Mobius Assembly for Plant Systems highlights promoter-coding sequences-terminator interaction in gene regulation

doi:10.21203/rs.3.rs-5118685/v1

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Mobius Assembly for Plant Systems highlights promoter-coding sequences-terminator interaction in gene regulation

https://doi.org/10.21203/rs.3.rs-5118685/v1

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Plants are the primary biological platforms for producing food, energy, and materials in agriculture; however, they remain a minor player in the recent synthetic biology-driven transformation in bioproduction. Molecular tools and technologies for complex, multigene engineering in plants are as yet limited, with the challenge to enhance their stability and predictivity. Here, we present a new standardized and streamlined toolkit for plant synthetic biology, Mobius Assembly for Plant Systems (MAPS). It is based on small plant binary vectors pMAPs, which contain a fusion origin of replication that enhances plasmid yield in both Escherichia coli and Rhizobium radiobacter. MAPS includes a new library of promoters and terminators with different activity levels; part sizes were minimized to improve construct stability and transformation efficiency. These promoters and terminators were characterized using a high-throughput protoplast expression assay. We observed a significant influence of terminators on gene expression, as the strength of a single promoter can change more than seven-folds in combination with different terminators. Changing the coding sequence changed the relative strength of promoter and terminator pairs, thus uncovering combinatorial gene regulation among all parts of a transcriptional unit. We further gained insights into the mechanisms of such interactions by analyzing RNA folding, with which we suggest a design principle for more predictive and context-independent genetic parts in synthetic biology of plant systems and beyond.

Biological sciences/Systems biology/Synthetic biology

Biological sciences/Plant sciences/Plant molecular biology

Biological sciences/Genetics/Gene regulation

Biological sciences/Biotechnology/Molecular engineering

Plant synthetic biology

context dependency

gene regulation

promoters

terminators

protoplast

interaction

RNA folding

Throughout the history of civilization, plants have been the primary culturing platform for food, energy, materials, and drugs. However, in synthetic and engineering biology applications, plants have remained a minor vehicle for bioproduction with untapped potential. Consequently, the molecular tools and technologies for rational engineering of plant systems are still limited compared to their microbial and mammalian counterparts. With the increasing world population and ongoing climate crisis, considerately designed plant engineering holds the key to a sustainable future, creating transformative solutions for a wide range of applications from agriculture to environmental regeneration^1–4. From enhancing crop yields, quality, and resilience to improving carbon fixation and biofuel efficiency, synthetic biology-based plant engineering is becoming a powerful catalyst to futureproof our society and planet^5–7.

Synthetic biology enables complex pathway engineering necessary for modifications of biological metabolisms, cellular and developmental pathways, or responses to pathogens and diseases. The foundation of synthetic biological genetic engineering is the engineering principles, such as standardization, modularity, and design-build-test cycles⁸. However, as the field matures past its first two decades, biology has often defied these fundamental assumptions. The concept of orthogonality – context-independent, universal functions of genetic parts – is routinely challenged, uncovering new insights and understanding of how biological systems interact⁹. As this can be the case for just a single gene, it poses a significant hurdle when working with the many genes necessary for pathway and network engineering.

The development of synthetic biology tools and technologies for multigene engineering starts with creating a collection (library) of standardized genetic parts. Standardized parts (e.g., Phytobricks)¹⁰ include promoters, coding sequences, terminators, and functional protein tags, which together form transcriptional units (TUs). Promoters, which contain the transcription start site (TSS) where RNA polymerase binds, are well established for their instructive roles in regulating gene activity. In comparison, terminators, typically found at the end of a gene, are often underestimated in their regulatory roles. However, they have been shown to influence transcription by controlling transcription arrest, mRNA stability, protecting genes against silencing and tuning other transcription functions¹¹.

The most commonly used promoters and terminators in plant genetic engineering originally stemmed from Cauliflower Mosaic Virus (35S), Rhizobium opine genes (NOS, MAS, and OCS), and more recently, plant genomes (e.g., UBQ10 promoter and HSP terminator from Arabidopsis thaliana or hereafter Arabidopsis). The shortage of well-characterized regulatory parts results in repeated use of the same sequences in multigene construction, increasing the possibility of homologous sequence-dependent recombination and gene silencing^12,13. In addition, there is a need for shorter standard parts, especially when multiple genes are delivered in a single vector, to reduce the size of the construct. Large constructs tend to make plasmids structurally unstable¹⁴ and decrease the efficiency of Rhizobium-mediated transformation¹⁵. Therefore, creating novel and shorter promoter and terminator parts will enrich the plant genetic engineering toolkit available to the community and increase our capacity to create genetic modifications.

Once standard genetic parts are made, their activity level should be assessed. This typically involves combining a promoter and terminator pair with a reporter protein coding sequence and quantifying the reporter expression as output. While promoters are generally more well characterized, recently, attention has also been directed towards terminators; diverse arrays of terminators have been characterized in bacteria¹⁶ and yeast¹⁷. For plant systems, the number of available terminator parts was limited for a long time, with their characterization typically performed using the Nicotiana benthamiana leaf infiltration system^18,19. It is worth noting that different species, or even different expression systems within the same species, may exhibit varying levels of expression for the same TU^18,19. Therefore, to achieve higher stability and predictability, new standard parts should be characterized in different combinations and chassis environments to account for potential context dependency.

As the number of constructs to build and characterize increases, molecular construction needs to become more efficient. Golden Gate is a widely used technology for DNA assembly that utilizes Type IIS restriction endonucleases that cut DNA outside their recognition sequences, allowing the introduction of short ‘sticky end’ sequences (syntaxis) that help orient and combine multiple DNA fragments in an intended order. Many Golden Gate frameworks have been developed for diverse species^20–23; recent developments in the plant field include Loop Assembly²⁴, an extension of the MoClo toolkit for plants²⁵, and Joint Modular Cloning¹⁸. We have previously developed a new Golden Gate framework²⁶, named Mobius Assembly, for its iterative cloning strategy between two vector sets; it allows theoretically infinite assembly of up to four DNA fragments each time. It is designed to be simple, versatile, and efficient (e.g., less need for domestication), complying with the universal and popularly employed the syntaxis design Phytobrick²⁷.

In plant transgenic engineering, binary vectors house transgenes and facilitate their delivery and incorporation into the recipient genomes. Although functional, these vectors could be improved to enhance transformation efficiency and stability. A typical Rhizobium-mediated transformation vector includes T-DNA borders²⁸, replication functions for E. coli and Rhizobium, and selection markers. Plasmid instability is a significant challenge, especially for plant systems, as vectors must be stable in both E. coli and Rhizobium. Factors such as size¹⁴, copy number²⁹, direct repeats12 and inverted repeats³⁰ affect plasmid stability. Traditional vectors are bulky (> 6 kb), but smaller backbones like pGreen (2.5 kb³¹), pLSU (4.6 kb³²), and pLX (3.3 kb³³) have been developed to reduce instability. The pLX vectors use a modular design with minimal functional parts and stability features, improving binary vector construction.

