MultiGreen: A multiplexing architecture for GreenGate cloning

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.


Introduction
Simple made-to-order gene expression units met research and commercial requirements for the first few decades of transformation technology.These expression units were assembled with traditional cut-and-paste-style molecular cloning [1].In principle, the only requirements are a backbone with a multiple cloning site, access to compatible restriction enzymes for the insert, the consumables for PCR and restriction ligation, and the result is a transcriptional unit with a minimum of four components: the promoter and 5' UTR, coding sequence, terminator and 3' UTR, and vector backbone [2].
Current needs increasingly require multiplexed guides for editing, or multiple enzymes for metabolic engineering or trait-stacking.While cut-and-paste cloning is efficient and largely the basis for many modern cloning strategies, its utility decreases as the number of transcriptional units to be assembled increases.A more streamlined approach to plasmid construction is the reliance on standardized components and the use of modular assembly.
Gateway cloning is one of the earliest methods for modular assembly and employs site-specific recombination for plasmid construction [3].Gateway cloning was commercialized by Invitrogen in the 1990s and relies upon recombinase enzymes from phage λ that can recombine DNA sequences flanked by Gateway recombination sites in a series of reactions-the BP and LR reactions recombining between attB-attP and attL-attR sites, respectively.Additional improvements led to MultiSite Gateway cloning, with which two to four DNA fragments can be combined in a single vector using the BP and LR Clonase enzymes [4].Besides cost, Gateway recombination leaves recombination sites in the final assembly, called scars, that can add 8 to 13 additional amino acids to final reading frames [5].
A more modern approach to modularity is Golden Gate cloning [5].Golden Gate cloning, which uses one step, one pot, rapid assembly, was proposed initially to address the major limitations of Gateway cloning.It relies on Type IIS restriction enzymes that cleave doublestranded DNA outside of their recognition sequence, leaving behind a minimal overhang.By deploying diverse Type IIS sticky overhangs, multiple DNA fragments can be combined unidirectionally in a one-pot reaction using only one restriction enzyme.As many as 52 parts have been assembled in a one-pot reaction with Golden Gate cloning, with approximately 50% efficiency [6].
There are several different Golden Gate cloning systems available that rely on Type IIS restriction enzymes, i.e., AarI/PaqCI, Eco31I/BsaI, and BsmBI/Esp3I, such as GoldenBraid [7], Mobius Assembly [8], GreenGate [9], and MoClo [10], Each of these systems provides varying leverage over the components necessary for gene expression and approach modularity in a similar fashion, with unique grammar.The grammar of each system is determined by four factors 1) the Type IIS enzymes being used, 2) the coordinated overhangs left behind after Type IIS enzyme digestion for directional assembly, 3) the number of units into which individual transcripts are split into for modularity and 4) organization of the different stages of assembly -such as in parallel, in series, or a combination of the two.
Modern modular assembly systems must fulfill two tasks.The first is assembling promoters, coding sequences, and other components into transcriptional units.Second, the resulting transcriptional units must be assembled into a single multigene vector.Hence, each modular cloning system typically relies on multiple stages of assembly.
The initial stage produces "base," "entry," or "level 0" plasmids, each of which contains one component for gene expression, such as a promoter, flanked by Type IIS restriction sites.Subsequent one-pot reactions enable freeing of the components and unidirectional production of individual transcriptional units in another plasmid.Initial assemblies of "level 0" parts are often referred to as "level 1" assemblies and are composed of individual transcriptional units.Level 1 assemblies can be joined together with other level 1 assemblies into a final, "level 2" assembly composed of multiple transcriptional units [7,8,10].Alternatively, level 1 assemblies can be iteratively combined for multiplexing before a final level 2 condensation.
Current modular cloning systems vary in complexity, the exact composition of overhangs, and the Type IIS enzyme responsible for freeing components.Except for GreenGate, all employ multiple Type IIS restriction enzymes to produce a final assembly.While using multiple enzymes can increase the burden of domestication, or the deactivation of naturally occurring Type IIS sites within parts, it enables efficient condensation of multiple transcriptional units either sequentially, or even by "braiding" of assemblies together in a cyclical fashion as in GoldenBraid [7].
