Golden Standard: a complete standard, portable, and interoperative MoClo tool for model and non-model proteobacteria

Abstract

Modular cloning has become a benchmark technology in synthetic biology. However, a notable disparity exists between its remarkable development and the need for standardization to facilitate seamless interoperability among systems. The field is thus impeded by an overwhelming proliferation of organism-specific systems that frequently lack compatibility. To overcome these issues, we present Golden Standard (GS), a Type IIS assembly method underpinned by the Standard European Vector Architecture. GS unlocks modular cloning applications for most bacteria, and delivers combinatorial multi-part assembly to create genetic circuits of up to twenty transcription units (TUs). Reliance on MoClo syntax renders GS fully compatible with many existing tools and it sets the path towards efficient reusability of available part libraries and assembled TUs. GS was validated in terms of DNA assembly, portability, interoperability and phenotype engineering in α-, β-, γ- and δ-proteobacteria. Furthermore, we provide a computational pipeline for parts characterization that was used to assess the performance of GS parts. To promote community-driven development of GS, we provide a dedicated web-portal including a repository of parts, vectors, and Wizard and Setup tools that guide users in designing constructs. Overall, GS establishes an open, standardized framework propelling the progress of synthetic biology as a whole.

Graphical Abstract

Figure 1.

Golden Standard fusion sites. Modules can be combinatorically assembled into a level 1 host vectors to make a transcription unit.

Figure 2.

Structure and nomenclature of GS pSEVA vectors (Supplementary Table S1). Following SEVA nomenclature, GS pSEVA vectors are named by 3 digits where the first number denotes the antibiotic resistance marker (blue), the second number identifies the origin of replication (orange), and the third number identifies the cargo (green). Golden Standard cargo is denoted with the number 19. In addition, vector level 1 (Lv1) receptors, level 2 (Lv2) receptors and level 3 (Lv3) receptor are named with the letter g followed of numbers, capital letters and Greek letters, respectively. The position of the fusion sites (FSs) of each specific vector and the basic structure of the cargos of each type of receptor vector (Lv1, Lv2 and Lv3) is indicated. Sequences of the BsaI FSs (blue) and BpiI FSs (red) are shown in the right tables.

Figure 3.

Hierarchy and graphical description of Golden Standard assembly. Hierarchy of Golden Standard assembly ranges from basic DNA parts (level 0), which are assembled to generate transcription units (level 1), which in turn can be assembled into more complex genetic circuits (level 2 and level 3). A Golden Standard reaction consists of a series of restriction/ligation cycles, whose operation is the same, regardless of the level under construction. Restriction steps release the genetic features flanked by the corresponding Type IIS enzyme (BsaI or BpiI), and removes the lacZ gene from the receptor vector. Ligation steps assemble the genetic features in a pre-defined order into the receptor vector.

Figure 4.

Portability of Golden Standard assembly in Proteobacteria. GFP fluorescence of cells carrying plasmids with RK2 (red), pBBR1 (blue) or pUC (green) origins of replication. Negative control is shown in black.

Figure 5.

Characterization of GS parts in E. coli W and P. putida KT2440. GS constitutive promoters (A), inducible expression systems (B), RBS (C) and terminators (D) characterization. For promoters and RBS, absolute part activity values are given. Inducible expression systems parameters were computed using Hill's equation, where y_max is maximum activity, EC50 is half maximal effective concentration, DR is dynamic range, and OR is operational range. For terminators, efficiency was measured by computing the ratio of GFPmut3/mRFP1 and normalizing it to a non-terminator linker (0% efficiency). Each point represents a single replicate well from two independent experiments. Error bars indicate standard deviation.

Figure 6.

(A) PHB operon construction combining Golden Standard with MoClo. Level 0 parts for PHB production previously constructed with the MoClo toolkit were assembled in level 1 MoClo vectors to obtain three TUs. Afterwards, these Tus were assembled in three level 2 GS pSEVA plasmids with RK2, pBBR1 or pUC origins of replication. (B) PHB granules present in E. coli DH10B cells harbouring plasmids pSEVA6219[gB] PHB (8% PHB), pSEVA63g19[gB] PHB (13% PHB) and pSEVA6819[gB] PHB (50% PHB). Image captured using phase contrast microscopy.

Figure 7.

Combinatorial assembly of the zeaxanthin biosynthetic pathway using the GS system. (A) Scheme of the heterologous zeaxanthin biosynthetic pathway in E. coli from glyceraldehyde 3-phosphate (G3P) and pyruvate using enzymes from P. agglomerans: FPP, farnesyl pyrophosphate; IPP, isopentyl diphosphate; GGPP, geranylgeranyl diphosphate; crtE, geranygeranyl synthase; crtB, phytoene synthase; crtI, phytoene desaturase; crtY, lycopene cyclase; crtZ, b-carotene hydroxylase. (B) Scheme of the assembly of level 0 parts and level 1 TUs for zeaxanthin production with multiple level 0 Anderson promoter parts (http://parts.igem.org/Promoters/Catalog/Anderson) into a polycistronic level 2 genetic device using Golden Standard assembly. (C) LB petri dish plate image showing colonies of E. coli cells transformed with the mix of level 2 parts for zeaxanthin production. Yellow colonies are positive colonies producing zeaxanthin, while the blue colonies are negative colonies carrying the empty vector. (D) Restreaking of different intensity yellow colonies selected by eyesight for subsequent analysis of the promoters used (1 to 5), 0, negative control colony. (E). Heat map showing the combination of Anderson's promoters for each CDS part in the five selected colonies.

Figure 8.

Schematic diagram and results of N- and C-terminal tag assemblies. (A) Graphical representation of individual MoClo assemblies of the OleD variants tested. (B) Combined plotted results (conversion of xanthohumol to 4′-O-β-d-glucoside of xanthohumol versus fluorescence of GFP-tag) in cell-free extracts from random clones obtained using mixed MoClo assembly with different N-terminal tags. (C) Graphical representation of MoClo assembly of 2xN- and 2xC protein with calculated MW masses of proteins; (D) SDS-PAGE analysis – on each well 1 ug of proteins was resolved. L – protein ladder; 1 – P. putida crude protein extact; 2 – eluted fraction after IMAC purification; 3 – HRV-3C digestion reaction; 4 – proteins not bound to Strep-Tactin column; 5 – proteins eluted from Strep-Tactin column. Photograph of fractions 3–5 irradiated with UV light are shown.