Subtopic: Fundamentals of Downstream processing (chromatography, filtration)
Question Statement:
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 lacT crp. Even in the absence of inducer IPTG, 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?
Options:
(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
Official Answer Key:
C
Step-by-Step Solution & Explanation
### **Official Solution & Step-by-Step Derivation (GATE BT 2017 Q.42)**
**1. Governing Concept & Question Classification**:
- **Section**: BT
- **Question Type**: **MCQ** (2 Marks)
- **Topic**: Genetic Engineering / Gene Expression Systems (*lac* Operon & IPTG Induction)
**2. Detailed Step-by-Step Analysis**:
- In *Escherichia coli* expression vectors driven by the *lac* promoter (or hybrid promoters like *tac* or *trc*):
- In the uninduced state, the **Lac repressor** (encoded by *lacI*) binds tightly to the *lac* operator ($lacO$), creating a steric DNA loop that physically blocks RNA polymerase from initiating transcription.
- To induce high-level recombinant protein expression, an inducer is added.
- **IPTG (Isopropyl $\beta$-D-1-thiogalactopyranoside)** is an allosteric structural analog of allolactose:
- Unlike lactose, IPTG is a **gratuitous inducer** (it is not metabolized or degraded by $\beta$-galactosidase).
- IPTG binds directly to the Lac repressor protein, triggering a conformational change that drastically reduces its affinity for the *lac* operator.
- The repressor dissociates from the operator, allowing *E. coli* RNA polymerase to transcribe the downstream recombinant gene at maximum velocity.
- **Option-by-Option Evaluation**:
- **Option (A) [INCORRECT]**: Glucose lowers cAMP levels (catabolite repression) and represses, rather than induces, the *lac* promoter.
- **Option (B) [INCORRECT]**: Lactose can induce expression, but it is rapidly metabolized by the host cells, leading to erratic and unsustainable induction compared to gratuitous inducers.
- **Option (C) [CORRECT]**: IPTG binds the Lac repressor, inducing its dissociation from the operator and driving high-level recombinant gene transcription.
- **Option (D) [INCORRECT]**: Arabinose induces the *araBAD* promoter ($P_{\text{bad}}$), not the *lac* promoter.
**3. Final Answer / Verification Summary**:
- **Official GATE Answer Key**: **C**
- **Scoring Criteria**: +2.0 Marks (Negative marking: -0.67 marks).
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