GreenGate strikes a balance between ease of use and modularity, enabling control over all the key elements of a cassette for plant expression: promoter, N-tag peptide, coding sequence, C-tag, terminator, and plant selectable marker.Each of these modules are flanked by BsaI sites, leaving seven, unique 4 base pair overhangs for unidirectional assembly-named the A, B, C, D, E, F, and G overhangs.The eighth, optional, H overhang can be introduced through a methylated oligoduplex for multiplexing and is the biggest pitfall of the GreenGate kit.
Therefore, we propose MultiGreen as a multiplexing solution, inspired by the original architecture set forth by GreenGate, to enable intuitive multiplexing while using only the syntax laid forth in the original GreenGate kit.MultiGreen is composed of two approaches to multiplexing: MultiGreen 1.0-assembly in series, and MultiGreen 2.0 assembly in parallel.Multiplexed assembly with MultiGreen is accomplished via a suite of additional vectors that enable condensation of multiple transcriptional units into one final plasmid.Both MultiGreen 1.0 and 2.0 can be combined for more complicated assemblies, or to insert transcriptional blockers [11] between transcriptional units without deviation from the original kit's grammar.MultiGreen also encompasses a suite of linker modules that bridge gaps in parallel designs that don't occupy all the conventional GreenGate overhangs, enabling assembly when some level 0 modules are absent.The suite of MultiGreen plasmids are available through Addgene.
All plasmids were isolated using standard protocols and reagents from the GenCatch plasmid DNA mini-prep kit (Epoch Life Science, Missouri City, TX).Amplification of target sequences under 5 kb were produced using Q5 high fidelity polymerase (NEB # M0491S) under the recommended three-step amplification conditions, while amplicons over 5 kb were produced using PrimeStar GXL polymerase (Takara Bio, San Jose, CA, #R050A) in a two-step amplification reaction.All NEBuilder HiFi assemblies were executed following standard incubation [12] and transformation procedures [13] into their respective destination strains.All products of amplification were sequenced with either Sanger sequencing (Genewiz, South Plainfield, NJ) for regions <1600 bp, or Oxford Nanopore long-read sequencing (Plasmidsaurus, Eugene, OR) for regions >1600 bp.

Transformation of bacterial strains
Commercially available cloning strains of E. coli were used for transformation of all assemblies.The same protocol was used for all chemically competent strains used, adjusting the resuspension medium, outgrowth duration, and antibiotic selection for the strain and resistances being used.Two μL of assembly mix were combined with 10 μL of freshly thawed competent cells and incubated on ice for 30 mins in a 2-mL Eppendorf tube.The bacteria were then heat shocked at 42˚C for 30s and immediately returned to ice for 5 minutes.Two hundred μL of resuspension medium (SOC for DH5α and OneShot cells; NEB 10-beta stable outgrowth medium for 10-beta cells) was added to the tubes.Plasmids selected on ampicillin were immediately plated on LB medium containing ampicillin while all other antibiotic selections were incubated in a 37˚C shaking incubator for a 1-hr outgrowth.E. coli transformations were plated on LB supplemented with 15 g�L -1 BactoAgar and appropriate antibiotics for the plasmid being selected-100 μg�mL -1 ampicillin, 50 μg�mL -1 kanamycin, 25 μg�mL -1 chloramphenicol, 100 μg�mL -1 spectinomycin.Colonies were isolated for liquid culture and sequencing following overnight incubation at 37˚C.

