DNA assembly is a vital process in biotechnology and synthetic biology research, during which DNA plasmids are designed and constructed using bioparts to engineer microorganisms for a wide range of applications. Here, we present an enzymatic homology-based DNA assembly method, SENAX (Stellar ExoNuclease Assembly miX), that can efficiently assemble multiple DNA fragments at ambient temperature from 30 to 37 °C and requires homology overlap as short as 12–18 base pairs. SENAX relies only on a 3′–5′ exonuclease, XthA (ExoIII), followed by Escherichia coli transformation, enabling easy scaling up and optimization. Importantly, SENAX can efficiently assemble short fragments down to 70 bp into a vector, overcoming a key shortcoming of existing commonly used homology-based technologies. To the best of our knowledge, this has not been reported elsewhere using homology-based methods. This advantage leads us to develop a framework to perform DNA assembly in a more modular manner using reusable promoter-RBS short fragments, simplifying the construction process and reducing the cost of DNA synthesis. This approach enables commonly used short bioparts (e.g., promoter, RBS, insulator, terminator) to be reused by the direct assembly of these parts into intermediate constructs. SENAX represents a novel accurate, highly efficient, and automation-friendly DNA assembly method.
DNA assembly is an important and routine process in biotechnology and synthetic biology research, during which plasmids are designed and constructed using DNA parts to build genetic circuits to reprogram the cells. Several DNA assembly methods have been developed over the years, and the methods can be categorized based on the operating conditions (in vivo or in vitro)[
Cloning using in vitro homology-based methods (or sequence-overlapping methods) (e.g., Gibson assembly and In-Fusion assembly) has gained popularity because these methods enable seamless assembly of multiple fragments with high efficiency. These methods generally utilize a mixture of enzymes, including polymerases, exonucleases and ligases, in an isothermal process that relatively eases the whole protocol. Unlike RE-based methods, homology-based methods are sequence-independent, which simplifies the design for DNA assembly. However, in comparison with RE-based methods, homology-based assembly methods offer lower modularity[
In this paper, we studied exonuclease type III (the XthA enzyme) from Escherichia coli Stellar cells for DNA assembly and developed a multifragment DNA assembly method (SENAX) that uses the XthA enzyme alone. XthA is known as a multifunctional DNA repair enzyme that has important biological roles in DNA metabolism (phosphatase, exonuclease, AP endonuclease, RNase H activities)[
Motohashi previously reported that DNA assembly can be performed using E. coli cell extract[
Graph: Figure 1 Purified XthA is sufficient for DNA assembly. (a) Purified XthA proteins were verified by 10.0% SDS PAGE. Lane 1 (left), protein marker; Lane 2, Purified XthA. (b) Efficiency of 3-fragment assembly by SENAX. Constructs A, B, C, D (2.8 kb); E (4.0 kb); F (5.0 kb); and G (6.3 kb) were used for the test (see Fig. S1 for details about the configuration of each construct). The size of the fragments used for assembly are presented on top of the plot (b): the red line represents RFP, and the green line represents GFP. The error bars represent the standard deviations (STDEVs) of three replicates. *p < 0.05, **p < 0.01 by paired t test against the control. Samples with no enzyme protein XthA were used as the control. (c) Transformation of plasmids (Construct B) assembled by SENAX using different competent cells, i.e., 10Beta (NEB), 5Alpha (NEB), and Stellar (Takara). The configurations of a replication origin (15A), antibiotic resistance (AmpR), and a green fluorescence gene were used for assembly. The obtained fluorescent colonies represented the efficiency of the method. The error bars represent the standard deviations (STDEVs) of three distinct replicates. *p < 0.05 by paired t test against the control. Samples with no enzyme protein were used as the control.
We first studied whether the enzyme XthA alone could assemble a small number of DNA fragments that would express green fluorescent protein (GFP) in vivo when assembled correctly (Fig. S3). To this end, we studied the efficiency of the mix with only XthA in assembling 3 typical medium-size fragments—an RSF origin of replication, a kanamycin resistance cassette, and a GFP reporter gene, which were PCR-prepared to have 18 bp overlapping regions to each other. Twenty nanograms of each fragment was used, and the reaction was performed at 37 °C for 15 min. Stellar cell extract, Stellar cell extract supplemented with XthA, and a mix without XthA were used as controls. From the results, we observed that the concentrated cell extract from E. coli Stellar had innate activity to assemble DNA parts as an obvious number of green fluorescent colonies grew on the screening plate (Fig. S3). This concurs with previously reported studies reporting SLiCE[
We further investigated the efficiency of SENAX for 3-fragment assemblies to produce a series of plasmids of varying sizes (2.8 kb-A/B/C/D; 4.0 kb-E, 5.0 kb-F; 6.3 kb-G) and with different bioparts, including different origins of replication and genes of interest (Figs. 1b and S1). The efficiency of assembly was evaluated based on either the number of fluorescent colonies on the antibiotic plate (all constructs) or the number of colonies grown on the plate (construct G). The results show that high efficiency was achieved as many fluorescent colonies appeared on the antibiotic plates (Fig. 1b). However, the number varied by template, ranging from 20 to 150. Nonetheless, the results demonstrated that SENAX is able to assemble common-size DNA fragments to generate a range of plasmid sizes.
