L5-6 microbial biotech

• what is a clone within a cDNA library context

  • cDNA molecule inserted into a vector and replicated in a host cell
• what is a cDNA library

  collection of complementary DNA clones synthesized from mRNA
• 2 types of DNA used as probes

  • cDNA
  • degenerate oligonucleotides
• enzymes used to make cDNA

  reverse transcriptase
• method used to detect the location of a radioactively labelled probe on a filter

  autoradiography
• reagent used in non-radioactive labelling

  digoxygenin
• enzyme conjugated to the antibody used in the non-radioactive detection method

  alkaline phosphatase
• process that produces light in the non-radioactive detection method

  chemiluminescence
• 2 methods used to screen cDNA libraries

  • colony hybridisation
  • immunodetection
• 2 enzymes used to label DNA probes

  • terminal transferase
  • taq polymerase
• 1. Why is digoxigenin used in the screening of cDNA libraries?

  • It provides a safe alternative to radioactive labelling, avoiding the need for special precautions and disposal procedures
  • Digoxigenin-labelled probes can be detected with high sensitivity and specificity using anti-digoxigenin antibodies
  • It allows for the stable and long-term storage of labelled probes, facilitating repeated use and verification of experiments
  • It offers flexibility in detection methods, including colorimetric, chemiluminescent, and fluorescent options, depending on the conjugated antibody used
• Producing recombinant proteins:

  • Collect DNA from gene A
  • Clone gene A into expression vector
  • Transfer vector into host
  • Host expresses gene A
  • Harvest protein A
• Why clone cDNA copies of genes:

  • To only clone genes
  • To remove introns (bacteria cannot splice introns out of the sequence)
  • mRNA cannot be cloned
  • To keep physical record of protein coding sequences
• making cDNA library


  sequence, identify and annotate
• 3 methods to screen cDNA libraries

  • hybridisation using labelling probes
  • immunodetection using antibodies
  • complementation using mutants
• homology probe is a probe that carries the cDNA sequence from a related organism
• degenerate oligonucleotides probe carriers degenerated DNA sequence inferred from a known protein sequence
• degenerate oligonucleotides as multiple codons make same amino acid
• dealing with degeneracy

  • prepare mix of 48 possible 20mer labelled oligonucleotides
  • use one long oligo with inosine based nucleotides (where several bases are possible)
• types of labels

  • radioactive
  • non-radioactive
• radioactive labels containing radioactive phosphorous
• non-radioactive labels contain nucleotides coupled to digoxygenin (DIG)
• incorporating labels into DNA

  • terminal transferase
  • Taq polymerase
• colony hybridisation steps

  • DNA stamp from each colony
  • denaturation of both plasmid DNA strands so probe can bind
  • colonies are tagged by labelled probe if they carry the corresponding cDNA
• immunodetection steps

  • make cDNA clone produce protein
  • produce antibody recognising this protein
  • clone of interest is where the antibody binds
• producing specific antibodies

  • known protein sequence
  • design antigen using monoclonal antibody technique
• making cDNA clones produce proteins by

  • cDNA in phage
  • infect E.coli
  • E.coli replicates phage and produces protein
  • phage ills E.coli which releases the protein
• immunodetection screening by

  • label primary antibody
  • use secondary antibody label to recognise the primary antibody
• complementation screening principle 


  use activity of protein to rescue the corresponding mutant, rescued clone easily detected among other clones
• complementation screening requires

  • loss of protein activity, leading to easily observable phenotype
  • cDNA library must be cloned into specific mutant background
• 3 elements needed adjacent to a cDNA for expression in a bacterial plasmid

  • promoter
  • ribosome binding sequence
  • transcription terminator
• 3 medically relevant recombinant proteins

  • human insulin
  • tPA (tissue plasminogen activator)
  • erythropoietin
• 3 reasons for expressing recombinant proteins as fusion proteins

  • greater stability
  • increased expression
  • simpler purification
• 3 fusion tags

  • Glutathione-S-transferase (GST)
  • maltose binding protein
  • polyhistidine
• 3 ligands used in purifying fusion proteins

  • nickel
  • amylose
  • glutathione
• protein sequence used to direct proteins 


  signal peptide
• natural enzyme used in biological washing powder

  subtilisin
• ice-nucleating bacterium 


  pseudomonas syringae
• precursor molecule for steroid production by microbial biotransformation

  diosgenin
• when are recombinant proteins used?

  • in development of vaccines and therapeutic drugs
  • in treatment of diseases through gene therapy
  • in production of enzymes for industrial processes
  • in agricultural biotechnology for developing pest-resistant crops