42. A recombinant protein is to be expressed under the control of the lac promoter and operator in a
strain of E. coli having the genotype lacI+ crp+. Even in the absence of inducer IPTC, low levels of
expression of the recombinant protein are seen (leaky expression). Which one of the following should
be done to minimize such leaky expression?
(a) Addition of lactose to the medium
(b) Removal of all glucose from the medium
(c) Addition of excess glucose to the medium
(d) Addition of allo–lactose to the medium
Leaky expression occurs when low levels of a recombinant protein leak out under the lac promoter in E. coli even without IPTG inducer, due to incomplete repression by LacI repressor in the lacI+ crp+ strain. The correct approach to minimize this involves catabolite repression via glucose. Option (c) Addition of excess glucose to the medium achieves tightest control.
Lac Operon Basics
The lac operon in E. coli regulates lactose metabolism genes through the lac promoter and operator. LacI repressor (from lacI+) binds the operator without inducer like IPTG or lactose, blocking transcription, but imperfect binding allows basal leaky expression. CRP+ (cAMP receptor protein) activates the promoter when bound to cAMP during glucose scarcity, enhancing any leakiness.
Option Analysis
Each option impacts lac operon differently in uninduced conditions:
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(a) Addition of lactose to the medium: Lactose (or allolactose) binds LacI, releasing it from the operator and inducing expression, worsening leakiness instead of minimizing it.
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(b) Removal of all glucose from the medium: Glucose absence raises cAMP levels, enabling CRP-cAMP to bind and activate the promoter, increasing leaky expression.
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(c) Addition of excess glucose to the medium: Glucose represses adenylate cyclase, lowering cAMP and preventing CRP activation (catabolite repression or glucose effect). This strengthens LacI repression, minimizing leakiness effectively.
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(d) Addition of allo-lactose to the medium: Allolactose, the natural LacI inducer, relieves repression like IPTG, causing high expression rather than control.
Why Glucose Works Best
Excess glucose triggers catabolite repression, prioritizing glucose metabolism over lac genes. Low cAMP blocks CRP binding upstream of the promoter, reducing basal transcription >1000-fold in lacI+ strains. This suits recombinant protein production in biotech, avoiding metabolic burden from leaks.
Applications in Biotech
In fermentation for molecular biology or biochemical engineering, glucose addition optimizes yields by enabling tighter control before deliberate induction. Users studying microbial biotechnology can apply this for experiments in Jaipur labs or similar setups.
