Contribution

Existing Part Description

BBa_J428028 repressor protein plays a crucial role in our system, as it mediates signal transmission from the sensor plasmid to the reporter plasmid. It was important for us to evaluate the activity of this protein in the presence of tetracycline and to determine the optimal tetracycline concentration required for proper system function.

We analyzed the TetR gene activity under varying promoter strengths and tetracycline concentrations to determine the threshold of antibiotic resistance. The maximum survival concentration was found to be 10 µg/mL, regardless of the promoter used.

Learn more in Part Documentation.

New Part: Inducible Chloramphenicol-Resistance Plasmid

As part of our project, we created a new inducible selection module — a plasmid carrying an antibiotic-resistance system controlled by a tetracycline promoter. This construct functions as a reversible selection or inverted screening system, expanding the utility of the Ptet/TetR regulatory framework.

The principle of operation of this new part is as follows:

• In the absence of TetR, the tetracycline promoter is active, the chloramphenicol-resistance gene is expressed, and cells survive on selective medium.
• In the presence of TetR, the promoter is repressed, chloramphenicol resistance is not expressed, and cell growth is inhibited.

This design enables real-time monitoring of TetR expression dynamics and provides a modular tool for assessing the repression efficiency of dual dCas-based systems. In our experiments, we used this construct to evaluate the efficiency of TetR inhibition mediated by paired Cas complexes. Testing in E. coli Top10 cells on LB medium containing chloramphenicol confirmed the correct on/off behavior of the system, both independently and in combination with a TetR-expressing plasmid.

Beyond our specific application, this part can be employed in various areas of synthetic-biology circuit design — for example:

• to monitor repressor stability,
• to construct logic-gated regulatory networks,
• or as a controllable clone-selection module in complex assemblies.

Learn more in Part Documentation.

Contribution to the iGEM Community

By developing this tetracycline-regulated chloramphenicol-resistance system, we provide a versatile tool for teams working with inducible control of antibiotic markers or feedback regulation based on the Ptet/TetR axis.
Our construct can serve as a modular selection backbone for diverse gene-regulatory studies, particularly where reversible antibiotic selection is needed.

This new plasmid is planned for submission to the iGEM Registry under a dedicated entry as a new Part, accompanied by full characterization data and documentation.
Its design and behavior were validated both in vitro and in vivo, demonstrating robustness and reproducibility.