SU 6 2(2)

• Untransformed bacteria


  Mixture of bacteria without plasmid

  
• Transformed bacteria


  Bacteria containing plasmid that can replicate and express antibiotic resistance gene

  
• Selecting transformed bacteria


  1. Grow bacteria in presence of antibiotic

  2. Normal bacteria killed

  3. Plasmid-containing bacteria survive

  
• Selectable marker


  Allows selection of transformed bacteria

  
• Gene cloning experiment


  1. Bacterium containing recombinant plasmid

  2. Grow liquid culture

  3. Purify plasmid DNA

  
• Analysing recombinant plasmid


  1. Digest with BamHI

  2. Run on agarose gel

  3. Observe 2 bands

  4. Estimate size of cloned fragment

  
• Insertional inactivation


  Cloning foreign DNA into antibiotic resistance gene disrupts gene and prevents resistance

  
• lacZ gene


  Encodes β-galactosidase enzyme that cleaves X-gal to produce blue colour
• Screening for recombinant plasmids using lacZ


  1. Transform plasmid mixture into cells

  2. Plate on agar with ampicillin and X-gal

  3. Blue colonies are non-recombinant, white colonies are recombinant

  
• Plasmid vectors


  • Limited in size of DNA fragments that can be cloned
  • Other vectors like phage, cosmids, BACs developed for larger DNA fragments

  
• Expression vector


  Plasmid with sequences for transcription and translation in bacterial cells

  
• Constructing expression vector

  1. Include RE sites, ori, selectable marker

  2. Add promoter, transcription/translation sequences

  
• Eukaryotic gene expression in bacteria


  Requires bacterial regulatory sequences, even if gene is eukaryotic
• Specialised cloning vectors


  • Yeast artificial chromosomes (YAC)
  • Ti plasmid of Agrobacterium for plants

  
• DNA library


  Collection of clones containing all DNA fragments from one source
• Genomic DNA library


  Contains clones representing all DNA sequences in the genome

  
• cDNA library


  Contains only DNA sequences transcribed into mRNA
• Creating a genomic library


  1. Partially digest genomic DNA with RE

  2. Join fragments to plasmid vector

  3. Transform into bacteria and plate