Here, we report the adaptation of Mobius Assembly for plant species engineering: Mobius Assembly for Plant Systems (MAPS). The MAPS toolkit includes new, compact plant binary vectors (pMAPs). These vectors are 3.6 kb in size, structurally stable through molecular construction, and suitable for transformation methods requiring high plasmid DNA amounts. MAPS also features bioluminescence and fluorescent protein reporters, along with new standardized promoter and terminator parts for a range of gene expression strengths. In high-throughput transient expression tests with Arabidopsis thaliana protoplasts, MAPS promoters effectively drove gene activation gradients. We also demonstrated that terminators can be used to control gene expression levels. The strength of the promoter and terminator parts depended on their combinations, and such interactions extended to the coding sequence selection. These findings facilitate a bottom-up understanding of gene regulation and the design of context-independent molecular constructs with more predictable outputs.

Development of Mobius Assembly for Plant Systems (MAPS)

MAPS is an extension of our molecular cloning framework Mobius Assembly²⁶ to enable transformation and expression of transgenes in plant systems. MAPS has a universal acceptor vector (mUAV) at Level 0 to house a standard part in the Phytobrick format. Level 0 parts are combined at Level 1, and up to four Level 1 constructs are then combined to make Level 2 constructs. MAPS follows a linear cloning strategy until Level 2 and then iterates between two cloning levels (Level 1 and Level 2) for quadruple augmentation of cloning units each time (Fig. 1a). Using the rare cutter AarI (PaqCI), as opposed to frequently used restriction enzymes that recognize shorter sequences (e.g., BsmBI or BpiI), reduces the need for removing internal restriction sites (i.e., domestication).

Initially, we developed pGreen-based vectors but encountered issues with large constructs consistent with reported instability issues^34,35. To address this, we created a new small plant binary vector called pMAP, based on the pLX architecture (Fig. 1b). This vector is suitable for transient expression in protoplasts, cell culture, tissues/organs, and whole-plant stable transformation. We devised a new origin of replication by fusing pWKS1 and pUC19 Ori. The pWKS1 Ori, derived from Paracoccus pantotrophus DSM 11072³⁶, is functional in Rhizobium but not in E. coli, so we fused it with the minimal stable pUC Ori from pUC19. pMAPS also has two Left Border (LB) sequences instead of one. Rhizobium gene transfer is from the Right Border (RB) to the LB, and having two LBs suppresses backbone transfer to the plant genome³⁷. Another feature of pMAPS is that terminators flank LB and RB sequences to isolate transgene activity from the plasmid backbone. Colourific markers, antibiotics selections, and restriction enzymes used for Mobius Assembly at different levels are summarized in Fig. 1c.

MAPS vector toolkit consists of a core set of pMAP cloning/destination vectors (Level 1 and Level 2 Acceptor Vectors, four variations Α-Δ for each level), which have a fusion origin of replication to replicate in E. coli and Rhizobium. The mUAV and the seven Auxiliary plasmids are also included, as described in the original Mobius Assembly kit²⁶. The MAPS toolkit also contains a selection of plant promoters, terminators, antibiotic resistance genes, and visible reporter genes (bioluminescence and fluorescent proteins). All MAPS plasmids are listed in Supplementary Fig. S1; Supplemental Tables S1, S2 and available through AddGene (https://www.addgene.org/browse/article/28211394/).

Reflecting on user feedback, two specific improvements were made to improve the Mobius Assembly vectors. A few users of the original Mobius Assembly kit indicated that the chromoprotein selection had been lost in their clones. Upon investigation, we observed independent events of transposon insertion in the promoter of the chromoprotein genes, which we hypothesized is a response to the stress imposed by chromoprotein production (Supplementary Fig. S2). To solve the problem, we replaced the marker chromogenic protein (spisPINK) with a red fluorescent protein (mScarlet-I). (Supplementary Fig. S2). We also noticed some Mobius Assembly clones showed growth retardation in the selection media and identified the instability was caused by plasmid dimerization. Therefore, we introduced a 240-bp cer domain, which recognizes dimers and triggers recombination help keep the plasmids in the monomeric state (Supplementary Fig. S3)³⁸.

Combinatorial DNA libraries are crucial for part characterization, as well as in applications such as biosynthetic pathway optimization³⁹, but their manual construction is time-consuming and resource-intensive. To aid combinatorial library construction, we developed the 'MethylAble' feature in Mobius Assembly, allowing standard part variants (Level 0) to be introduced to specific sites in single or multi-gene constructs (Supplementary Fig. S4). MethylAble utilizes the DNA methylation sensitivity of BsaI to mask its recognition sites by cytosine methylation during Level 1 cloning. We designed an amilCP expression cassette with divergent and convergent BsaI recognition sites, where CpG methylation blocks BsaI digestion only at divergent sites, allowing insertion of Level 0 parts into premade Level 1 constructs. Correct constructs show a purple color from amilCP until the Level 0 parts replace the cassette. As a proof of concept, the MethylAble protocol was used to build the library of the three inducible promoters (see below), each of which was combined with the 14 terminator coparts, making 42 constructs in total (Supplemental Table S3). MethylAble presents a novel strategy to create construct libraries and can be implemented in all Golden Gate frameworks in which BsaI enzyme is used, not only in Mobius Assembly.

Designing, building, and testing MAPS promoter/terminator standard parts

To select new ‘constitutive’ promoter and terminator parts, we chose ubiquitously expressed genes that are likely to have strong expression in different tissue types (Supplementary Table S2). The promoters and terminators were characterized with a transient gene expression assay based on Arabidopsis mesophyll protoplasts and PEG transformation. We optimized the parameters throughout the protocol based on⁴⁰ to improve the transformation efficiency and reproducibility. We were able to reach up to 70% transformation efficiency consistently (Supplementary Fig. S5), which was high enough to adapt to plate reader measurements in a 96-well format.

To evaluate the promoter/terminator activity levels, we used a dual luciferase system with highly sensitive nano luciferase (NLuc) as the reporter and firefly luciferase (FLuc) for normalization⁴¹. To account for possible batch-to-batch differences in overall protoplast transformation rates/efficiency, we included FLuc gene (UBQ10-FLuc:UBQ5) in each construct and calculated the NLuc/FLuc ratio as RLU (Relative Light Unit).