Level 0 entry vector construction
Level 0 entry vectors were created through a scaled down NEBuilder (NEB, Ipswich, MA) reaction.For single insert entry vectors, individual PCR amplicons were produced using Q5 high fidelity polymerase (NEB # M0491S) and isolated from a 0.5X TBE gel by centrifugation of a gel core through an EconoSpin column (Epoch Life Science, Missouri City, TX), at 10000 x g for three minutes.One μL of the eluate was immediately combined with 1 μL of BsaI-HFv2-digested level 0 entry vector normalized to 50 ng�μL -1 and 1 μL of NEBuilder HiFi DNA Assembly Master Mix.Single-insert assemblies were incubated at 50˚C for 20 minutes prior to transformation into chemically competent DH5α cells.A list of primers used for assemblies is in S1 Table .Domestication of Esp3I sites in level 0 inserts was addressed via site-directed mutagenesis with degenerate PCR primers.Briefly, overlapping primers for NEBuilder assembly were designed with a minimum of 15-bp-overlap spanning the restriction site to be domesticated.A single point mutation was introduced within the overlap to disable the site.If the domestication site was within a coding sequence, synonymous mutations were introduced.Following amplification of the two fragments spanning the site, the level 0 entry vector construction proceeded as described with NEBuilder.Components requiring multiple restriction site domestications per insert were incubated at 50˚C for 1 hour prior to transformation, rather than 20 minutes.Further, 0.5 μL of NEBuilder HiFi DNA Assembly Master Mix was added for each additional insert.
As the default GreenGate entry vector plasmids contain two Esp3I sites within 40 bp of each other in the backbone, we created a derivative suite of base level 0 entry vectors eliminating the Esp3I sites.Domestication of these sites is not essential for successful MultiGreen assembly using pre-made level 0 entry vectors, as the overhangs resulting from Esp3I digestion of the entry vector backbone are incompatible with the overhangs for GreenGate assembly.Initial attempts of single-stranded oligo assembly of the digested vector failed, so we combined inverse PCR with single-stranded oligo assembly to disable the two Esp3I sites.The sequences 5'-AGACGAAAGGGCCTCGTGAT-3' and 5'-AGACGGTCACAGCTTGTCTG-3' were used as the forward and reverse primers, respectively, to amplify the entire entry vector using Q5 high fidelity polymerase.The amplicon was isolated as described for level 0 amplicons and combined with 1 μL of a 1:250 dilution of 100 μM single-stranded oligo for recircularization (5'-CAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGA AACGCGCGAGACGAAAGGGCCTCGTGAT-3').One μL of NEBuilder HiFi DNA Assembly Master Mix was then added, and the mix incubated for 1 hour at 50˚C before transformation into E. coli strain DB3.1.
Level 0 entry clones for linker modules not requiring PCR were assembled via a sense/antisense single stranded oligo NEBuilder hybridization.Briefly, single-stranded oligos were designed to contain 20 bp of complementarity to their desired vector backbone, the required Type IIS recognition sequence and overhang, and 15-20 bp of complementarity to the 5' oligo for hybridization.Then, 1:250 dilutions of 100 μM oligos (Millipore Sigma, Burlington, MA) were prepared with type I water.One μL each of the sense and antisense oligo dilutions were then combined with 1 μL of cut level 0 entry vector at 50 ng�μL -1 and 2 μL of NEBuilder HiFi DNA Assembly Master Mix.The reaction mix was then incubated at 50˚C for 1 hour prior to transformation.A list of oligos used for producing linker modules can be found in S1 Table.

Level 1 MultiGreen intermediary vector construction
Level 1 MultiGreen intermediary vectors were made through successive PCR amplification and assembly using NEBuilder.Destination vector pGGP-AG [14] was domesticated of its three Esp3I sites as described, generating pVP076, which was then used as the template in a second round of PCR amplification and NEBuilder assemblies to generate the initial suite of MultiGreen level 1 acceptor vectors.
