Iptg induction

An overview of IPTG induction and the commercially used lac based systems

1. IPTG INDUCTION
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2. Lac operon
•Lac operon is an inducible operon containing the promoter, operator and 3 genes for
metabolising lactose.
•The 3 genes are Lac Z, Lac Y and Lac A encoding enzymes beta galactosidase,
permease and transacetylase respectively.
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3. •LacZ - Beta-galactosidase : Breaks down lactose into glucose and galactose. LacZ is
also responsible for the production of allolactose, the natural inducer of the lac
operon.
•LacY - Lactose permease : Lactose uptake by the cell through symport; creates a
proton gradient to transport lactose in to the cell.
•LacA - Beta-galactosidase transacetylase: The enzyme acetylates other beta
galactosides other than lactose and protect the cell from creating toxic products by
the non-specific cleavages.
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4. Lac Repressor
•Protein of 360AA - associates into a homo-tetramer.
Each subunit of 38KDa
•Encoded by LacI gene
•4 amino terminal fragments (∼60 residues) - Bind
specifically to operator DNA
•Carboxyl terminal tetrameric ‘core’ - Binds inducers.
•Hinge region - extremely susceptible to proteolytic
cleavage and lacks structure.
•The last 30 amino acids of core - responsible for the
oligomeric state of the repressor and essential for
tetramerization.
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5. Inducers of Lac Operon
•Lactose and its analogs that allosterically inactivate the repressor and lead to
expression of the lac genes are termed inducers.
•Each tetrameric Lac repressor molecule contains four inducer-binding allosteric
sites.
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6. Allolactose
•Allolactose is an isomer of lactose. Small amounts of
allolactose are formed when lactose enters E. coli.
•a combination of galactose and glucose with an α-1,6
rather than an α-1,4 linkage.
•Allolactose binds to an allosteric site (core region) on
the repressor protein causing a conformational change.
•The repressor can no longer bind to the operator region
and falls off.
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7. IPTG
•Isopropyl β-D-1-thiogalactopyranoside
•Molecular mimic of allolactose
•used to induce protein expression in E.coli where the gene is
under the control of the lac operator.
•IPTG binds lac repressor and releases the tetrameric repressor
from the lac operator in an allosteric manner, thereby allowing the
transcription of genes in the lac operon.
•Used in conjunction with X-Gal or Bluo-Gal in blue-white
selection of recombinant bacterial colonies.
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8. •IPTG bind to the lac repressor similar to
allolactose and reduces the affinity of lac
repressor for DNA 1000 times.
•The binding of IPTG induces structural changes
that alter the relation between the two DNA-
binding domains so that they cannot interact
effectively with DNA.
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9. •IPTG is a gratuitous inducer, which cannot be hydrolysed by the enzyme β-
Galactosidase. It induces the E. coli lac operon activity by binding and inhibiting the lac
repressor without being degraded.
•Genes controlled by the lac or tac promoter/operator sequences are expressed to high
levels in the presence of IPTG.
•For induction, a sterile, filtered 1 M solution of IPTG is typically added by 1:1000
dilution into an exponentially growing bacterial culture, to give a final concentration of
1mM. However, different concentrations of IPTG may also be used.
•It is recommended for blue/white screening by lacZ α-complementation with
appropriate vectors and host strains, and for protein expression with BL21 (DE3) cells
and other lac promoter-derived expression systems.
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10. Expression of the Lac Operon also determined by levels of glucose and lactose in
the cell.
•If glucose is present, the cell will prefer to use it as an energy source.
•For the RNA Polymerase to properly attach, the CAP-cAMP complex must be attached to
the DNA, and the LacI repressor must not be attached to the operator site. therefore,
transcription only occurs when lactose, but not glucose, is present.
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11. Lac Promoters
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12. Lac Promoters
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14. Commonly used hosts for IPTG induction
•BL21(DE3) E.coli strains
•carries a λDE3 lysogen that carries the gene for T7 RNA polymerase
under control of the lacUV5 promoter.
•direct high-level expression of cloned genes under the control of the
T7 promoter
•BL21(DE3)pLysS E.coli strains
•Additional gene coding for T7 lysozyme which inactivates the T7
promoter.
•This keeps background expression levels of T7 RNA polymerase low.
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16. Applications
•Blue/white screening
•Induction of lac operon for protein
expression
•Genes controlled by the lac or tac
promotor/operator sequences are
expressed to high levels in the
presence of IPTG
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