Synthetic Assembly DNA Cloning of Multiplex Hextuple Luciferase Reporter Plasmids

Authors

Alejandro Sarrion-Perdigones
Yezabel Gonzalez
Koen J T Venken

Publication Date

1-1-2022

Journal

Methods in Molecular Biology

DOI

10.1007/978-1-0716-2453-1_32

PMID

35821490

PMCID

PMC10155188

PubMedCentral® Posted Date

5-3-2023

PubMedCentral® Full Text Version

Author MSS

Published Open-Access

yes

Keywords

Cloning, Molecular, DNA, Genes, Reporter, Luciferases, Plasmids, Transcription Factors, Synthetic assembly DNA cloning, Goldenbraid cloning, Type IIs restriction enzymes, BsaI, BsmBI, T4 DNA ligase, luciferase assaying, multiplex

Abstract

Multiplex hextuple luciferase assaying allows monitoring the activity of five experimental pathways against one control at the same time. To perform multiplex hextuple luciferase assaying, six orthogonal luciferase reporter units are needed of which five are pathway-specific and one acts as a control for normalization. To ensure stoichiometric delivery of all six luciferase reporters in every transfected cell, synthetic assembly DNA cloning is used to stitch together all six luciferase reporter units into a single vector. Here, we provide a detailed three-step synthetic assembly DNA protocol to generate multiplex hextuple luciferase reporter plasmids for any five cellular signaling pathways of interest, against a control normalization pathway. A first protocol is provided on how to generate plasmids that contain novel transcription factor-binding motifs for specific transcription factors. A second protocol details on how to couple these novel transcription factor-binding motifs to one of five orthogonal luciferases to obtain specific luciferase reporters for cellular signaling pathways acting upstream of those transcription factor-binding motifs. Finally, a third protocol provides details on how to assemble orthogonal luciferase reporters for five cellular signaling pathways acting upstream of five unique transcription factor-binding motifs together with a control constitutive pathway luciferase reporter that will be used for normalization to obtain a final multiplex hextuple luciferase vector.