Since XthA is expressed using the gene derived from Stellar E. coli, we sought to examine whether its activity is dependent on specific components of Stellar E. coli. Hence, we performed the 3-fragment assembly of construct B using different competent cells, including DH5-alpha and 10Beta (NEB), for the transformation step. The results show that DNA assembly activity based on the use of XthA was present even when different types of competent cells were used, although the cloning efficiency differed among the competent cells used (Fig. 1c). This suggests that the DNA assembly activity does not depend on specific components of Stellar E. coli. Taken together, the results demonstrated that XthA alone is sufficient for DNA assembly, as other enzymes (e.g., polymerase and ligases) are not present in the mix.
To evaluate the performance of SENAX in assembling multiple fragments, we performed assembly using constructs of varying sizes (2.8 kb and 10 kb) (Figs. 1b, 2) and with a varying number of joining fragments (
Graph: Figure 2 SENAX was tested with different numbers of DNA fragments. (a) A reporter plasmid (15A + AmpR + GFP) was separated by PCR into several linear fragments (3–4–5–6) with 18 bp homology. Top of the plot, an illustration of the configurations used in assembly tests. Fragments coloured in grey are the fragments used for assembly. The plot shows the efficiency of the assembly tests with an increasing number of fragments involved. (b) A naringenin-producing plasmid (10.5 kb) was separated by PCR into several linear fragments (3–4–5–6–7) with 18 bp homology arms. The fragments were then used for assembly reaction. Top left, an illustration of the configurations used in the assembly tests. Top right, a plot showing the efficiency of assembly along with an increasing number of fragments involved. Bottom left, DNA fragments with homology arms prepared by PCR verified by agarose gel electrophoresis. Bottom right, assembly mixes after incubation were verified by agarose gel electrophoresis, where the arrows indicate the expected intermediate assembly products. The error bars represent the standard deviations (STDEVs) of three distinct replicates. *p < 0.05, **p < 0.01 by paired t test against the controls. Control samples were prepared with the same amount of input DNA fragments but without enzyme protein XthA.
We then investigated the multifragment assembly using a larger plasmid construct (10.5 kb) to gain further insight into the ability and limitation of SENAX (Fig. 2b). A gene cluster for naringenin synthesis under the control of constitutive promoters was cloned into the RSFori/AmpR backbone, resulting in a 10.5 kb plasmid (Fig. 2b). The plasmid was separated into 3, 4, 5 or 6 fragments using PCR, and these fragments were treated with DpnI to remove the circular template. The control samples were prepared by using a similar amount of input DNA without supplementation with the XthA enzyme. The negative colonies were mainly from the undigested vector, which was used as the PCR template but was incompletely digested by DpnI. Several hundred colonies were obtained from the plate of 3-fragment assembly, and the results revealed that the efficiency of assembly decreased exponentially with an increasing number of DNA fragments involved. This is a common observation, as reported for other assembly methods[
To test the ability of SENAX to perform short fragment assembly, a library of short fragments (size varied—200 bp, 150 bp, 100 bp, 88 bp, 70 bp) made up of a specific set of promoter and RBS pairs was assembled with different template linear plasmids (backbone) and transformed into E. coli. The promoters and RBS were selected from the Anderson collection[
Graph: Figure 3 Short-fragment assembly by SENAX in comparison with commercial DNA assembly enzyme mixes. Short fragments with different lengths (200–150–100–88–70 bp) were introduced into variants of backbone templates by SENAX, In-Fusion (Takara), and Gibson (NEB). Do note that the two commercial kits were used according to the manufacturer recommended protocol and optimal conditions (e.g., at a temperature of 50 °C for at least 15 min). (a) Short fragments were introduced to the GFP reporter plasmid (2.8 kb). (b) Short fragments were introduced to the dCas9 expression plasmid (6.3 kb), and (c) short fragments were introduced to a naringenin-producing plasmid (9.0 kb). The error bars represent the standard deviations (STDEVs) of three distinct replicates.