For the promoter testing, seventeen promoters drove NLuc expression, with termination by either the NDUFA8 or HSP terminator (Promoter:NLuc:HSP/NDUFA8). The UBQ10 promoter exhibited by far the highest expression activity among the promoters, followed by MAS (Fig. 2a,b). The HSP terminator increased gene expression for all promoters except TUB9. Two of the newly isolated promoters, UBQ11 and UBQ4, matched or exceeded the activity of the 35S and OCS promoters. Furthermore, the newly isolated promoters ACT7, TUB2, TUB9, APT1, ACT2 and LEC2 outperformed the commonly used the NOS promoter. The FAD2 and NDUFA8 promoters had the lowest expression.

For the terminator evaluation, the NLuc expression was driven by a strong (UBQ10) or weak (NDUFA8) promoter and one of the 14 terminators (UBQ10/NDUFA8:NLuc:Terminators). The luciferase expression varied by 5.3–6.3 fold for the UBQ10 and NDUFA8 promoters, respectively, depending on the terminators they were paired with (Fig. 2d,e). For the strong UBQ10 promoter, the FAD2 terminator had the highest activity (547.9 RLU), while the NOS terminator had the lowest (103.9 RLU). For the weak NDUFA8 promoter, the HSP terminator led to the highest expression (0.327 RLU) and APT1 to the lowest (0.052 RLU).Since it was surprising to see a wide range of gene expression levels led by different terminators under the same promoter, we extended terminator characterization with the three chemically inducible systems popularly used in plant sciences: dexamethasone (Dex), estradiol, and ethanol inducible systems^42–44. They are all based on two-component mechanisms involving at least two transcriptional units. The exogenously applied chemical activates the transcription factor that further transactivates the downstream target genes. The target genes are activated by the specific promoters that contain binding sites for the transactivator (pOp6, lexA, and alcSynth), and hence promoters cannot be changed, while terminators can be.

Interestingly, the different terminators resulted in more uniform NLuc expression, with a 2.2- and 2.9-fold range in expression levels for the Dex and estradiol inducible promoters (pOp6-35S and lexA-35S), respectively (Fig. 2h,i). For the Dex system, RLU was spread between 37.9 and 111.6 in combination with the UBQ5 and 35S terminators. For the estradiol promoter (lexA), the E9-RbcS and 35S terminators had RLU counts of 11.6 and 25.1, respectively. In contrast, the ethanol inducible promoter (alcSynth), showed a much wider range, with the HSP terminator driving sevenfold higher expression than the LEC2 terminator (Fig. 2j). Both the Dex and estradiol systems showed a basal expression of around 10 RLU; with Dex inducing a ~ 11-fold and estradiol a ~ 3-fold activation. The ethanol system had high basal expression (i.e., it was leaky), leading to only 30% increase in luminescence upon chemical induction.

Promoter-coding sequence-terminator interactions in gene regulation

Reflecting on the observed promoter-terminator interactions, we investigated whether changing the coding sequence also influences promoter/terminator activity. Fluorescent proteins are an alternative visible reporting system to luciferases. However, using fluorescent proteins in a plant chassis can be challenging as plants emit red and green-range autofluorescence from their chloroplasts, and stress-induced blue-range autofluorescence from their cytoplasm⁴⁵. Over the years, fluorescent proteins with improved brightness and expression dynamics have been developed, but their quantitative efficacy was not comprehensively characterized in protoplasts. Therefore, we screened fluorescent proteins from four spectrums (green, red, yellow, and blue) to examine their compatibility with a protoplast system using a plate reader or microscopy (Supplementary Table S4). Generally, expression of fluorescent proteins was detected 6 hours after transformation and plateaued around 15 hours (Supplementary Fig. S6). Informed by this screening, we selected the brightest two fluorescent proteins from different spectra: sfGFP and mScarlet-I. sfGFP was the main reporter, while mScarlet-I was used as the normalizing gene (similar to FLuc above) and expressed using the UBQ10 promoter and UBQ5 terminator.

Initially promoters were evaluated with the HSP terminator for sfGFP expression. Unlike luciferase reporters, not all promoters drove strong enough expression that could be detected with a plate reader (Fig. 2c). The highest expression was driven by the UBQ10 and MAS promoters, followed by the 35S promoter. Readouts from the rest of the promoters could not be distinguished from the background autofluorescence. Therefore, the UBQ10 and MAS promoters were analyzed in combination with all the MAPS terminators (Fig. 2f,g). The HSP terminator was the strongest with both promoters (9.0 and 12.1 RFU for UBQ10 and MAS, respectively). The FAD2 terminator was on the high-expression side for both promoters, along with the UBQ10 promoter-35S terminator and MAS promoter-NOS terminator combinations. The Rbsc2b terminator resulted in the lowest expression with both promoters. Overall, a 3-fold expression difference was observed by using different terminators with the UBQ10 promoter, and the difference was 6-fold with the MAS promoter.

Our part characterization revealed that although promoter choice could dominantly determine gene expression strength in some cases (e.g., pOp6 and NDUFA8) (Fig. 3a), TU activation by a promoter, terminator or coding sequence is not independent or additive, but combinatorial and even synergistic (Fig. 3b). A clear example of such all-part interactions is seen with the NOS terminator, which in combination with the UBQ10 resulted in weak, NDUFA8 strong, lexA medium, pOp6 weak and alcSynth mid-level expression in the luciferase system. When we switched to a fluorescence-based reporter, the combination of the NOS terminator with the UBQ10 and MAS promoters drove medium and strong expression, respectively. Among the 14 terminators characterized, only four exhibited stable behaviours with both coding sequences: FAD2 and HSP were consistently on the strong side, while LEC2 and RbcS2b tended towards the weak side.

Dissecting the mechanism of the combinatorial gene regulation

Next, we sought for insights into how the promoters, coding sequences, and terminators interacted in gene regulation. The interactions may regulate gene expression by changing the transcript abundance or with post-transcriptional modifications affecting translation. To distinguish these two possibilities, we performed qPCR to examine how the transcript (mRNA) level correlates with the reporter readout. Because transforming enough protoplasts for RNA extraction is laborious, we selected key constructs to test. The HSP and FAD2 terminators were chosen for consistently strong expression regardless of different promoters and reporter protein sequences. For consistently weak expression, the NOS and Rbcs2b terminators were selected for NLuc, whereas the Rbcs2b, APT1 and E9-RbcS terminators were chosen for sfGFP. The NOS terminator was chosen because its relative strength varies the most depending on the partnering promoters or coding sequences. Similarly, the 35S terminator was selected for its variability in strength, although it tends to be on the strong side.