Six sets of overlapping PCR primers were designed to include both homology to the backbone, pairs of flanking Esp3I and BsaI sites, GreenGate-compatible overhangs, and homology to the ccdB/chloramphenicol cassette to retain the counterselection used in all GreenGate base vectors.Plasmid VP076 was digested in a 10 μL digest using 1 μg of plasmid, 0.5 μL of BsaI-HF-v2, and 1 μL of CutSmart Buffer (NEB #B6004S) for one hour at 37˚C.Following incubation, 2 μL of loading dye were added, the digestion was run on a gel, and the backbone isolated as before.One μL each of PCR amplicon, backbone, and NEBuilder were assembled and transformed into OneShot ccdB survival cells as outlined above, generating pVP078, pVP079, pVP080, pVP081, pVP082, and pVP083 corresponding to MultiGreen level 1 acceptor vectors with final GreenGate-compatible AB, BC, CD, DE, EF, and FG overhangs, respectively.
To make another destination vector, and progenitor to a second suite of MultiGreen intermediary vectors, kanamycin resistance was introduced using restriction ligation of pGGP-AG and NEBuilder assembly with an amplified aph(3')-Ia kanamycin resistance gene from p201NCas9 [15].Destination vector pGGP-AG was digested as above, except with Ava II instead of BsaI, ejecting a 641-bp fragment of the spectinomycin resistance gene from the vector.The kanamycin resistance gene was then amplified using 5'-CGTATGCGCTCACGCAACT GGATGAGCCATATTCAACGG-3' as the forward primer and 5'-AAAGAGTTCCTCCGCCGCT GTTAGAAAAACTCATCGAGC-3' as the reverse primer, sharing a 20-bp overlap with the cut vector, and maintaining the reading frame established by the remaining 5' end of the spectinomycin resistance gene.The NEBuilder assembly was performed and transformed into OneShot ccdB survival cells, and selected on LB kanamycin for successful assembly, generating pGGPK-AG2.
The Esp3I sites of pGGPK-AG2 were domesticated as in pVP076, with an additional pair of overlapping degenerate primers for the Esp3I site within the kanamycin resistance gene to generate pVP077.Plasmid VP077 was subsequently used for its kanamycin-resistant and Esp3Idomesticated backbone to generate a second suite of MultiGreen level 1 acceptor vectors, pVP311, pVP312, pVP313, pVP314, pVP315, pVP316, following the same steps and amplifications as with pPV078-pVP083.pVP076 was also digested and assembled via NEBuilder with a PCR amplicon containing mRFP1E chromoprotein (Addgene #160442) in place of ccdB/CmR, generating pVP096.

Level 1 and Level 2 GreenGate assembly with NEBridge
Level 1 and level 2 assemblies were performed using NEBridge ligase master mix described previously unless otherwise noted [16].Level 1 assemblies with solely GreenGate level 0 entry vectors require only BsaI as the restriction enzyme.Assemblies using previously made MultiGreen level 1 assemblies also required Esp3I in the reaction cocktail to facilitate freeing of level 1 modules.Recurrent level 1 and level 2 assemblies were performed in a one-pot reaction with a total volume of 15 μL.Each overhang in the assembly was be used, either directly through the compatible level 0 module or level 1 assembly, or indirectly via a MultiGreen linker module spanning the unused overhangs.
Three biological replicates of the MultiGreen 2.0 assemblies were constructed and transformed on independent days into NEB 10-beta chemically competent cells.Three serial dilutions of the bacterial culture were plated on LB kanamycin plates and incubated for 40 hours prior to imaging to allow for pigment production.Serial dilutions with discernable colony separation were used for imaging and assembly efficiency quantification.Undigested destination vector carryover (pVP096) was scored for fluorescence of the mRFP1E visual reporter situated between assembly sites.Three random colonies failing to express both the protoviolaceinate pigment and the mRFP1e reporter were sent for whole plasmid sequencing via Plasmidsaurus to evaluate assembly fidelity.Colony counts were tallied, and efficiency calculated in Microsoft Excel for Mac version 16.81 (Microsoft Corporation, Redmond, WA).