Among the tested short fragment sizes, 88 bp appeared to be a good candidate size to harbour bioparts such as promoters and RBSs, which are routinely used for fine-tuning gene expression. Within this fragment, we can incorporate a unique spacer, the full sequence of an Anderson constitutive promoter, a short spacer between promoter and RBS, and a common size RBS. To take advantage of SENAX's ability to assemble short fragments directly, we created a library of 88 bp fragments comprising promoters of varying strength (Bba_23119, Bba_J23100, Bba_J23101, Bba_J23106) layered with an RBS (RBS0034) (see supplementary Table S2), which can be reused using SENAX to be assembled into different backbone templates (Fig. S5a–g). Based on the sequencing results that we obtained (a total of 18 colonies from 7 plates), an average success rate of 88.9% per plate was achieved (see Fig. S5—Table S3). This further shows that 88 bp fragment assembly is reliable and has the potential to be used as a standardized assembly framework. On the other hand, as we sought to determine the limitation in the size of the short fragment that can be assembled by SENAX, we performed an additional test with the 70 bp fragment and a 60 bp fragment using the ho1 template (3.0 kb) (Fig. S5h). Tests of the 70 bp fragment with the ho1 template were successful, achieving a high number of colonies (
To further test SENAX assembly capability, we also used SENAX successfully to create a small combinatorial library of naringenin-producing plasmids (Fig. S6). While the different plasmids vary in promoter/RBS driving the respective GOIs, each plasmid consists of multiple repeated regions, including terminators, promoters, RBS, and spacers near the junctions, making assembly challenging. Nonetheless, correct constructs were obtained with reasonable accuracy, although efficiency was not high (data not shown).
XthA is also commercially available for research use, such as for the preparation of single-stranded DNA for dideoxy sequencing or site direct mutagenesis. A test using the commercial product (M0206 L—NEB) (with 30 times dilution) for 3-fragment assembly generated a number of fluorescent colonies (Fig. S7), although the number of colonies was lower compared with SENAX in the parallel experiment. This could be due to chemicals present in the storage buffer by the manufacturer.
We investigated the assembly efficiency using different amounts of XthA. We obtained similar efficiency when using 10–30 ng of XthA for a single reaction. In contrast, no fluorescent colonies were obtained when more than 50 ng of purified XthA was used in a single 10 µL reaction. The control sample with 0 ng of XthA showed no colonies, as expected. We further verified the assembly product by agarose gel electrophoresis. The faint bands that represented the final assembly product (approximately 3 kb) only appeared in the sample with 20 or 30 ng of XthA (Fig. 4a), which was consistent with the transformation-based results. Hence, 20 ng of XthA was found to be optimal for assembly, while 50 ng was the upper limit of the enzyme amount needed for a single 10 µL assembly reaction.
Graph: Figure 4 Optimization of SENAX. The configurations of a replication origin (15A), antibiotic resistance (AmpR), and a green fluorescence gene (Construct B) were used for assembly for all the tests. (a) Effect of Enzyme XthA amount on SENAX. Assembly mixes of the 3-fragment assembly reaction after 15 min with different XthA amounts were verified by agarose electrophoresis (below). Plot showing the efficiency of assembly along with the different amounts of XthA (above). The sample without XthA supplementation was used as the control. Arrows indicate the expected intermediate assembly product. The error bars represent the standard deviations (STDEVs) of three distinct replicates. *p < 0.05, **p < 0.01 by paired t test against the control (without XthA). (b) Effect of temperature on SENAX. Assembly mixes of 3-fragment SENAX assembly after 15 min incubation at different temperatures were verified by agarose gel electrophoresis (above). Plot showing the efficiency of assembly by SENAX at different temperatures after transformation (below). Arrows indicate the expected intermediate assembly product. The error bars represent the standard deviations (STDEVs) of three replicates. *p < 0.05, **p < 0.01 by paired t test against the control (without XthA). (c) Effect of incubation time on SENAX. 3-fragment assembly with different incubation times (0 min, 5 min, 10 min, 15 min, 30 min, 60 min). The error bars represent the standard deviations (STDEVs) of three replicates. *p < 0.05, **p < 0.01 by paired t test against the control (0 min incubation). (d) Effect of Mg2+ concentration on SENAX. 3-fragment assembly with different amounts of Mg2 + supplemented in the reaction. The error bars represent the standard deviations (STDEVs) of three distinct replicates. *p < 0.05, **p < 0.01 by paired t test against the control (without supplementation of Mg2 +).
