GB3.0: a platform for plant bio-design that connects functional DNA elements with associated biological data

Abstract

Modular DNA assembly simplifies multigene engineering in Plant Synthetic Biology. Furthermore, the recent adoption of a common syntax to facilitate the exchange of plant DNA parts (phytobricks) is a promising strategy to speed up genetic engineering. Following this lead, here, we present a platform for plant biodesign that incorporates functional descriptions of phytobricks obtained under pre-defined experimental conditions, and systematically registers the resulting information as metadata for documentation. To facilitate the handling of functional descriptions, we developed a new version (v3.0) of the GoldenBraid (GB) webtool that integrates the experimental data and displays it in the form of datasheets. We report the use of the Luciferase/Renilla (Luc/Ren) transient agroinfiltration assay in Nicotiana benthamiana as a standard to estimate relative transcriptional activities conferred by regulatory phytobricks, and show the consistency and reproducibility of this method in the characterization of a synthetic phytobrick based on the CaMV35S promoter. Furthermore, we illustrate the potential for combinatorial optimization and incremental innovation of the GB3.0 platform in two separate examples, (i) the development of a collection of orthogonal transcriptional regulators based on phiC31 integrase and (ii) the design of a small genetic circuit that connects a glucocorticoid switch to a MYB/bHLH transcriptional activation module.

Assessment of Cas12a‐mediated gene editing efficiency in plants

“…After the first wave of tools based on Cas9, which was shown capable of producing edited mutants in virtually every species tested, a second generation of refined tools is being progressively incorporated into the genome editing toolbox. These new tools are emerging in parallel with a number of modular cloning methods that facilitate the customization of often multiplexed editing constructs (Engler et al ., ; Peccoud et al ., ; Sarrion‐Perdigones et al ., ; Vazquez‐Vilar et al ., ). Thus, following the same design principles adopted earlier to integrate CRISPR/Cas9 into GB modular cloning (Vazquez‐Vilar et al ., ), we adapted Cas12a tools to the so‐called phytobrick standard, adopting both crRNA and Cas12a TUs as level 1 structures to maximize the exchangeability while preserving the combinatorial potential.…”

“…To this end, we mixed equal volumes of Agrobacterium cultures of the P19 suppressor of silencing, the RGEN and the crRNA/sgRNA. These cultures were first grown from glycerol stock for 2 days to saturation; then, 10 μL was subcultivated for 16 h. Next, the cultures were pelleted, resuspended in agroinfiltration buffer (10 m m MES, pH 5.6, 10 m m MgCl 2 and 200 μ m acetosyringone) and adjusted to an optical density of 0.2 at 600 nm [estimated 8 active T‐DNA copies per cell (Vazquez‐Vilar et al ., )]. Finally, the three cultures (P19, RGEN and crRNA/sgRNA) were mixed at equal parts to prepare the agroinfiltration mixture.…”

“…These assembly protocols are detailed in Vazquez‐Vilar et al . (). The generated constructs are included in Table S1.…”

“…Transient expression experiments were performed to test the mutagenesis efficiency at several loci as described in Vazquez‐Vilar et al . (, ). Each locus was assessed separately for the three RGENs (SpCas9, AsCas12a and LbCas12a).…”

Multigene Engineering by GoldenBraid Cloning: From Plants to Filamentous Fungi and Beyond

“…GB comprises a library of DNA parts (phytobricks) used to compose multigene constructs for metabolic engineering, genome engineering, and other synthetic biology applications. Among other elements, this library includes (i) a collection of plant promoters and terminators with documented transcriptional activities (Sarrion‐Perdigones et al., 2013), (ii) a selection of inducible gene expression systems, such as dexamethasone, estradiol, or ethanol (Vazquez‐Vilar et al., 2017), (iii) a number of Cas9 and Cas12a phytobricks for genome editing (Bernabé‐Orts et al., 2019; Vazquez‐Vilar et al., 2016), and (iv) a collection of dCas9‐based programmable transcriptional activators for gene regulation (Selma et al., 2019).…”