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Massively Parallel Reporter Assays in Cultured Mammalian Cells

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Validating a Promoter Library for Application in Plasmid-Based Diatom Genetic Engineering.

Erin A Garza¹, Vincent A Bielinski¹, Josh L Espinoza¹

¹J. Craig Venter Institute, La Jolla, California 92037, United States.

ACS Synthetic Biology

|October 19, 2023

Summary

Researchers developed a new method to discover genetic parts for diatoms, enabling faster and more flexible synthetic biology applications. This expands the available tools for engineering these promising microorganisms.

Keywords:

diatom episomal gene expression forward genetics genetic engineering parts registry promoter characterization

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Published on: November 29, 2024

Area of Science:

Synthetic biology
Microbial engineering
Algal biotechnology

Background:

Diatoms are valuable for synthetic biology but lack sufficient genetic regulatory parts.
Traditional DNA transformation methods (biolistic particle bombardment) are inefficient and lead to variability.
Episomal plasmids delivered by bacterial conjugation offer a more stable and flexible alternative for diatom engineering.

Purpose of the Study:

To expand the genetic toolbox for plasmid-based engineering in the diatom *Phaeodactylum tricornutum*.
To develop a high-throughput method for discovering novel promoter elements.
To characterize and rank promoter-terminator pairs for reporter gene expression.

Main Methods:

Developed a conjugation-based forward genetics screen for promoter discovery.
Applied the screen to a diatom genomic DNA library to identify promoter elements.
Delivered 40 promoter/terminator pairs via conjugation on episomal plasmids.
Characterized reporter gene expression in vivo and ranked elements across four orders of magnitude.

Main Results:

Identified 252 *P. tricornutum* promoter elements from a genomic DNA library.
Successfully delivered and characterized 40 promoter/terminator pairs using episomal plasmids.
Demonstrated a wide range of reporter gene expression levels, spanning four orders of magnitude.
Validated the utility of conjugation and episomal plasmids for rapid genetic part discovery.

Conclusions:

The developed conjugation-based screen significantly expands the available genetic regulatory parts for diatom engineering.
Episomal plasmids combined with modular DNA assembly offer a flexible and rapid platform for diatom synthetic biology.
This work provides a valuable resource for advancing genetic engineering in diatoms like *P. tricornutum*.