We also investigated the effect of temperature on SENAX efficiency. The results show that SENAX produced colonies harbouring the assembled construct with almost similar efficiency when the reactions were performed in the temperature range of 30–42 °C (Fig. 4b). The number of fluorescent colonies significantly decreased when the incubation temperature was 50 °C or when the temperature was lower than 28 °C. The result suggests that the highest efficiency can be obtained at 32 °C, as the highest number of fluorescent colonies was obtained. At 35–37 °C, we obtained a similar number of fluorescent colonies compared with that at 32 °C. However, there were only a few colonies without fluorescence when the temperature was 32 °C, while the number of nonfluorescent colonies gradually increased when the temperature was reduced to 30 °C and lower. We performed sequencing analysis for some of the nonfluorescent colonies and found that these colonies had incorrect constructs with missing small DNA parts (data not shown). At 50 °C, few or no fluorescent colonies grew on the plate. For further analysis, an aliquot of assembly solution was verified by agarose electrophoresis (Fig. 4b). The DNA bands at approximately 1 kb represented the linear input DNA fragments. The DNA bands found from 1.5 to 2.0 kb probably represented the linear assembled product, in which only 2 DNA fragments were concatenated. Above those bands, we found bands at approximately 3.0 kb, representing the intermediate circular construct. As we only found these intermediates in the profile of samples at 30–42 °C, this is consistent with what we obtained from the screening of colonies on plates after transformation. The remaining linear input fragments after reaction in samples 30–42 °C were also much less than those of samples incubated at 25 °C, 28 °C, and 50 °C. We concluded that these temperatures inhibit enzyme activity and that 50 °C would likely deactivate XthA. In summary, the temperature for assembly using XthA was optimal between 32 °C and 37 °C.
To test the influence of reaction time on XthA assembly activity, we performed parallel assembly reactions joining the 3 DNA fragments for different incubation times. The results show that 10 to 30 min is the best incubation duration for cloning efficiency (Fig. 4c). Shorter than 10 min of incubation greatly decreased the efficiency, as approximately 2 times less activity was observed. The 60 min incubation time sharply decreased efficiency. The percentage of fluorescent colonies was reduced by more than 70% compared with the experiment using a 15 min incubation time. These results suggest that 10–30 min are suitable for DNA assembly by XthA.
Structural analysis of XthA revealed that this enzyme has single divalent metal ion and nucleotide binding sites at the active site of the enzyme[
The typical length sequence needed for annealing in a PCR is 18 bp. Therefore, the length of the cloning primer, which should include the homology arm shorter than 20 bp, can be approximately 33–38 bp. This length is generally accepted as a fine balance between specificity and amplification efficiency. The long homology region (e.g., 30–40 bp homology as in the typical Gibson method) will require more cost and increase the chance of DNA mispriming and more likely result in an unexpected construct. Therefore, to reduce the possibility of mispriming and the presence of unexpected constructs due to the long homology arm, we designed the length of the homology region in our bioparts to be shorter at 18 bp. From most of the experiments performed, we demonstrated that 18 bp homology works well for SENAX. We also tested using 15 bp homology arms (e.g., for the Naringenin plasmid assembly and the overhang test) (Fig. S8b) and using 16 bp homology arms for short-fragment assembly. We found that the homology length can be as low as 15 bp without affecting cloning efficiency. Since the length of the homology arm will affect the annealing of the exonuclease-generated overhangs, the short homology is also suitable for the temperature used in SENAX (30–37 °C) rather than the 50 °C used in Gibson and In-Fusion. To determine the lower limit of the homology arm size using SENAX in-vitro DNA assembly, the DNA assembly of 3 fragments (Amp, 15A, GFP) with different overlapping lengths between the fragments (18 bp, 15 bp, 12 bp, 10 bp) was investigated (Fig. S10). The results show that the efficiency of the reaction decreased with the decrease in the size of the homology arm. However, the number of colonies obtained by the 12 bp homology arm was reduced by approximately 70% compared with the fluorescent colonies obtained with the 15 bp homology arm. The 10 bp homology arm produced only a few fluorescent colonies. Hence, 10 bp homology can be considered the lower limit for SENAX design. Nonetheless, using the 12 bp and 10 bp homology arm sizes still yielded fluorescent colonies, thereby showing that the SENAX method works even with smaller homology arm sizes. Overall, 15–18 bp can be considered the optimized length of the homology arm for SENAX assembly.