The qPCR assay revealed that the consistently strong FAD2 and HSP terminators have significantly higher mRNA levels; similarly, the weak E9-Rbcs2b terminator had significantly lower mRNA levels than the other terminators (Fig. 4a). Generally, NLuc displayed a linear correlation between the measured reporter expression levels and mRNA levels (Fig. 4b), while sfGFP exhibited a looser correlation (Fig. 4c). Surprisingly, the 35S terminator yielded higher reporter expression in both reporter combinations compared to its mRNA levels (Fig. 4b,c). Taken together, the transcript level analysis suggests that the combinatorial gene regulation is partially explained by transcript abundance and likely to involve post-transcriptional (post-mRNA formation) processes in some constructs. The 35S terminator for both NLuc and sfGFP suggested a post-transcriptional enhancer effect, while NLuc:NOS, together with sfGFP:RbcS2b and sfGFP:APT1 constructs indicated post-transcriptional repression.

Transcriptional regulation is mediated by specific DNA sequences that recruit functional proteins to activate or repress processes from transcriptional initiation to stable mRNA formation. Surprised by how effective terminators are in controlling gene expression, we investigated the nucleotide sequence features possibly influencing terminator strength, such as the GC content and presence of likely functional sequences (e.g., the canonical poly-A signal AAUAAA and UGUA motifs) (Fig. 5, Supplementary Fig. S7).

A GC content of approximately 30% has been shown to be optimal for synthetic terminator functions, and the average GC content in natural Arabidopsis terminators is about 32.5 ⁴⁶.The GC content in the MAPS terminators had no clear correlations between the strength and GC content of the terminators; consistently or predominantly strong terminators [HSP (29.1%), FAD2 (36.5%), and 35S (32%)] had a similar range of GC content as weak or variable terminators [RbcS2b (30%), E9-RbcS (32.8%), APT1 (35%), and NOS (38.7%)] (Fig. 5a). One apparent feature in the strong terminators is that the GC content is apparently lower at a 50 bp window, which is located around their dominant Poly-A signal, and then the GC content goes back up. The presence of the canonical AAUAAA poly-A signal tends to enhance terminator strength⁴⁶.

We also searched for sequence motifs that may link to the consistently high strength of HSP and FAD2 terminators. The presence of the UGUA motif around 30–40 bp upstream of the RNA cleavage site is thought to enhance the cleavage and thus increase terminator strength in the 35S terminator ⁴⁶. The HSP terminator has two UGUA motifs at locations 119 bp and 206 bp, while FAD2 lacks the motif. Interestingly, the XSTREME motif discovery tool identified four motifs that putatively have functional roles a piriori (Fig. 5b,c,e). One of them is the 15-bp motif (CAAAUGUUUUGUGUC) found around 145 bp in both the HSP and FAD2 terminators, which correspond to the transcript cleavage site. Three other motifs - CUCAUUAUGUUA, UUGUUGUGUUAUGAC, and UUUUUCUAAUAUUA - were found at similar locations in both terminators but around 10–20 bp apart. Only the CUCAUUAUGUUA motif is present in the UBQ5 terminator at 167 bp (Fig. 5b). The effects of the UUUUU motif are complicated; it decreases terminator strength in maize protoplasts, especially if they surround a UGUA motif, while U-rich sequences increase terminator strength in tobacco leaves⁴⁶. Many of these AT/U-rich motifs reside inside the 50 bp low GC domains described above.

We examined similar potentially functional features in the ‘outlier terminators’ – the terminators performing stronger or weaker than expected for their transcript levels (i.e., likely post-transcriptionally regulated) (Fig. 4a,b). The GC content of outlier terminators is moderate and varies between 30.5–35.0% (Fig. 5a). RbcS2b is the only outlier terminator that does not possess the canonical poly-A signal. The 35S terminator has three repeats of the UGUA motif (x3 starting from 96 bp separated by UU). The UUUUU motif is present in the NOS, RbcS2b and APT1 terminators (Supplementary Fig. S6). Therefore, no distinct sequence signatures were identified to differentiate the terminators that are primarily regulated transcriptionally from the outliers.

We then wanted to experimentally probe how the above-identified sequence features influence the terminator activity by generating a deletion series of the HSP and FAD2 terminators. Five terminator variations were made to sequentially delete possible regulatory elements, such as Poly-A signals, putative destabilization signals, and Musashi binding elements (Fig. 5c-f). Statistical analysis of the results showed only a significant difference between the full-length terminator (240 bp) and FAD2 Sequence 5, which is the shortest variation (70 bp) with no Poly-A site, as well as the deletion series Sequence 1 (200 bp). The rest of the HSP series, as well as the FAD2 series, showed no statistically significant difference in reporter expression (Fig. 5c-f). This result suggests short sequences (30–50 bp) at the 5’ end of terminators might determine the gene expression strength, and terminator functional elements remain unresolved, especially in plant contexts.

We also investigated a sequence feature likely enhancing promoter activity. UBQ10 is by far the strongest promoter in the MAPS toolkit (Fig. 2,3), and its TSS structure is predicted to be unstructured and single-stranded (Fig. 6). In the survey of the Arabidopsis genome, translation efficiency was found higher for transcripts with unstructured 5’UTR⁴⁷. To test if the strength of the UBQ10 promoter is dependent on the open loop structure of its 3’ UTR, which consists of mostly TSS, three mutated versions were created. The first version, Mutated Control (MUTC), involves point mutations that preserve the predicted loop structure; therefore, it is expected to behave similar to the original UBQ10 TSS sequence. Additionally, two other versions, a tight stem-loop structure Mutated 1 (MUT1) and a slightly more branched but more loosely stemmed Mutated 2 (MUT2), were designed with point mutations (Fig. 6a,b). TSSPlant and Softberry software were used to confirm the mutations do not interfere with the identified motifs or introduce new motifs compared to the WT.

When the TSS variants were used to drive sfGFP with five different terminators (HSP, FAD2, 35S, NOS, and RbcS2b), the results revealed no statistically significant difference in expression between WT and MUTC, as expected (Fig. 6c). However, MUT2 exhibited a reduction in expression by approximately 20% for the HSP and FAD2 terminators. Additionally, MUT1 resulted in a 60% decrease in expression with 35S, while resulting in a 30% increase with the NOS terminator. There was no difference between the three mutated versions and the WT for the RbcS2b samples, where the WT expression is already very weak (Fig. 6c). With the strong terminators, reporter expression was reduced in MUT1 and MUT2 compared to WT and MUTC variants, suggesting that conversion from open loop to stem-loop decreases gene expression.

To gain insights into how promoters, coding sequences, and terminators interact post-transcriptionally, we studied RNA folding. RNA is single-stranded and extensively forms secondary and tertiary structures via hydrogen bonds bringing together (nearly) complementary sequences. Such 2D and 3D structures (e.g., G-quadruplex) can strongly influence translation and protein expression^48,49. The combination-dependent regulatory function among the three TU parts may be explained by direct physical interactions through RNA folding.