Competent cells of an in-house disarmed thyA-recA-variant of Agrobacterium rhizogenes strain K599 (aka NCPPB2659) [20] were prepared as specified in the BioRad MicroPulser™ (Biorad Laboratories, Hercules CA, US) manual.Cells were recovered in liquid YP medium [21] containing 150 mg�L -1 thymidine and no antibiotics for 4 h at 28˚C with shaking.After recovery, the bacterial suspension was plated onto solid YP medium containing 150 mg�L -1 thymidine and 50 mg�L -1 kanamycin.Single kanamycin-resistant colonies growing on the electroporation plate were inoculated into 25 mL liquid YP cultures for overnight incubation at 28˚C with shaking.Agrobacterium suspensions and Nicotiana benthamiana infiltrations were conducted as described previously [11], only deviating by addition of thymidine to all media containing Agrobacterium.The three youngest, expanded leaves were infiltrated with bacterial suspension of OD600 of 0, 0.1, 0.2, 0.5, and 1.0.
Three days after infiltration, betanin production was quantified based on previously outlined experimental procedures [22].Individual 8-mm leaf discs were collected using disposable biopsy punches, one leaf per infiltration site, three leaves per plant, and added to 2-mL Eppendorf tubes containing 2 mL of 100% ethanol for chlorophyll bleaching for a minimum of 24 h.Following bleaching, leaf discs were individually removed, rinsed with fresh 100% ethanol, and added to a 96-well plate (VWR, Radnor, PA, # 10861-562) containing 150 μL of type I water per well.The 96-well plates were allowed to incubate on the bench top for 24 hours, upon which leaf discs were removed and absorbance at 538 nm was measured on a Biotek Syn-ergy2 plate reader (BioTek Instruments Inc., Winooski, VT, USA).Absorbance readings were imported to GraphPad Prism 10 for macOS version 10.2.0 for plotting and analysis.

MultiGreen 1.0-Multiplexing in series
The original GreenGate cloning kit is composed of a suite of entry modules containing convergent pairs of BsaI sites flanking components of interest for one-pot assembly [9].For Golden Gate assembly reactions, it is important to domesticate, i.e., remove all the unnecessary restriction sites for the Type IIS enzyme used for assembly to avoid unintended mis-assemblies.Fortunately, many of the Type IIS restriction enzymes have recognition sequences >5 bp, reducing their frequency of occurrence.
Often, fragments for Golden Gate assemblies are plasmids containing Type IIS sites flanking the insert, or PCR amplicons with compatible cut sites within or near the amplification primers.If plasmids are used as the DNA source, it is common to use alternating antibiotic selections between the parent and child vectors to circumvent carryover, or persistence, of the parent plasmid in subsequent reactions [7,9,10,23].
The canonical entry vectors for GreenGate cloning include A, B, C, D, E, F, and G overhangs in ampicillin-resistant level 0 plasmids to enable unidirectional assembly (Fig 1A).Multiplexing as proposed with the original GreenGate kit introduces the optional H overhang via methylated oligos and linker modules between F and G, thereby multiplexing in series from a 5' !3' direction.For duplex assemblies with GreenGate, one additional overhang can be introduced with a specifically methylated oligoduplex, such that the first transcriptional unit assembled becomes the destination vector for the second transcriptional unit by exploiting the methylation sensitivity of BsaI, effectively blocking digestion of the hidden overhangs in the first iteration of cloning [9].Multiplexing via the methylated oligoduplex strategy is not only cost-prohibitive, but results in a heightened screening burden, as there is no intrinsic counterselection between assemblies.
