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Gerbang Emas (Golden Gate Jerusalem) yang berdiri saat ini mungkin dibangun pada tahun 520 Masehi, sebagai bagian dari program pembangunan Justinianus I di Yerusalem, di atas reruntuhan gerbang sebelumnya. Using the traditional overhang design standards, we could identify high-fidelity overhang sets containing approximately 10–12 overhang pairs (Fig 5B). Importantly, we did not observe any colonies upon transformation of these control reactions. To determine if DAD could be used to increase the capacity of these cloning systems, we repeated our overhang set analysis for assemblies with T4 DNA ligase and BsmBI-v2 (Fig 5B). Golden Gate assembly uses a series of DNA fragments generated in this way by a single type IIS endonuclease (in each assembly step), such that each fragment has unique non-palindromic cohesive ends designed to anneal only to the correct end of the next DNA fragment in the series. Yes https://doi.org/10.1371/journal.pone.0238592.g007. Thus, the choice of Type IIS restriction enzyme and the reaction conditions could significantly impact the assembly yield, and it is advisable to optimize the assembly reaction conditions, especially for assemblies with many fragments. In contrast to Golden Gate assembly with four-base overhangs, we found that non-palindromic self-mismatches were among the most frequently observed mismatch pairs (Table 1). (B) GetSet was used to estimate assembly fidelity for overhangs sets with up to 40 overhang pairs in an assembly reaction with T4 DNA ligase and BsmBI-v2. email or call 1-800-NEB-LABS. To ensure the observed transformants were the result of in vitro assembly and not assembly of the DNA fragments within the E. coli by cellular DNA repair mechanisms, we also carried out control reactions lacking SapI and T4 DNA ligase. Once inserted into a backbone, BioBrick2.0 allows cloning through Golden Gate assembly and Gibson assembly. Reactions were cycled between 37°C and 16°C for 5 minutes at each temperature for 30 cycles, and then subjected to a heat-soak at 60°C for 5 minutes before being incubated at 4°C prior to transformation. Practically, it should be noted that both the 13-fragment and 35-fragment assembly reactions contained many overhang pairs anticipated to join with relatively low efficiency, and we still obtained an ample number of transformants for both reactions. Using these tools, we demonstrate how to troubleshoot and expand sets of overhangs for modular cloning systems as well as estimate the limits of high-fidelity assembly. The hairpin substrates were combined with T4 DNA ligase and a Type IIS restriction enzyme and assembly reactions were carried out using a thermocycling protocol. Your credentials are incorrect or you are trying to login with a non-existing webshop account. 2094, 185th Street, Golden Gate Building, Suite #22 (778.24 mi) Fairfield, IA, IA 52556 Conceptualization, Writing – review & editing, Roles Research Department, New England Biolabs, Ipswich, Massachusetts, United States of America, Roles Writing – original draft, We found that 81% of the assembly products are predicted to be error-free when using this overhang set (Fig 4A). https://doi.org/10.1371/journal.pone.0238592.g003. Importantly, these tools can support the design of assembly reaction using Type IIS restriction enzymes that generate three-or four-base overhangs. For example, 5′-CTG/5′-CTG is a frequently observed mispair that significantly decreases the anticipated assembly fidelity for reactions using the 5′-CTG/5′-CAG Watson-Crick pair. Software, In a recently published report from our lab, we found DNA ligation fidelity could be used to estimate the fidelity of Golden Gate assembly reactions [29]. However, the utility of this methodology has been limited by a lack of resources to guide experimental design. here. Creating the targeting vectors using Golden Gate cloning is accomplished by either 1) by amplifying the homology arms (600 bp to 1 kb) using oligonucleotides with SapI sites on the 5′ end of the oligonucleotides and performing a 4 part Golden Gate Reaction using the vector pSapI, the two arm PCR products and the landing site construct, or 2) first amplifying the arms using oligonucleotides that have BsaI sites on the 5′ ends, then performing a BsaI Golden Gate … We thank Nilisha Pokhrel, Lexi Gehring, Kelly Zatopek, and Jennifer Ong (New England Biolabs) and Karen Lohman for careful reading of the manuscript. Formal analysis, For example, 5′-AAT/5′-ATT is among the highest efficiency overhang pair, whereas 5′-TTA/5′-TAA is joined inefficiently (Table 1). Briefly, transformations