Calvache, C. A. | bioRxiv Science

bioRxiv2023

A quantitative autonomous bioluminescence reporter system with a wide dynamic range for Plant Synthetic Biology

Engineered autonomous bioluminescence (EAB) offers many potential applications in Plant Synthetic Biology, notably as in vivo reporter system. Current EAB reporter configurations are limited for quantitative applications due to low dynamic range. We reconfigured the Neonothopanus nambi fungal bioluminescence (NeoLuc) pathway to serve as a high-throughput and inexpensive reporter for quantitative analysis of gene expression. We showed that by configuring the first committed step in the pathway (HispS) as the transcriptional entry point instead of the fungal luciferase, the dynamic range of the output increased dramatically, equaling that of the FLuc/RLuc reporter, and outperforming it in high throughput capacity. Furthermore, the inclusion of an enhanced GFP as normalizer allowed transient ratiometric measurements in N. benthamiana. Fast and rich datasets generated by the NeoLuc/eGFP system enabled us to undertake the optimization of new challenging synthetic gene circuits, including a complex agrochemical/optogenetic dual input switch for tight control of engineered metabolic pathways.

synthetic biology↗

bioRxiv2022

GB_SynP: a modular dCas9-regulated synthetic promoter collection for fine-tuned recombinant gene expression in plants.

Programable transcriptional factors based on the CRISPR architecture are becoming commonly used in plants for endogenous gene regulation. In plants, a potent CRISPR tool for gene induction is the so-called dCasEV2.1 activation system, which has shown remarkable genome-wide specificity combined with a strong activation capacity. To explore the ability of dCasEV2.1 to act as a transactivator for orthogonal synthetic promoters, a collection of DNA parts was created (GB_SynP) for combinatorial synthetic promoter building. The collection includes (i) minimal promoter parts with the TATA box and 5UTR regions, (ii) proximal parts containing single or multiple copies of the target sequence for the gRNA, thus functioning as regulatory cis boxes, and (iii) sequence-randomized distal parts that ensure the adequate length of the resulting promoter. A total of 35 promoters were assembled using the GB_SynP collection, showing in all cases minimal background and predictable activation levels depending on the proximal parts used. GB_SynP was also employed in a combinatorial expression analysis of an auto-luminescence pathway in Nicotiana benthamiana, showing the value of this tool in extracting important biological information such as the determination of the limiting steps in an enzymatic pathway.

synthetic biology↗