As an alternative, MultiGreen 1.0 introduces the additional H overhang via two new Level 0 entry vectors, the FH and HG modules (Fig 1B).This approach is possible by incorporating a second Type IIS restriction enzyme, Esp3I.Therefore, all components in an assembly must be domesticated for Esp3I or adequately screened for correct assembly from one reaction to the next.Domestication of existing components can be readily performed by inverse PCR with degenerate primers destroying the undesired cut site followed by restriction-ligation or Gibson assembly.Alternatively, single-stranded oligos have been successfully used in conjunction with digested vectors in a Gibson assembly reaction for site domestication in an oligo-stitching reaction [24].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.https://doi.org/10.1371/journal.pone.0306008.g001 Like BsaI, Esp3I is has a 6-base pair recognition sequence, leaving a 4-base pair sticky overhang.The MultiGreen 1.0 FH module is reserved for a transcription blocker, or alternatively can contain a spacer sequence (Fig 1B).We preconfigured several HG multiplexer modules with visually distinct chromoprotein reporters including meffBlue [25], mRFP1e [26], and spisPink [25].With each iteration of multiplexing, correctly assembled clones possessing the multiplexer module for the next round of assembly can be selected for expression of the respective chromoprotein reporter.The availably of multiple reporters enables recurrent passage of assemblies through visual screening for the reporter included in each assembly step.The chromoproteins that come with MultiGreen 1.0 can be swapped for other reporters, or even antibiotic resistance genes by performing a standard PCR and either restriction-ligation or Gibson assembly.
The chromoproteins within the HG multiplexer were amplified directly from Addgene bacterial expression plasmids.The chromoproteins in the HG multiplexer modules are flanked by convergent BsaI sites, enabling assembly alongside other level 0 entry vectors, as is the case in a conventional GreenGate reaction.Just internal to the BsaI sites, but still external to the chromoprotein, are a pair of divergent Esp3I sites.These remain undigested in the initial assembly, as BsaI is the only restriction enzyme present, effectively providing a future pair of compatible A-G destination overhangs in a subsequent reaction with Esp3I (Fig 1C ).A correct clone expressing the chromoprotein can then be used as the destination vector for a subsequent assembly by digestion with Esp3I, freeing the receptive destination A-G overhangs for another transcriptional unit assembly.
MultiGreen 1.0's time for assembly scales at an approximate rate of 2n +1 days, where n is the number of transcriptional units, and assumes all level 0 components and reagents are available when assembly is started, and that diagnostic screens occur the same day colonies appear.This rate of assembly is tolerable for simple assemblies but draws out when additional transcriptional units are desired.A unique advantage of MultiGreen 1.0 is that if there is a gene cassette containing Esp3I sites that cannot be easily domesticated, such as in regulatory sequences that may need additional validation after domestication, assembly can be structured such that the non-Esp3I domesticated components are in the final reaction, where only BsaI is the active restriction enzyme.

MultiGreen 2.0-Multiplexing in parallel
While MultiGreen 1.0 enabled efficient transcriptional unit stacking following the GreenGate multiplexing grammar, its efficiency of assembly lags for constructs with many transcriptional units.For example, assembling six cassettes with MultiGreen 1.0 would require a minimum of 13 days of cloning to produce the one final vector.Additionally, since MultiGreen 1.0 is multiplexing in series, and each successive reaction becomes the destination vector of the next, if it is desirable to exchange an individual transcriptional unit after assembly, it would be necessary to revert to the assembly just before the unwanted unit and restart incorporating the remaining transcriptional unit.To circumvent the limitations of overall time efficiency of assembly, maximize the compatibility of level 1 assemblies across constructs, and maintain compatibility with the original GreenGate syntax, we propose MultiGreen 2.0 -multiplexing in parallel (Fig 2).
MultiGreen 2.0 takes inspiration from the other modular cloning standards that support multiplexing, such as GoldenBraid [7], Mobius Assembly [8], and MoClo [10] by parallelizing multiplexing with a suite of intermediary level 1 acceptor vectors.MultiGreen 2.0 assemblies are performed into a suite of level 1 acceptor vectors mirroring the overhang syntax of Green-Gate assembly, allowing for concatenation of up to six level 1 assemblies in parallel.Alternation of antibiotic selection allows this to occur indefinitely with adequate planning of which 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.https://doi.org/10.1371/journal.pone.0306008.g002level 1 acceptor vector complement to use, and strategic reversion to level 0 GreenGate entry vectors.Using the same assumptions as in MultiGreen 1.0 for assembly constraints, Multi-Green 2.0 assembly time scales at a rate of 2*dlog 6 ne+3 days, where n is the number of transcriptional units.When comparing the time efficiency between MultiGreen 1.0 and 2.0, that is a time savings of nearly two weeks for a six transcriptional unit assembly, taking a minimum of 5 days with MultiGreen 2.0.By cloning with MultiGreen 2.0, the assembly is also better suited for future proofing, since transcriptional units can be more easily exchanged later.