We tested the cloning of the blunt end and sticky end (3′-prime overhang and 5′-prime overhang inserts) using SENAX (Fig. S8). The inserts were amplified by PCR with specific primers that harbour either restriction site of XbaI with BamHI or XbaI with KpnI at the 2 terminals of the inserts. The amplicon was then treated with the corresponding restriction enzyme to release the 5′–5′ overhang fragment (XbaI-BamHI) and 5′–3′ overhang fragment (XbaI-KpnI) (Fig. S8a). The results showed that the efficiency of blunt-end insert cloning was the highest, followed by 5′–5′ overhang insert cloning, while there were no colonies in the sample with a 5′–3′ overhang (Fig. S8a). It can be assumed that the 3′-overhang fragment remained undigested upon completion of the incubation time. The same phenomenon was obtained when we performed a test of overhang's influence on short-fragment assembly (Fig. S8b), in which we obtained 37, 33, and 0 colonies for samples of the blunt end, 5′-overhang and 3′-overhang short fragments, respectively. This is consistent with the literature reporting on the activity of exonuclease III, in which the enzyme was described to not actively work on single-stranded DNA, as the 3´-protruding termini (over 4 bp) are resistant to cleavage[
XthA is known as a multi-functional enzyme, and its homologues are involved in the DNA repair system in various bacterial species. Nonetheless, XthA has been applied to several in vitro applications, including the analysis of protein-DNA complexes[
We demonstrated that SENAX can assemble up to 6 DNA fragments, and the length of the final construct can vary from 0.1 to 10 kb. This method has succeeded in producing a high success rate of correct colonies with design matched sequences, demonstrating the overall accuracy of the developed method. Importantly, SENAX allows short fragments from 200 bp down to 70 bp to be inserted into the template construct (from a few kb to 9.0 kb) in a single step. This overcomes the difficulty faced by the use of currently available homology-based assembly techniques for short fragment assembly. When we applied SENAX for promoter-RBS short fragment assembly, although the efficiency was relatively not as high as that of the medium-sized fragment assembly, correct colonies were obtained in the tested cases, while Gibson and In-Fusion gave almost no colonies. The difficulty of other homology-based assembly technologies in dealing with these fragments could be due to the short DNA and/or the nicked DNA being degraded much faster by T5 exonuclease (in Gibson mix)[
A series of repetitive steps are usually required using the current homology-based methods (e.g., Gibson or In-Fusion) to make the desired construct with the gene of interest accompanied by a specific promoter. As illustrated in Fig. 5, using the current methods, the primer that will include the whole short biopart (e.g., promoter) sequence upstream of the gene of interest will first need to be synthesized. After this, PCR will be performed to generate amplified products that harbour the desired short bioparts. However, this requires the use of long primers (usually 50–100 bp or more), resulting in a higher cost of DNA synthesis. In addition, because the Gibson assembly technique requires a longer overlapping region than other homology-based methods, even longer primers would be necessary. This could be considered a drawback of the Gibson assembly. If a fragment longer than 60 bp would be targeted, the length of the primer would not be suited for short oligo synthesis or would be difficult for PCR optimization. Alternatively, the intermediate template can be created by inserting the short target fragments directly into the original template using SENAX instead of resynthesizing the whole plasmid to achieve the complex construct. If only one plasmid is to be constructed, the cost between current homology methods and SENAX would be similar. However, in the event when the short fragments are commonly used and/or to be used in multiple constructs, using SENAX would enable the researcher to avoid the need to resynthesize the short fragment (considering that the amount of synthesized short fragment provided by the company is adequate for ~ 2000 reactions) but only need to synthesize the new short primer (~ 35 bp) to link the short fragment to the backbone. Therefore, the short fragments are considered "reusable", which is where the cost is being saved compared with other typical methods. For example, using current methods, researchers would need to repeatedly synthesize new long primers (over 100 bp) to include the short fragment in advance. In other words, the more different constructs that the researcher needs to assemble using the short fragments, the more synthesis cost savings can be attained using the SENAX method.
Graph: Figure 5 Illustration of SENAX versus commonly used homology-based methods to generate variants of short-fragment assembly constructs. n = the number of short parts to be incorporated. Because of the ability of SENAX to assemble short fragments directly into the backbone, the need for PCR long fragments to include the short fragment before assembly can be avoided. As a result, SENAX enables easier reuse of short fragments.