Using the RNAFold software⁵⁰, the folding energy of the whole mRNA sequence was calculated for the transcript species we selected for the qPCR analysis above. The transcription start site (TSS) was identified based on the TSSPlant software⁵¹, and the downstream promoter sequence was incorporated, along with the protein-coding and terminator sequences. The lower the holding energy, the tighter the transcript folds, and the less likely for translation to occur. No direct correlation was found between the predicted transcript folding energy and mRNA expression levels or between the folding energies and reporter gene expression (Fig. 4d,e). We therefore proceeded to examine the RNA folding structure and local interactions among the nucleotide sequences.

We then visually examined the RNA folding structures in 2D. RNA secondary structure formation was predicted using the ViennaFold2.0 software⁵⁰, in which the whole transcript (identified as described above) was used as the input. Within the transcript, the strong UBQ10 and MAS promoters are likely to have little interaction with the other parts: no interaction with the HSP and RbcS2b terminators and minimal to medium interactions with the FAD2 terminator were predicted by the software (Fig. 7). On the contrary, NOS, a highly variable terminator, may have strong interactions with the other parts (UBQ10:NLuc) when it drives weak reporter expression. Interestingly, there was no apparent correlation between inducible promoter-terminator structures and reporter output, except for pOp6:NLuc:NOS (Supplementary Fig. S8). In strong and consistent terminators like HSP, we tended to find loops bigger than 20 bp (Fig. 7). When the variable strength-terminator NOS is paired with strong promoters (UBQ10 and MAS), it also may form a large loop structure, though not when combined with other promoters (e.g., NDUFA8) (Fig. 6, Supplementary Fig. S9). RNA sequence-mediated cross-part interactions among the promoters, coding sequences, and terminators could explain the combinatorial gene regulation.

MAPS: A new synthetic biology toolkit for engineering with plant systems

In the present work, we established the Mobius Assembly for Plant Systems (MAPS), which is an adaptation of the Mobius Assembly²⁶ for plant genetic programming (Supplemental Figure S1, Supplemental Table S1). MAPS is a stand-alone yet highly versatile and functional DNA assembly platform and genetic toolkit collection. It is based on small binary vectors for all levels of cloning and comes with a well-characterized, Phytobrick-compatible standard part library for gene expression control in plant species, including a suite of reporter protein cassettes. We have also introduced a new feature for Mobius Assembly to facilitate the generation of a series of constructs with part variations; MethyAble exploits in vitro DNA methylation to directly feed Phytobricks in single- or multi-TU constructs.

For plant genetic engineering, small binary vectors are instrumental in enhancing efficiency in cloning and transformation. As we wanted a reliable core vector in our kit for both cloning and transformation applications, we developed a new binary vector pMAP. This new vector has a unique dual-mode origin of replications for E. coli and Rhizobium backgrounds, which enables amplification in high copy numbers for experiments demanding high DNA yields, such as the protoplast assay (4 ug DNA per 75 ul transformation) used in this study. The plasmid dimerization phenomenon we have observed is not unique to Mobius Assembly vectors; in fact, with the increasing availability of whole-plasmid sequencing technologies, more dimers are being detected. Therefore, including a cer site in the backbone provides an effective method for preventing dimerization and growth defects (Supplementary Fig. S3)³⁸.

MAPS delivers a collection of short promoters (17 constitutive and three inducible) and terminator (14 constitutive) standard parts for plant expression. Ten of the promoters and six of the terminators were newly isolated, and they are short in length: 300–600 bp (promoters) and 200bp (terminators). We confirmed the short size of the promoters did not compromise their strength. The Actin 2 promoter characterized in MoClo and GB2.0 toolboxes had comparable activity to the NOS promoter^52,53; the shorter version in the MAPS toolkit, ACT2 (340bp) and NOS promoter, exhibited similar expression activity (Fig. 3). In general, the size of the available standard promoter or terminator parts for plant engineering (e.g., MoClo plants, GB plants, and GreenGate) can be as long as 4 kb. Shorter parts are desirable as large constructs can reduce plasmid stability in bacteria, while also lowering transformation efficiency and causing incomplete/truncated transformation in planta^14,15.

Here, we reported the first characterization of the MAPS toolkit, which was done exclusively in Arabidopsis leaf mesophyll protoplasts with a plate reader-based high-throughput assay. Most plant standard part characterization to date has been conducted in either Arabidopsis protoplasts or Nicotiana benthamiana (leaf expression system), which are both dicot plants. The standard parts can behave differently in different backgrounds. In monocots, for example, the 35S and NOS promoters drive low activity in monocot Setaria viridis^18,54. Even within dicots, the same part can have different levels of expression^52,53. The OCS promoter had strong activity in our study but low expression in N. benthamiana⁵². Genetic parts can behave differently in different chassis, and we should not generalize characterization results among different plant species or expression systems (e.g., whole plants, leaves, protoplasts, or cell cultures). We recommend not making assumptions and testing the activity levels of standard parts in the specific context in which users want to apply them.

Terminators influence gene expression, in part through interaction with promoters

Our characterization of the MAPS terminators underscored the strong influence of the terminators cast on gene expression. It is a common practice to rely on promoters to control gene expression level and overlook the impact of terminators. For example, when the domestication of the synthetic plant promoter G10-90 promoter resulted in loss of expression, researchers replaced it with the AtRPL37aC promoter to rectify⁴⁴. They were using the psE9-RbcS terminator, which in our assay had low activity; the gene expression could potentially have been restored with a stronger terminator instead. Even though promoters generally have a strong influence on gene expression (Fig. 3a), terminators could alter the gene expression up to 8-fold in this study (Fig. 2,3). Alternative to constitutive expression, chemically inducible systems are powerful tools to control the timing, localization, and amplitude of transgene expression. Dex-, estradiol-, and ethanol-inducible systems were developed 20 years ago and have been used in numerous studies since then⁴². The extent of target gene induction can be modulated by exchanging the terminators (Fig. 3).