The level 1 intermediary vectors of MultiGreen 2.0 use an additional pair of flanking Esp3I sites to free assembled transcriptional units for additional rounds of assembly (Fig 3).Each MultiGreen 2.0 level 1 acceptor vector contains a pair of Esp3I sites externally flanking the BsaI sites necessary for a GreenGate single transcriptional unit assembly.When a GreenGate reaction is performed using a MultiGreen 2.0 level 1 acceptor vector as the destination vector, it can then be digested with Esp3I to liberate the assembled transcriptional unit for another GreenGate reaction, since Esp3I digestion of MultiGreen 2.0 level 1 assemblies leaves behind either AB, BC, CD, DE, EF, or FG overhangs, depending on the MultiGreen level 1 intermediary destination vector chosen (Fig 2).The freed overhangs enable level 1 assembly products to act as pseudo-level 0 modules.
At its core, MultiGreen 2.0 enables recursive assembly of GreenGate modules.In other words, it enables performing GreenGate reactions into MultiGreen level 1 acceptor vectors, which when digested with Esp3I, function as level 0 parts (Fig 2).Should transcription blockers be a desirable feature between stacked gene cassettes, the dedicated FH transcription blocker 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.https://doi.org/10.1371/journal.pone.0306008.g003module and HG filler module from MultiGreen 1.0 can be incorporated in each level 1 assembly, resulting in transcription blockers or other spacers being placed between cassettes.The HG filler module allows for GreenGate assembly with the additional H overhang when multiplexing in parallel.
In MultiGreen version 2.0, level 1 acceptor vectors are derived from the pVS1 backbone of pGGP-AG [14] for use as binary vectors directly in Agrobacterium, and each complement of level 1 intermediary vectors has a different antibiotic marker-either spectinomycin or kanamycin.Two antibiotic complements enable the cycling of level 1 assemblies.By alternating kanamycin and spectinomycin selection between reactions, undigested plasmid carryover from one assembly to the next is eliminated.Since Esp3I digestion of level 1 MultiGreen 2.0 assemblies frees level 0-compatible overhangs, the individual transcriptional units released during Esp3I digestion can also be readily converted back to true level 0 modules with T4 ligase.Should a level 0 reversion be desired for a particular assembly, it can be efficiently performed as outlined in S1 Method.A list of all plasmids in the MultiGreen kit and sequence maps can be found in Table 1 and S1 Dataset, respectively.

MultiGreen 2.0 cloning validation in bacteria
As a proof of concept for MultiGreen 2.0 and to quantify the efficiency of a dual restrictionenzyme assembly, we reconstructed the partial violacein operon from Chromobacterium violaceum for production of protoviolaceinate in E. coli.C. violaceum is a species of gram-negative bacterium present various tropical and subtropical areas of the world [27].A unique feature of the bacterium is that it produces the purple antimicrobial compound, violacein.Violacein is the end-product of a five-enzyme cascade beginning with L-tryptophan.Truncations of this operon result in visually distinct pigments [28].Previously, Mobius assembly used protoviolaceinate as a functional validation of multiplexing, but only in validation of a level 1 reassembly of the polycistron [28].