Standardization of the assembly process is among the necessities for high-throughput DNA assembly. For sequence homology-based methods, one approach is to standardize the overlapping regions that are basically independent of the sequence of DNA parts. This will also allow the higher modularity of homology-based DNA assembly methods. In our study, the spacer (18 bp) was designed to facilitate the easy reuse of the bioparts. All constructs A, B, C, and D and their variants that differed from each other by only the promoter region were produced using this approach (Fig. 1b, Fig. S1, and Fig. S5a–d). Having 18 bp spacers for assembly also provides a means to positionally validate the assembled construct because these sequences can be used to design PCR primers. This PCR profiling approach provides a good marker to demonstrate the correct direction and order of bioparts in the final construct (Fig. S9). This also facilitates screening through colony PCR before sequencing, which is more convenient and cost-effective when applied in high-throughput assembly. It is worth noting that the number of spacers to guide assembly is not limited to 3 (as in our current 3-fragment vector design) but can be expanded for convenient use as long as more fragments are involved in the assembly.
As it is common practice to fine-tune gene expression by replacing promoters or RBSs, we developed a library of well-defined reusable DNA fragments of 88 bp to take advantage of SENAX's capability to assemble short fragments. Each fragment is made up of commonly used constitutive promoters of varying strengths and RBSs. The specific set of promoters and RBSs in the proposed format can be reused in multiple constructs for various purposes (e.g., fine-tuning and combinatorial assembly). By using this approach, we were able to directly generate a library of construct variants that differ from each other by only the promoter region. This is advantageous, as the common homology-based technique would require starting over the whole plasmid construction in an ad hoc manner (Fig. 5). Overall, SENAX allows a standardized framework for reusing bioparts and improves the modularity for homology-based assembly (Fig. 5).
In this study, we designed our experiments such that we could compare the results within one experimental group when we varied one factor while keeping all the other factors fixed. However, it is difficult to keep all the factors fixed among the different experimental groups, including the competency of cells and the quality of DNA, due to the large number of samples required in the multiple experimental groups. It is known that cloning efficiency is critically affected by transformation efficiency, which is dependent on the competency of cells. Nonetheless, our experiments have enabled us to identify the optimal value of the factor studied. As a result, we elucidated several parameters required for SENAX. In particular, the SENAX workflow can be carried out flexibly with good efficiency for only 15 min from 32 to 37 °C, which is the temperature range used in common incubators widely available in laboratories. Notably, most of the commonly used homology-based methods will require a working temperature of 50 °C. Therefore, routine DNA assembly would be more convenient using SENAX for any laboratory, as water baths and hot blocks are no longer necessary. This temperature range would also be more compatible with a high-throughput automation system. In addition, SENAX comprises only a single exonuclease, while Gibson requires a polymerase, a T5 exonuclease, and a T4 ligase, and In-Fusion relies on a polymerase with exonuclease activity[
The strains and plasmids used in this study are listed in Table S1. Cells were cultured in LB medium (Axil Scientific Pte Ltd) containing the appropriate antibiotics at the designated temperature of 37 °C. In some experiments, the cultures were incubated at different temperatures for optimization purposes. The final concentrations of antibiotics ampicillin (Amp) (100 µg/mL), kanamycin (km) (50 μg/mL), chloramphenicol (Cm) (35 μg/mL), and spectinomycin (Spc) (50 μg/mL) were used for screening and maintaining plasmids in E. coli.
All the plasmids, DNA fragments, and primers used in this study were designed in silico using SnapGene (GSL Biotech) and Benchling (https://benchling.com). The primers used for the preparation of assembly fragments were designed to have a 12–18 bp homology region and are listed in Table S2. For the multifragment DNA assembly, 18-bp overlaps between fragments were designed. For the short fragment DNA assembly, a 16–18 bp overlapping region was designed. Genes and primers were obtained as gene fragments (gBlocks) or single-stranded oligos from Integrated DNA Technologies (IDT). GFP (green fluorescent protein), RFP (red fluorescent protein), and sfGFP (superfolding GFP) were used as reporters for gene expression characterization. Illustrations of the plasmids were prepared using SnapGene (GSL Biotech). The plasmids were constructed using the commercial enzyme mix Gibson (NEB), In-Fusion (Takara Bio), and the SENAX assembly method developed in this study. All constructed plasmids were chemically transformed into either E. coli Stellar (a brand derived from strain HST08) (Takara Bio), DH5alpha, or 10Beta (NEB). All protocols for transformations, PCR, and DNA manipulation used in this work were performed with reference to the manufacturer's manual and were optimized when necessary.