Terminators influence gene expression through multiple mechanisms⁵⁵, including post-transcriptional regulation via the 3' UTR, which can affect mRNA stability through specific sequence motifs. For instance, certain motifs at the 3' UTR in Arabidopsis can stabilize (e.g., TTGCTT) or destabilize (e.g., AATTTT) mRNA⁵⁶. Poly(A) tails also play a role, with short tails often linked to strong gene expression ⁵⁷. In Arabidopsis, the poly(A) tail can prevent RDR6 from converting aberrant mRNAs into degradation substrates⁵⁷. Unpolyadenylated transcripts derived from terminator-less constructs or readthrough mRNAs from transgenes with strong promoters are subjected to RDR6-mediated silencing⁵⁹, which might explain the increased gene expression observed with double terminators^60–62. However, the synergistic effects of terminators, particularly when combined with weak promoters, suggest interactions beyond these mechanisms. Direct interactions between terminators and promoters, potentially involving gene looping mediated by DNA-binding proteins, can influence gene expression⁶³. These interactions might affect transcriptional memory⁶⁴, intron-mediated modulation of transcription⁶⁵, transcription directionality⁶⁶, reinitiation of transcription⁶⁷, and transcription termination^68,69. Additionally, terminators may impact epigenetic regulation, as the absence of a terminator can lead to increased DNA methylation on promoter regions, silencing transgene expression⁷⁰.

Promoter-coding sequence-terminator interactions in gene regulation

In addition to the interactions between terminators and promoters, we observed that protein-coding sequences can also modify the strength of promoter-terminator function. The coding sequence may impact gene expression through factors such as codon usage bias, gene length, GC content, correct 5' cap, and mRNA folding⁷¹. Both the NLuc (516 bp, GC 52.7%) and sfGFP (714 bp, GC 61.3%) have been optimized for plant expression in terms of codon usage. In an orthogonal system where part performance does not depend on the context, any factors related to codon usage, gene length, GC content, or correct 5' cap may affect the magnitudes of the reporter readout, but the comparative profiles of gene expression across the promoter-terminator pairs should remain the same. However, the relative strengths of promoters and terminators changed when the reporter protein was replaced (Fig. 3,4), indicating that the coding sequence interacted with the promoter and the terminator presumably directly and physically.

We wanted to understand how the promoter-coding sequence-terminator interactions occur. There are many possible mechanisms (some examples outlined above), which likely to influence gene activity in combinations. However, to simplify, such interactions can be explained either at the DNA or RNA level – or transcriptional or post-transcriptional mechanisms. The qPCR assay indicated that the reporter genes activity was regulated transcriptionally by controlling the stable transcript abundance, but also post-transcriptionally as the transcript abundance alone did not always correlate with the reporter activity (Fig. 4).

To explore the mechanisms behind the transcriptional regulation, we surveyed DNA sequence features potentially involved in determining terminator or promoter activity. The terminators with consistently strong reporter protein readout (HSP and FAD2) also had high mRNA levels (Fig. 5a). These two terminators contain a local drop (50 bp) in the GC content, that was absent in most other terminators. It is likely that this low GC region holds other structural features that aid the recognition of transcription termination and cleavage. When other possible motifs were searched for a priori in HSP and FAD2 but not in other MAPS terminators, the XSTREME motif discovery tool identified four motifs. Among the four, CAAAUGUUUGUGUC motif at location 145 bp in HSP and 146 bp in FAD2, may aid with cleavage site recognition and cleavage efficiency, enhancing overall terminator strength (Fig. 7b,c).

With our deletion series, we have seen that only the shortest FAD2 terminator (70 bp) has significantly reduced strength compared to the full-length version (240 bp). The first 35 bp of the HSP terminator has a strength statistically nondifferent compared to the full-length (Fig. 5c,d); this is consistent with Felippes et al.⁷², which showed that the first 32 bp of the HSP terminator is a transferable element that enhances the strength of other terminators. A very short (< 50 bp), minimal terminator domain may govern much of the terminator activity, especially in transient expression systems, whereas the specific features identified (Fig. 5) may play clearer roles in longer timescales or certain physiological or developmental contexts.

Recent studies revealed the roles of RNA structures in gene expression control. Lower mRNA folding energies, typically associated with greater structural stability, impact ribosome function and translation in organisms such as E. coli and S. cerevisiae^73,74. Beyond global folding, local secondary structures can also play a pivotal role in shaping gene expression. Pseudoknots, hairpins, loops, and the presence of hydrogen bonding patterns upstream of the transcription start site have been shown to reduce ribosome sequestration and translation rates^75,76. A study with 224,000 synthetic sequences in E. coli has highlighted the importance of secondary structures at the 30 bp region upstream and downstream of the start codon (referred to as STR_-30:+30)⁷⁷. While comprehensive studies in plants are still pending, in vivo genome-wide profiling of Arabidopsis RNA secondary structures revealed that unstructured regions upstream of the start codon are enriched in high translation efficiency mRNAs⁴⁷. In the strong promoter UBQ10, we found an unstructured TSS (loop) where altering its structure affected reporter output (Fig. 6).

RNA secondary structures also point to a similar regulatory influence in terminator-dependent gene regulation. When the consistently strong terminators HSP and FAD2 were combined with various promoters, loops of > 20 bp were predicted (Fig. 7). This might be a structural feature of strong-acting terminators, since a similar > 20 bp loop was predicted when the variable NOS terminator was combined with strong promoters (MAS, UBQ10); yet the loop is thought to re-configure to a stem-loop structure when combined with weaker promoters. Expanding on these structural themes, we examined the folding structure of the terminators from Felippes et al.⁷¹ that showed increased strength when combined with the first 32 bp of the HSP terminator. The predicted folding of the transcript showed that the addition of the 32 bp led to an increase in the size of the loops > 20 bp for the NOS and ACS2 terminators, as well as higher base-pairing probability structures for Rbcs1A (Supplementary Fig. S9).

RNA folding patterns predicted the consistent activity promoter and terminator parts tend to be isolated in terms of secondary structure, displaying a low level of physical interactions from the other parts, regardless of their sequences (Fig. 7, Supplementary Fig. S8). Additionally, in the case of the NOS terminator, cross-part interactions could explain the high variability. It is plausible that certain promoters and terminators possess currently unknown regulatory elements that enhance their activity while physically restricting their interactions with other parts. Meanwhile, there may be TU parts that may require physical interactions with other parts to function effectively. For example, in mammalian systems, terminator elements require specific secondary structures to interact between the polyadenylation complex and mRNA⁷⁸. Synthetic parts with known elements, like the pOp6 and LexA inducible promoters, likely hold key to context-independency. Even though their transcripts were predicted to fold, generating cross-fold interactions, such interaction did not seem to have a strong effect on the reporter output (Supplementary Fig. S8).

We developed a new, simple and versatile synthetic biology toolkit for plant genetic engineering, Mobius Assembly for Plant Systems. In so doing, we unravelled new insights into gene regulation using a bottom-up approach and have shown that gene expression is regulated by all parts of a transcriptional unit combinatorically. Uncovering the relationship between the structure and function of RNA (and DNA) will help establish the principles for designing consistent and predictive standard parts that are independent of the nucleotide context-independent and more orthogonal.

