• what must be upstream of cDNA cloned in E.coli expression vector?

  • ribosome binding site (required to translate mRNA arising from the insert)
  • promoter (required for RNA polymerase binding)
• in a vector it doesn't matter where the:

  origin of replication is, or the antibiotic resistance gene (as long as it doesn't disrupt the cDNA sequence)
• why might we express a eukaryotic protein as a fusion protein in bacteria, the fusion protein:

  • can have greater stability in bacteria
  • may be easier to purify that the native protein
  • may be expressed at higher levels than the native protein
• improved characteristics can be engineered using recombinant proteins by the _ technique:


  site directed mutagenesis