Episome-Based Gene Expression Modulation Platform in the Model Diatom Phaeodactylum tricornutum.

Abstract

Chemically inducible gene expression systems have been an integral part of the advanced synthetic genetic circuit design and are employed for precise dynamic control over genetically engineered traits. However, the current systems for controlling transgene expression in most algae are limited to endogenous promoters that respond to different environmental factors. We developed a highly efficient, tunable, and reversible episome-based transcriptional control system in the model diatom alga, Phaeodactylum tricornutum. We assessed the time- and dose-response dynamics of each expression system using a reporter protein (eYFP) as a readout. Using our circuit configuration, we found two inducible expression systems with a high dynamic range and confirmed the suitability of an episome expression platform for synthetic biological applications in diatoms. These systems are controlled by the presence of β-estradiol and digoxin. Addition of either chemical to transgenic strains activates transcription with a dynamic range of up to ∼180-fold and ∼90-fold, respectively. We demonstrated that our episome-based transcriptional control systems are tunable and reversible in a dose- and time-dependent manner. Using droplet digital polymerase chain reaction (PCR), we also confirmed that inducer-dependent transcriptional activation starts within minutes of inducer application without any detectable transcript in the uninduced controls. The system described here expands the molecular and synthetic biology toolkits in algae and will facilitate future gene discovery and metabolic engineering efforts.