The partial violacein operon from Chromobacterium violaceum was reassembled to produce protoviolaceinate via four independent transcriptional units using MultiGreen 2.0 (Fig 4B).To simplify the level 1 assemblies, level 0 modules were constructed with AC, CD, DF, FG overhangs representing the promoter, coding sequence, terminator, and filler sequence.Simplification of the modules was performed to avoid interruption of the gene products or distances between ribosome binding site, transcriptional start site, and start codon for each of the transcripts.Further, initial challenges with producing level 1 assemblies for vioA and vioD motivated the characterization of four promoter variants using a GFP expression assay (S1 Fig) .Of the four promoter variants characterized, one (pGG-A-PJ23119::PGLPT-C/pVP217) highly expresses eGFP relative to that of a DH5α negative control, while the remaining three all express eGFP at low levels relative to that of the DH5α control (S1 Fig).
Overall efficiency of the parallelized MultiGreen 2.0 assembly for production of protoviolaceinate using both Esp3I and BsaI to concatenate level 1 modules was 95 ± 3% across three replicates (Fig 1D ) as noted by the percentage of colonies expressing the pigment relative to the total of non-carryover colonies.Misassemblies that occurred appeared to consist of escapes resulting in vectors that lack the full vioABDE complement (S3 Table ), potentially indicating a metabolic cost of vioABDE expression in E. coli in this fashion.Further, correctly assembled colonies required approximately 40 h for growth to reach a size comparable to that of an overnight E. coli colony without the vioABDE assembly.

In planta validation through deconstruction of the RUBY reporter
To further validate MultiGreen as a viable strategy to stack genes in one construct for plant transformation, we deconstructed the RUBY visual reporter [17] using MultiGreen 2.0.RUBY is a three-gene polycistronic transcript, encoding three separate peptides on one transcript.RUBY reconstitutes the biosynthetic pathway from Beta vulgaris that converts L-tyrosine into betanin.Each enzyme in the polycistron is separated by self-cleaving P2A peptides [17], using only one promoter and terminator to coordinate expression of all three genes simultaneously.Through a series of MultiGreen parallelized assemblies, we deconstructed the polycistronic transcript for independent transcript expression.The individual transcriptional units of RUBY were combined with the GmUbi3L promoter and the StPinII terminator into spectinomycin-resistant MultiGreen 2.0 level 1 acceptor vectors.They were later concatenated with a linker module to produce the final reconstituted RUBY marker in a kanamycin-resistant pVS1-replicon destination vector (Fig 5A ) and evaluated in Nicotiana benthamiana leaf infiltration (Fig 5C).The results of these infiltrations support MultiGreen 2.0 being an effective strategy to assemble multiplexed plasmids for in planta expression.

Conclusion
MultiGreen expands the original GreenGate cloning architecture by enabling a rapid and efficient means of multiplexing.MultiGreen 1.0 and MultiGreen 2.0 enable multiplexing in series and parallel using components that strictly adhere to the architecture established in the original GreenGate kit.MultiGreen 1.0 takes inspiration from the original GreenGate approach to multiplexing in series, adding the optional H overhang through two new level 0 entry vectors.Alternating the multiplexer modules in MultiGreen 1.0 assemblies rapidly screens out carryover from one concatenation of level 0 modules to the next.Furthermore, MultiGreen 1.0 incorporates the ability to add transcription blockers or spacer sequences between transcriptional units to help minimize interference between successive gene cassettes.
MultiGreen 2.0 introduces multiplexing in parallel within the confines of the default Green-Gate assembly architecture to further boost speed of assembly.Parallelized assembly also enables easier exchange of cassettes as needs for a particular gene stack change.In addition, by incorporating two complements of level 1 acceptor vectors for assembly with different antibiotic resistances, and reversion to level 0 modules, transcriptional units can be iteratively stacked in multiples of six.An infinite number of cassettes can therefore be stacked with Multi-Green, only limited by the carrying capacity of the host E. coli cloning strain.
The MultiGreen kit encompassing all the vectors and linkers presented in this report for cloning in series and in parallel are available through Addgene.

Fig 1 .
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 1.0expansion 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 .
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 .
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 .
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.https://doi.org/10.1371/journal.pone.0306008.g004

Fig 5 .
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/.https://doi.org/10.1371/journal.pone.0306008.g005