Microbial biotechnology

• Clone


  cDNA piece inserted into a vector which expresses it
• Types of libraries

  • cDNA
  • Genomic
• cDNA is used to express a given protein
• How to produce cDNA from mRNA

  1. ssDNA production

  2. RNA degradation

  3. dsDNA production

  4. Termination
• How to produce a cDNA library

  1. isolate and collect mRNA from sample
  2. Use reverse transcription to produce a double-stranded cDNA
  3. Ligate into plasmid
  4. Transform into bacterial cell
  5. culture
  6. plasmid isolation and purification
• 3 methods to screen cDNA libraries

  • Hybridization (using labelled probes)
  • Immunodetection (using antibodies)
  • Complementation (using mutants)
• Hybridization probe


  DNA (or RNA) fragment, usually 100-1000 bases, specific to target cDNA sequence, labelled
• Homology probe


  The probe carries the corresponding DNA sequence from a related organism
• Degenerate oligonucleotides


  The probe carries a degenerated DNA sequence inferred from a known PROTEIN sequence
• How to design degenerate oligonucleotides


  Choose all possible cDNA sequences that codes for that protein

  Choose a sequence that has the fewest number of possibilities, shorter sequence
• Radioactive labels

  Incorporation of nucleotides containing radioactive phosphorus (32P or 33P)
• Non-radioactive labels


  Incorporation of nucleotides coupled to digoxygenin (DIG)
• Incorporating labels into DNA

  Using terminal transferase or Taq polymerase
• Immunodetection: clone identification using antibodies


  Make the cDNA clone produce protein, produce an antibody recognising this protein, the clone of interest is where the antibody binds
• How to produce specific antibodies


  Take gene of interest, immunisation of animal, tumour cells fused, forming a hybridomas, screened for desired antibody, selected antibody is mass produced
• How to make cDNA clones produce proteins

  cDNA in phage, infect E. coli, E. coli replicates phage and produces the protein, phage kills E. coli, which releases the protein
• Nitrocellulose

  Binds to any protein
• Immunodetection screening

  Label primary antibody, label secondary antibody that recognize the primary antibody
• Complementation screening


  Use the activity of the protein to rescue the corresponding mutant
• Recombinant proteins

  • Insulin
  • Tissue Plasminogen Activator (tPA)
  • Human growth hormone (hGH)
  • Factor VII
  • Granulocyte Colony Stimulating Factor (G-CSF)
  • Erythropoietin (EPO)
  • Vaccine proteins
• Uses of recombinant proteins

  • Management of blood sugar levels (diabetes)
  • Dissolving blood clots, pulmonary embolism, heart attack
  • Short stature/hGH deficiency
  • Haemophilia
  • Boosting white blood cells (infections, chemotherapy)
  • Anaemia, chronic kidney failure, doping (cyclists)
  • Immunisation against bacteria or viruses
  • Disease management
• Important considerations for expression system

  Size of the target protein, Solubility of the target protein, Post-translational modifications
• Genetic optimization for E.coli protein expression

  • Enhancing the codon usage to match E.coli preferred codons
  • Optimizing promotor and operator sequences for stronger expression
• Why E. coli?


  Simple, cheap, and fast to grow, genetics (many strains and mutants available), high yield (high-copy plasmids: many DNA templates per cell)
• What can be optimised?

  • The E. coli strain
  • The plasmid vector
  • The coding cDNA sequence
• Considerations when choosing expression system

  • Size of the target protein
  • Solubility of the target protein
  • Post-translational modifications required
• Bacteria won't produce very large molecules or protein complexes, CHO cells would
• Membrane proteins won't be easily recovered from bacteria, a eukaryotic system may be necessary
• Bacteria cannot perform some post-translational modifications like glycosylation, porcine cells are favoured for human proteins because they use similar sugar groups
• Genetic optimization for E.coli protein expression

  • Enhancing the codon usage to match E.coli preferred codons
  • Optimizing promotor and operator sequences for stronger expression
• What can be optimised for E. coli expression

  • The E. coli strain
  • The plasmid vector
  • The coding cDNA sequence inserted
• Genetic optimisation for e.coli expression


  1. Optimising protein expression

  2. Optimising purification
• Optimising protein expression


  • Protease deficient strain (increases protein yield)
  • Promoter optimisation (inducibility, expression level)
  • Codon usage optimisation (improves translation rate)
  • PolyA signal (promotes mRNA stability, protection from endonucleases)
  • Selection marker (increases template number)
  • Replication origin (increases plasmid copies per cell)
  • N-terminal fusions (promotes protein stability)
• Optimising purification


  • Fusion tags (affinity purification)
  • Signal peptides (secretion)
  • Chaperone co-expression (solubility)
• Expression plasmid design


  • Promoter (for transcription)
  • Ribosome binding sequence (for translation)
  • N-terminal fusion (optional)
  • Signal peptide for secretion (optional)
  • Cloning site (for cDNA insertion)
  • Fusion tag for purification (optional)
  • Transcription terminator
  • PolyA signal (mRNA stability)
  • Chaperone-encoding gene (optional)
  • Selection gene (often antibiotic resistance)
  • Replication origin (controls copy number)
• Lac operon


  Lacl produces a mRNA that becomes a proton that binds to operator that stops activation
• Lac promotor


  • Protein expressed only when analogue of lactose added (ex: IPTG)
  • Expression products can be toxic, amplifying bacteria before expressing cDNA increases yield
• Codon usage optimizationto improve translationation rate


  Genetic code degeneracy leads to species-specific codon bias, some amino acids are used more than others in E. coli
• Means of optimising codon usage

  • Changing of one nucleotide base to change amino acid because it is better for e.coli
• describe incorporating N- terminal fusion


  Can improve translation rate, increase protein stability, facilitate purification