Bacterial strains and growth conditions

E. coli strains JM109, and NEB stable were used. E. coli chemically competent cells were prepared in-house using the TSS preparation described by Chung & Miller⁷⁹. E. coli cells were incubated at 37°C (30°C for NEB stable), 200 min-1 either in 5 ml (for a high copy) or 10 ml (for a low copy), or 100 ml (for midi prep) in LB growth medium supplemented with antibiotics. Cells bearing the LhGR-pOp6 and sXVE-lexA inducible systems were grown for 24 h instead due to their slower growth rate.

Bacterial transformations and plasmid isolation

For E. coli transformation, 5 µl of the plasmid DNA was incubated with 100 µl of the competent cells on ice for 30 min, followed by a heat shock at 42°C for 90 sec (30 sec for TOP10) and re-cooled on ice for 5 min. S.O.C medium (400 µl) was added, and after 1 h incubation at 37°C, 100 µl of the cell suspension was plated on LB agar plates with antibiotic selection. Plasmids were isolated using Monarch (NEB) Plasmid Miniprep Kits. The GeneJET Plasmid Midiprep Kit (ThermoFisher) were used for higher yields.

MAPS vector construction

MAPS Level 1 and Level 2 Acceptor Vectors were built using Gibson Assembly. The Mobius Assembly cassettes were amplified from the corresponding E. coli plasmids in the Mobius Assembly Vector toolkit²⁶ and fused to the plant binary vectors. The pGreen-based Level 1 vectors were constructed using pGreen0029³¹. The NptI gene was replaced with the spectinomycin resistance gene amplified from the pCR8 vector (ThermoFisher) to generate Level 2 vectors. The pLX-based Level 1 and Level 2 vectors were built using pLX-B3 and pLX-B2³³, respectively.

WKS1 Ori was synthesized (Twist Bioscience) into two parts due to repetitive sequences, and pUC Ori was amplified from pUC19, both flanked by BsaI recognition sites. The minimum sequence requirement of pUC for stable replication in E. coli was found to include RNA I/RNA II transcripts on the 5′-side and dnaA/dnaA′ boxes on the 3′-side, while co-directional transcription of two different replicons in the same plasmid was shown to increase transformation efficiency and DNA yield³². A pLX Level 1 A vector with the construct NOS:BglR:NOS-UBQ10:nluc:HSP was amplified outside the BBR1 Ori with primers harbouring BsaI recognition sites and fused with pUC and WKS1 Ori. The resulting plasmid was used as the template to amplify the pUC-WKS1 fused Ori, which then replaced BBR1 Ori from the pLX-based Level 1A and Level 2A vectors with Gibson Assembly, resulting in the pMAP Level 1A and Level 2A vectors. The rest of the pMAP vectors were constructed again using isothermal assembly and as a template for the Mobius cloning cassettes, the plasmids from the original Mobius Assembly kit²⁶.

MethylAble modules were devised with the Gibson Assembly using mUAV as a template. Overlapping primers bearing two outward-facing BsaI sites prone to CpG methylation and suitable standard overhangs were used to amplify mUAV into two parts. The resulting parts were purified and digested with DpnI (ThermoFisher) to eliminate the template DNA, and they were fused in an isothermal reaction.

Mobius Assembly and standard part library construction

A detailed protocol on Mobius Assembly can be found in⁸⁰. Briefly, the assembly was performed in a one-tube reaction with a total volume of 10 µl, with ~ 50 ng Acceptor Vectors and double amounts of inserts. Reagents added were 1 µl of 1 mg/ml BSA (diluted from 20 mg/ml - NEB), 1 µl T4 DNA ligase buffer (ThermoFisher/NEB), 0.5 µl AarI/PaqCI (ThermoFisher/NEB) and 0.2 µl 50x oligos of the AarI recognition site for Level 0 and Level 2 cloning or Eco31I/BsaI-HFv2 (ThermoFisher/NEB) for Level 1 cloning, and 0.5 µl T4 DNA ligase (ThermoFisher/NEB). The reactions were incubated in a thermocycler for 5–10 cycles of 5 min at 37°C and 10 min at 16°C, followed by 5 min digestion at 37°C and 5 min deactivation at 80°C (5 cycles for Level 0 and the first round of Level 1 cloning – 10 cycles for Level 2 and large constructs > 10 kb).

PCR amplification

All PCR amplifications for plasmid construction and cloning were performed using Q5® High- Fidelity DNA Polymerase (NEB), followed by purification with Monarch® PCR & DNA Cleanup Kit (NEB). Successful DNA assembly was verified first by colony PCR using GoTaq® Green Master Mix (Promega) and then with double restriction digestion with EcoRI-HF (NEB) and PstI- HF (NEB). The constructs were further verified by Sanger sequencing (GATC Biotech-Eurofins, Edinburgh Genomics and Source BioScience) and whole plasmid sequencing (Full Circle).

Promoter and terminator part design and generation

Likely constitutive promoters and terminators were selected from the literature, especially the genes commonly used as positive controls in qRT-PCR⁸¹. Their sequences were retrieved from the TAIR webpage⁸² (http://www.arabidopsis.org/index.jsp) and blasted in NCBI to find the untranslated regions flanking the genes. A 1.5 kb sequence upstream of the start codon was run through the online prediction software and TSSPlant⁵¹ (http://www.softberry.com) to identify TATA and TATA-less promoters or enhancer sites. In the promoter selection, it was also considered, when possible, to include the initiator (INR) elements (YYA(+ 1)NWYY- TYA(+ 1)YYN-TYA(+ 1)GGG)) and downstream promoter (DPE) element (RGWYV). Potential promoter elements linked to increased gene expression were identified using PlantCare⁸³ and PLACE⁸⁴ software. Terminators were selected with PASPA, a web server for poly(A) site prediction in plants and algae⁸⁵ (http://bmi.xmu.edu.cn/paspa/interface/run_PASPA.php). A sequence 300bp downstream of the stop codon was input into PASPA, and the end of the terminator was set at 10bp after the second polyadenylation site, resulting in a sequence of around 200bp. They were then analyzed in RegRNA2.0 to identify other RNA functional motifs⁸⁶ (http://regrna2.mbc.nctu.edu.tw).

The parts designed in the first round were short promoters (~ 300bp) and short terminators (~ 200bp) from the genes ACT2, FAD2, TUB9, APT1, NDUFA8 and LEC2. As the ~ 300 bp promoters were very low in activity (Data not shown), we designed a new set of promoters derived from the genes TUB2, UBQ11, UBQ4, ACT7, and the longer versions of the previous promoters (~ 500 kb). Appropriate primers compiling to the Phytobrick standard overhangs were designed for both promoters and terminators for cloning into mUAV.