Several plasmid variants were designed for the testing of the DNA assembly (Fig. S1). The plasmid format of the variants mainly comprises a configuration of DNA parts, including a replication origin (REP), an antibiotic resistance cassette (AbR), and a target gene of interest (GOI). Bioparts are linked by a random-sequence 18 bp spacer and can be produced by PCR amplification using either Q5 DNA polymerase (NEB) or KOD One PCR Master Mix (TOYOBO). Primers for amplification of the bioparts were designed based on junction sequences between spacers and bioparts. For the GOI of constructs A–D, we placed either a GFP or RFP reporter gene under the control of a constitutive promoter (e.g., J23101 from the Anderson promoter collection) and RBS0034, while REP and AbR were varied. These constructs were used for multifragment assembly and short-fragment assembly tests. A 2.8 kb reporter plasmid (construct B) was separated into 3, 4, 5, and 6 fragments by PCR for the multifragment assembly test. The DpnI-treated PCR-derived fragments were reassembled with SENAX. The sizes of the fragments were 750–1116–1029 bp (3 fragments), 750–719–415–1019 bp (4 fragments), 750–719–415–555–492 bp (5 fragments), and 750–719–415–249–324––492 bp (6 fragments). Constructs E (4 kb) and F (5 kb), which carried RFP and GFP, respectively, were used as templates for PCR preparation of 3 linear fragments for assembly to reproduce the original construct. Construct G (6.3 kb), which is a dCas9 expression plasmid, was used as a template for PCR preparation of fragments to produce the original plasmid and used for the short-fragment assembly test to produce its promoter variants. Construct H (10.4 kb), which is a naringenin-producing plasmid, was used as a template for PCR preparation of multifragments to produce the original plasmid and used for the short-fragment assembly test to produce its promoter variants. For the multifragment assembly test, this construct (H) was separated into 3, 4, 5, 6, and 7 fragments using PCR. The resulting amplicons from PCR preparation were treated with the restriction enzyme DpnI (NEB) to reduce the background of the circular DNA template, followed by purification in a gel (QIAGEN) or per aliquot by column (MACHEREY–NAGEL, Takara Bio USA).
The transformants were screened on antibiotic screening plates, and the extracted plasmids from several positive colonies were sent for sequencing (1st-BASE, Axil Scientific—Sanger sequencing service) to confirm the match to the designed constructs. The colonies with fluorescence reporters were also screened based on fluorescence that could be visualized with a trans-illuminator (GeneDireX, Inc.). The nonfluorescent colonies were screened by colony PCR.
The complete XthA gene sequence was directly cloned from a single colony of E. coli Stellar. The fully amplified 807 bp DNA fragment was purified using a gel extraction kit (Qiagen) and cloned into the linear blunt-end cloning vector pColdI, which was amplified by PCR, to yield plasmid pColdI::XthA (Fig. S2). The construct was introduced into E. coli Stellar, and the plasmid was isolated from the cells using a Miniprep kit (Qiagen). The inserted XthA and junctions were verified by nucleotide sequencing to confirm that the cloning was in frame. The correct plasmid was introduced into E. coli BL21 for protein expression. The cold-shock expression procedure using the pCold system allowed continuous translation of the histidine-tagged XthA gene product. The expressing culture was incubated at 37 °C until its absorbance at 600 nm reached 0.5. The culture was then placed on ice for 30 min. Isopropyl β-d-1-thiogalactopyranoside (IPTG) was added for induction at a final concentration of 1 mM for the next 16 h at 16 °C. The cells were then harvested and resuspended in PBS buffer. Cells were chemically disrupted by incubation with Tris–HCl-based lysis buffer with Triton X-100 (MERCK) for 30 min. The cell debris was removed by centrifugation (17000×g for 20 min) and filtration through a 0.22-µm membrane. The obtained cell extract was concentrated through 10-kDa cut-off filters (Millipore) by centrifugation at 5500×g until it reached an appropriate volume. An aliquot of concentrated cell extract was applied to a Ni–NTA spin column (Qiagen) for purification under the designated native conditions. In the last step, the buffer containing the purified protein fraction was changed to 50 mM Tris–HCl pH 7.5. The protein concentration was examined by NanoDrop One using Bradford reagent (BioRad).
Approximately 1 µg was loaded onto SDS–PAGE (Tris–glycine 10% polyacrylamide gel). The single protein band appeared in the gel was then excised and dried using Vacuum Concentrator Plus (Eppendorf). The proteins were extracted from dried gel pieces and digested with trypsin, and the resulting peptide sequences were subjected to analysis (MALDI-TOF MS/MS—Proteomics International Laboratories Ltd, Australia).
