Researchers have established a rapid and robust method for quantifying genetic parts in plants utilizing relative promoter units (RPUs). This method addresses the challenges of high variability in transient expression systems, specifically protoplast transfection.
The study began by setting up the Arabidopsis leaf mesophyll protoplast transfection system, quantifying various native promoters with firefly luciferase (LUC) as the reporter. They tested truncated versions of the 35S promoter, discovering that a 200-bp version maintained high strength while others showed reduced activity.
To minimize output variations, a normalization module with a β-glucuronidase (GUS) protein was integrated. This allowed for a consistent trend across seven promoters and reduced variations significantly. However, batch variations remained evident.
The researchers then introduced the concept of RPUs, defining the LUC/GUS value of the 200-bp 35S promoter as 1 RPU. This standardization greatly reduced experimental variations, facilitating reproducible analyses.
To expand the library of genetic parts, the team designed synthetic promoters that can be repressed by specific repressors, forming the basis for building NOT gates in genetic circuit design. They evaluated various repressor-promoter pairs and identified those with the highest dynamic range.
Additionally, the study involved the development of sensors to measure input-output characteristics for genetic parts, demonstrating the system's capability to construct complex Boolean genetic circuits. The researchers successfully predicted circuit performance and verified their models through in vivo applications in Arabidopsis plants.
Furthermore, the research explored the potential for cross-species applications by testing the circuits in Nicotiana benthamiana, showing promising results in controlling cell death in response to pathogen attacks.
This innovative approach to genetic circuit design and quantification in plants may pave the way for advanced genetic engineering applications, enhancing our ability to manipulate plant traits for agricultural improvement.