Scientific Context: TEM-1 β-Lactamase Expression and Inhibitor Screening

Overview

This experiment asks a sequential scientific question:

Can we produce an active TEM-1 β-lactamase enzyme, identify compounds that inhibit it, and use the first experimental results to design a more informative second round?

The experimental pipeline contains five linked stages:

1. Express the protein using cell-free protein synthesis.
2. Confirm protein expression using the sfGFP fluorescent reporter.
3. Confirm enzyme activity using nitrocefin.
4. Screen compounds for reduced TEM-1 activity.
5. Characterize promising compounds using dose-response experiments and estimate their IC₅₀ values.

Each assay answers a different question. GFP fluorescence indicates that protein was produced, but does not prove that the TEM-1 portion of the fusion is enzymatically active. Nitrocefin cleavage establishes activity. Reduced nitrocefin cleavage in the presence of a compound provides evidence of inhibition.

Biological background

TEM-1 is a β-lactamase: an enzyme that hydrolyzes the β-lactam ring found in penicillins and related antibiotics. This prevents the antibiotic from reaching its bacterial target. Inhibiting TEM-1 can restore the activity of otherwise ineffective β-lactam antibiotics.

Nitrocefin is a chromogenic β-lactam substrate used to measure β-lactamase activity. Before cleavage, nitrocefin is predominantly yellow. Hydrolysis by TEM-1 produces a red product that can be measured through increased absorbance.

The rate at which absorbance increases is proportional to enzyme activity:

• Fast increase in absorbance: high TEM-1 activity.
• Slower increase: partial inhibition or reduced enzyme activity.
• Little or no increase: strong inhibition, no active enzyme, or an assay failure.

DNA constructs