Protoplast isolation and transformation

Arabidopsis (Wildtype, Col-0) seeds were sown on the soil. After a 2-day pre-treatment at 4°C in darkness, they were sawn and grown under long-day conditions (21°C; 16h light / 8h dark cycles; light intensity ~ 100µmol/m2s-1; humidity 40–65%) until harvest. The protoplast isolation and transformation protocol was developed based on Chupeau et al., (2013)⁴⁰ and Faraco et al., (2011)⁸⁷. The optimized protocol is descripted in Supplementary Information, with representative images of consistently high transformation (Supplementary Fig. S4).

Luciferase assay

The 96-well plates containing the transformed protoplasts were let to sediment and 60 µl of supernatant was discarded. The protoplasts were resuspended, and 40 µl was transferred to white optical plates in a grid pattern with empty spaces between wells to reduce luminescence bleed-through. Luciferase activity was assayed in an Omega luminescence plate-reader (Fluostar) with four different gains following the instructions of the Nano Dual-Luciferase® Reporter kit (Promega, N1620). A further correction for luminescence bleed-through and background signal was applied using the software developed by Mauri et al.⁸⁸ Then NLuc signal was divided by the FLuc signal to normalize for the transformation efficiency.

Fluorescence assay

For fluorescent protein assays, TECAN Spark plate reader was used with either 96-well plates (Greiner) or black Thermo Scientific Nunc F96 MicroWell. Excitation and emission for the super-folder green fluorescent protein (sfGFP) was 485 nm and 520 nm, respectively. For mScarlet-I, excitation and emission wavelengths were 560nm and 620nm. Measurements were taken at two different gains to ensure signals don’t overshoot beyond saturation. The sfGFP values were divided by the mScarlet-I values to normalize for the transformation efficiency. Since fluorescence was not always active, unlike luminescence and also because black-walled plates were used to prevent signal bleed-through, deconvolution correction was not applied to the fluorescent samples.⁸⁸

RNA isolation and real-time PCR (qRT-PCR)

Isolation and transformation were performed as described above, with reaction and solution volumes upscaled by 26 times. 24 h after transformation, the supernatant was discarded, and sedimented protoplasts were harvested for RNA extraction with GeneJET Plant RNA Purification Kit (ThermoFisher). Extracted RNA was treated with DNA-free DNA Removal Kit (Invitrogen). Then, reverse transcription was performed with UltraScript 2.0 cDNA Synthesis Kit (PCR Biosystems). This cDNA synthesis kit uses optimum amounts of oligo (dT) and random hexamers for unbiased amplification of RNA variants. It is advised to use 2 µM of both oligo (dT) and random hexamers for cDNA synthesis when using a different kit.

qPCR reaction was set up with primers and probe sequences listed in Supplementary Table S4. Sso Advanced Universal Probes Supermix (Bio-Rad) was used following the manufacturer’s instructions. with the reactions were prepared in MicroAmp Endura 96-well plate and sealed with MicroAmp Optical Adhesive films, and the optical output was measured with StepOnePlus qPCR machine (all Applied Biosystems). A standard curve was included on the plate for each gene target tested; this standard curve was used to calculate cDNA copy number of the samples on the same plate. The FLuc or mScarlet-I cDNA copy number was used for normalization for the transformation efficiency. The resulting values from all the constructs were then normalized to the NLuc cDNA copy number from the UBQ10:NLUC:NOS in the same experiment, to account for any batch-to-batch variations.

Functional dissection of the HSP and FAD2 terminators

Five length deletion series were constructed for HSP and FAD2 terminators. The deletions removed putative motifs that could affect their function to influence the gene expression level. Putative Stabilization/Destabilization Motifs⁸ while the rest were identified using RegRNA2.0¹ and PASPA online tools². Primers were designed to gradually remove functional sequence elements from the 3' end of each terminator through PCR and subsequently cloned to mUAV. Site-directed mutagenesis by Gibson Assembly was employed to mutate the poly-A signal of the HSP Part3, while the same method was used to build HSP Part 5. Transformation of the construct was performed with PEG-transformation of Arabidopsis leaf mesophyll protoplasts in 96-well plates. The expression activity of the constructs was assayed using a plate reader, measuring the Nano luciferase activity normalized by Firefly luciferase. The construct we used was UBQ10:Nluc:Terminator Part:UBQ10:Fluc:UBQ5, housed in a pMAP vector.

RNA folding prediction

For mRNA folding predictions, the transcription start site was predicted for each promoter by using TSSPlant⁵¹. The nucleotide sequence downstream of the TSS to the end of the terminator was used as the mRNA sequence. mRNA folding was predicted by using RNAFold software that uses Vienna RNA package.⁵⁰ Thermodynamic ensemble prediction was used for folding energy predictions which accounts for all possible RNA secondary structures weighted by their Boltzmann probabilities, allowing the calculation of base pairing probabilities and ensemble free energy. The centroid algorithm was used for secondary structure prediction which is the structure with minimal base pair distance to all other secondary structures in the Boltzmann ensemble⁵⁰.

ACKNOWLEDGEMENTS

This project was funded by the Biotechnology and Biological Sciences Research Council (BBSRC) High-Value Compound from Plants (HVCfP) Network Proof-of-Concept Award (POC- NOV16-04), Royal Society University Research Fellowship (UF140640 and URF\R\201035), and Schmidt Science Polymath Award to NN, as well as the University of Edinburgh Principal's Career Development PhD Studentship to AIA, the IBioIC CTP PhD Studentship to JN, and Imperial College Bioengineering Studentship to EGK.

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Mobius Assembly for Plant Systems highlights promoter-coding sequences-terminator interaction in gene regulation

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Abstract

Figures

INTRODUCTION

RESULTS

Development of Mobius Assembly for Plant Systems (MAPS)

Designing, building, and testing MAPS promoter/terminator standard parts

Promoter-coding sequence-terminator interactions in gene regulation

Dissecting the mechanism of the combinatorial gene regulation

DISCUSSION

MAPS: A new synthetic biology toolkit for engineering with plant systems

Terminators influence gene expression, in part through interaction with promoters

Promoter-coding sequence-terminator interactions in gene regulation

CONCLUSION

MATERIALS AND METHODS

Bacterial strains and growth conditions

Bacterial transformations and plasmid isolation

MAPS vector construction

Mobius Assembly and standard part library construction

PCR amplification

Promoter and terminator part design and generation

Protoplast isolation and transformation

Luciferase assay

Fluorescence assay

RNA isolation and real-time PCR (qRT-PCR)

RNA folding prediction

Declarations

ACKNOWLEDGEMENTS

References

Additional Declarations

Supplementary Files

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