Controlled expression of click beetle luciferase using a bacterial operator-repressor system

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• Bioluminescence using luciferase genes is a versatile tool for microorganism detection.
• Stable genetic constructions are needed for reliable microbial tagging and monitoring.
• Controlling reporter gene expression in environmental conditions is crucial for sensitive detection.

Purpose of the Study:

• To construct a genetic system for stable introduction and inducible expression of the click beetle luciferase gene (lucOR) in Gram-negative bacteria.
• To enable sensitive microorganism detection through controlled bioluminescence.
• To ensure stable maintenance of the luciferase marker in various bacterial strains.

Main Methods:

• Development of miniTn5-based suicide vectors carrying the lacIq repressor gene and a Ptrc::lucOR fusion.
• Transposition of the miniTn5-lucOR construct into various Gram-negative recipient bacteria via mating with Escherichia coli.
• Induction of luciferase expression using Isopropyl β-D-1-thiogalactopyranoside (IPTG).

Main Results:

• Successful transposition of the miniTn5-lucOR construct into recipient bacteria with frequencies ranging from 10(-7) to 10(-5).
• Demonstrated IPTG-inducible luciferase activity in tagged bacterial strains.
• Observed strain-dependent stringency of regulation and light emission intensity.

Conclusions:

• The developed system allows for stable maintenance of the luciferase marker in Gram-negative bacteria.
• Tight control over luciferase expression is achieved through IPTG induction.
• This inducible bioluminescent system offers a sensitive and versatile method for microorganism detection and monitoring.