For medium size DNA fragments, 18-bp homology arm length was designed; twenty (constructs A,B,C,D,E,F,G) or fifty nanograms (construct H) (ng) of each part was subjected to the mix for each assembly reaction. Except for short-fragment DNA assembly tests, an equal ratio of each part was used for each assembly reaction. For the short fragment, 500–1000 ng in 1 µL was supplied for each reaction. This depends on the original concentration of short-fragment stock synthesized by the company (IDT). Together with DNA parts, 20 ng in 1 µL of concentrated protein was mixed with 1 µL of buffer solution (100 mM MgCl
To study the effect of the amount of XthA, temperature, reaction time and the effect of Mg2 + on the efficiency of the assembly, 3 fragment assemblies were performed using different amounts of XthA (0–100 ng) for each 10 µL reaction. The 3 fragments include one with a GFP placed downstream of the constitutive promoters J23101 and RBS0034 (GFP reporter), one with an antibiotic resistance gene (AmpR), and one with an origin of replication 15A (15A ori).
To identify the optimal temperature for the reaction, the reaction was performed at different temperatures (25 °C, 28 °C, 30 °C, 32 °C, 35 °C, 37 °C, 42 °C, and 50 °C), and the efficiency of the assembly was studied. A range of amounts of XthA, i.e., 5, 10, 20, 30, 50, and 100 ng, were also tested to optimize the method. The times evaluated for optimization were 0, 5, 10, 15, 30, and 60 min.
The resulting assembly mixtures (up to 10 µL) were verified by electrophoresis in a 1% agarose gel or chemically transformed into competent stellar cells (Takara), DH5 alpha (NEB), or 10beta (NEB). The transformed cells were preincubated at 37 °C for 1 h, plated on antibiotic screening plates, and incubated overnight. The resulting colonies were picked from overnight plates, and plasmid extraction (MiniPrep QIAGEN) was performed using 5 mL of fresh culture derived from a single colony.
The short DNA fragments (single-stranded DNA oligos) were designed using SnapGene and purchased via IDT. The delivered dry oligos were suspended to a final concentration of 100 µM in water as the storage stock, and the two complementary oligos were mixed at a final concentration of 20 µM each. The obtained mixture was heated to 95 °C for 5 min and lowered to 4 °C at 0.1 °C/sec to allow annealing. The resulting duplex DNA solution was then kept at − 20 °C and was used for subsequent multiple different DNA assembly constructs. Approximately 500–1000 ng was used for each assembly reaction. Five short fragments of different lengths (200 bp, 150 bp, 100 bp, 88 bp, 70 bp) were designed (Table S2). All of the short fragments consist of spacer S1 at the 5′ terminus, a promoter, and an RBS. The capability and efficiency of assembling the short fragments into variants of the backbone template of different lengths (2.8 kb, 6.3 kb, and 9.0 kb) were investigated.
Linearized vectors/inserts are recommended to be generated by PCR, while chemically or enzymatically synthesized DNA fragments (e.g., gBlock) are also suitable for SENAX. To achieve successful assembly, the primers to generate a fragment via PCR must be designed in the way that its 5′-end contains bases (15–18 bp) of homology to the other fragment and its 3′-end must contain the sequence that can complementarily bind to the DNA template. Desalted oligonucleotide primers are suitable for PCR for SENAX. DpnI treatment of PCR products followed by gel purification is recommended to reduce the background derived from circular PCR template. In a final reaction mix of SENAX, two (
The authors would like to thank the support from Singapore National Research Foundation (NRF SBP-P6 and SBP-P5), the Synthetic Biology Initiative of the National University of Singapore (DPRT/943/09/14), Summit Research Program of the National University Health System (NUHSRO/2016/053/SRP/05), and NUS startup grant (R-397-000-257-133). The authors also like to acknowledge the support from Singapore Biofoundry at the National University of Singapore.
V.L.D. and C.L.P. conceived the study. V.L.D. designed and performed the experiments, and processed the data. N.R. performed the cultures and validated the protein expression. C.S. and J.Z. designed and performed combinatorial library construction. V.L.D. and C.L.P. wrote the paper. All authors reviewed and approved the final manuscript. C.L.P. supervised the study.
A non-provisional patent application pertaining to the method developed in this study has been filed. All authors have no other conflicts of interest to declare.
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By Viet Linh Dao; Sheena Chan; Jingyun Zhang; Russell Kai Jie Ngo and Chueh Loo Poh
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