MultiGreen: A multiplexing architecture for GreenGate cloning

Abstract

Genetic modification of plants fundamentally relies upon customized vector designs. The ever-increasing complexity of transgenic constructs has led to increased adoption of modular cloning systems for their ease of use, cost effectiveness, and rapid prototyping. GreenGate is a modular cloning system catered specifically to designing bespoke, single transcriptional unit vectors for plant transformation-which is also its greatest flaw. MultiGreen seeks to address GreenGate's limitations while maintaining the syntax of the original GreenGate kit. The primary limitations MultiGreen addresses are 1) multiplexing in series, 2) multiplexing in parallel, and 3) repeated cycling of transcriptional unit assembly through binary intermediates. MultiGreen efficiently concatenates bespoke transcriptional units using an additional suite of level 1acceptor vectors which serve as an assembly point for individual transcriptional units prior to final, level 2, condensation of multiple transcriptional units. Assembly with MultiGreen level 1 vectors scales at a maximal rate of 2*⌈log6n⌉+3 days per assembly, where n represents the number of transcriptional units. Further, MultiGreen level 1 acceptor vectors are binary vectors and can be used directly for plant transformation to further maximize prototyping speed. MultiGreen is a 1:1 expansion of the original GreenGate architecture's grammar and has been demonstrated to efficiently assemble plasmids with multiple transcriptional units. MultiGreen has been validated by using a truncated violacein operon from Chromobacterium violaceum in bacteria and by deconstructing the RUBY reporter for in planta functional validation. MultiGreen currently supports many of our in-house multi transcriptional unit assemblies and will be a valuable strategy for more complex cloning projects.

Fig 1. MultiGreen 1.0—Multiplexing in series.

A) Schematic overview of a standard GreenGate reaction condensing six entry vectors, AB Promoter, BC N-tag, CD GOI, DE C-tag, EF terminator, FG resistance, into a destination vector using a one-pot BsaI mediated restriction ligation reaction. B) MultiGreen 1.0 expansion modules introduce the H overhang as a split of two new Level 0 entry vectors, the FH module is reserved for transcription blockers and the HG module is reserved for the multiplexer. The HG multiplexer module contains a pair of Esp3I sites internal to the BsaI sites for conventional GreenGate assembly, but external to the chromoprotein reporter. Each assembly that incorporates the level 0 HG and FH modules for multiplexing can be visually selected for integration of the multiplexer module. Subsequent MultiGreen 1.0 reactions should alternate visual reporters to efficiently screen one assembly round from the next as the chemical selection is set by the initial destination vector. The assembly for the final transcriptional unit should either include the FH transcription blocker and HG filler modules to terminate the multiplexing, or alternatively a level 0 FG plant selectable marker module can be included. C) Detailed view of how the multiplexer operates by incorporating an external set of Esp3I sites flanking a visual reporter. Inclusion of the FH and HG modules in lieu of the FG followed by selection for colonies expressing the visual reporter enables iterative stacking in series.

Fig 2. MultiGreen 2.0—Multiplexing in parallel.

A schematic overview of multiplexing in parallel with MultiGreen 2.0. MultiGreen 2.0 uses a suite of level 1 acceptor vectors to produce initial concatenations of entry vectors. The level 1 acceptor vectors are not ampicillin resistant, allowing for chemical counterselection from level 0 entry vectors. Level 1 acceptor vectors are binary vectors themselves and can be used directly in Agrobacterium for (co)infection. Up to 6 Level 1 assemblies can be combined per reaction. By alternating selection choice of level 1 acceptor vectors, assemblies can be iteratively combined in multiples of 6. Note that alternating selection of level 1 acceptor vectors is only necessary for assemblies with >6 transcriptional units. Conventional GreenGate level 0 entry modules can be incorporated in any level 2 assembly with compatible overhangs, provided both Type IIS enzymes are included in the one-pot reaction cocktail.

Fig 3. Precise mechanism of MultiGreen 2.0 cloning and reversion to level 0 modules from level 1 assemblies.

A series of level 1 acceptor vectors are made with complimentary overhangs for each GreenGate entry module (AB, BC, CD, DE, EF, FG). A) The above example is for a MG2.0 AB assembly. A standard GreenGate cloning reaction is performed using the MG2.0 AB intermediary vector as denoted in A. The product of that assembly will hold the first gene cassette in the series. By the nature of the flanking Type IIS restriction enzyme sites, the recognition sequence for BsaI is dropped out, but the 4bp overhangs are retained. This intermediary MG2.0 AB vector containing Gene cassette 1 can be used directly in a level 2 assembly incorporating Esp3I, be digested and used as an AB fragment in a subsequent GreenGate reaction or may be B) ligated into a GreenGate AB entry vector backbone effectively converting the level 1 assembly into a level 0 module.

Fig 4. MultiGreen 2.0 cloning validation through protoviolaceinate biosynthesis.

A) The full violacein operon and its product alongside two truncations producing different pigmented metabolites. B) Example Level 0 entry vectors for assembling the vioABDE operon using MultiGreen 2.0. Not pictured are the filler sequences to bridge overhang gaps (FG filler for level 1; FG eGFP cassette and CD filler for Level 2). Two different synthetic promoters and the bacteriophage T1 rrnB terminator were chosen to drive expression of the four genes from Chromobacterium violaceum to produce protoviolaceinate. C) A representative DH10B plate of the Level 2 assembly producing protoviolaceinate as noted by the dark colonies. D) Replicated colony counts of the assembly in independent one-pot reactions produced on different days.

Fig 5. Use of the deconstructed RUBY reporter re-assembled via MultiGreen to confirm vector functionality.

A) Workflow for infiltrating and extracting betanin pigments from Nicotiana benthamiana. 3–4 week old Nicotiana benthamiana cv. TW17 plants were infiltrated with Agrobacterium suspension at OD600 ranging from 0, 0.1, 0.2, 0.5, 1.0. Single leaf punches were removed from the infiltration sites. One punch per leaf, three leaves per plant, in three biological replicates performed on different days. Leaf discs were cleared of chlorophyll by soaking in 100% ethanol overnight. Betanin were bled out of the punches by transferring to a 96 well plate containing type I water prior to measuring absorbance at A538 on a Biotek Synergy 2 plate reader. B) Deconstructed RUBY plasmid made using MultiGreen 2.0 stacking the three transcripts of RUBY in tandem. C) Absorbance values for all leaf discs collected. Compact letter display of Tukey HSD comparisons across OD. microtube-open-translucent icon by Servier https://smart.servier.com/ is licensed under CC-BY 3.0 Unported https://creativecommons.org/licenses/by/3.0/.