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Synthetic Assembly DNA Cloning of Multiplex Hextuple Luciferase Reporter Plasmids.

Alejandro Sarrion-Perdigones¹, Yezabel Gonzalez¹, Koen J T Venken^2,3,4,5,6

¹Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, USA.

Methods in Molecular Biology (Clifton, N.J.)

|July 13, 2022

Summary

This study introduces a three-step synthetic DNA cloning protocol for multiplex hextuple luciferase assays. This method enables simultaneous monitoring of five signaling pathways and one control pathway using a single DNA vector.

Keywords:

Luciferase assaying Multiplex Synthetic assembly DNA cloning T4 DNA ligase Type IIs restriction enzymes

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Area of Science:

Molecular Biology
Biotechnology
Cellular Signaling

Background:

Multiplex assays enable simultaneous monitoring of multiple biological processes.
Current methods for multiplex luciferase assays require multiple vectors or lack pathway specificity.
Efficient delivery of multiple reporter units is crucial for accurate multiplex assays.

Purpose of the Study:

To develop a streamlined protocol for generating multiplex hextuple luciferase reporter plasmids.
To enable simultaneous monitoring of five distinct cellular signaling pathways alongside a control.
To facilitate the study of complex cellular signaling networks through a single, unified reporter system.

Main Methods:

Synthetic assembly DNA cloning was employed to construct a single vector containing six orthogonal luciferase reporter units.
A three-step protocol was established: 1) generating transcription factor-binding motif plasmids, 2) coupling motifs to luciferases for pathway-specific reporters, and 3) assembling five pathway reporters with one control reporter.
Orthogonal luciferases and unique transcription factor-binding motifs were utilized for pathway specificity and normalization.

Main Results:

A detailed three-step protocol for generating multiplex hextuple luciferase reporter plasmids was successfully established.
The protocol allows for the creation of vectors capable of monitoring five user-defined signaling pathways and one constitutive control pathway.
Synthetic assembly DNA cloning ensures stoichiometric delivery of all six luciferase reporters into transfected cells.

Conclusions:

The developed protocol provides a robust and efficient method for constructing multiplex hextuple luciferase reporter vectors.
This system simplifies the simultaneous analysis of multiple cellular signaling pathways, enhancing experimental throughput and data accuracy.
The generated reporter plasmids are versatile tools for investigating complex biological pathways and their interactions.