were performed using 2 μL of each assembly reaction added to 50 μL of competent T7 Express cells. Contact your local subsidiary or distributor. These tools enable users to check the estimated assembly fidelity of overhang sets, generate customized high-fidelity overhang sets, and divide a target sequence into high-fidelity assembly fragments. Briefly, we generated hairpin DNA substrates containing Type IIS restriction enzyme recognition sites and Pacific Bioscience (PacBio) single-molecule, real-time sequencing (SMRT)-bell adapter sequences (Fig 1A). Set up 20 µl assembly reaction as follows: Fill out our Technical Support Form, This analysis suggests the traditional overhang design rules offer a clear improvement over random overhang selection; however, DAD can be used to identify much larger sets of high-fidelity overhangs, with the added advantage of eliminating the laborious task of selecting overhangs by hand. During examination of the high-fidelity overhang sets generated by the GetSet tool, we noticed that many overhang sequences in these sets violate the traditional rules for designing overhang sets by hand. Thus, using comprehensive data sets to calculate predicted assembly fidelity and select fusion sites demonstrably leads to efficient assembly with few erroneous products, even when assembly complexity is much greater than current typical one-pot assemblies. BsaI, BbsI, and BsmBI (along with its isoschizomer Esp3I) each have a distinct six-base pair recognition sequence and cleave DNA to generate four-base overhangs with a 5′-phosphate. Several recent reports have attempted to improve assembly design by utilizing experimental data to inform overhang selection. Yes broad scope, and wide readership – a perfect fit for your research every time. Three. Citation: Pryor JM, Potapov V, Kucera RB, Bilotti K, Cantor EJ, Lohman GJS (2020) Enabling one-pot Golden Gate assemblies of unprecedented complexity using data-optimized assembly design. If you don't see your country above, please visit our We have also developed a binary vector that uses SapI, a Type IIS enzyme that uses a 7-bp recognition sequence and allows for additional flexibility when performing Golden Gate … Strap Tali Jam Tangan Kulit buaya asli golden gate Harga diatas sudah TERMASUK BUCKLE. All assembly products were transformed into T7 Express chemically competent E. coli cells, and the assembly fidelity was scored as described previously [29]. Golden Gate parts were generated for T7-based expression in E. coli. If the new combination of overhangs improves the computed fidelity score (s > s0), it is accepted and used as a starting combination in the new iteration; otherwise (s < s0), the new combination is accepted according to the acceptance probability , where T is the temperature. The Golden Gate protocol allows for convenient and rapid cloning in a single-tube, one-step coupling restriction digestion and ligation . Taken together, these data verify the GetSet prediction that >10 fragments can be accurately assembled in a one-pot Golden Gate assembly reaction with SapI and T4 DNA ligase. (A) The normalized overhang ligation frequencies for all 120 non-palindromic Watson-Crick pairs were plotted for DNA assembly reactions containing T4 DNA ligase and the indicated Type IIS restriction enzyme. Is the Subject Area "Ligases" applicable to this article? We found that the choice among commonly used Type IIS restriction enzymes that generate the same overhang structure did not considerably impact assembly fidelity and bias, suggesting that DNA cleavage is robust and not dependent on the restriction site sequence under standard Golden Gate assembly reaction conditions. Contact our Customer Service Team by The overhang pairs are color-coded according to their frequency relative to the average in terms of the number of standard deviations. Adding products to your cart without being signed in will result in a loss of your cart when you do sign in or leave the site. The P lux promoter was PCR-amplified from pSB1A2-P lux-GFP using primers A15 and A16, which add flanking SapI sites, and inserted into pACYC-SapI-SapI-dCas9 using Golden Gate cloning to yield pACYC-P lux-dCas9. In addition, users can exclude specific fusion site sequences to ensure compatibility with pre-existing modular cloning systems or include fixed sites by setting a narrow search window to cover which site or sites must be used. Golden Gate assembly is a DNA assembly methodology that has been particularly useful in these applications as it supports assembly of multiple DNA fragments in a single reaction and is amenable to automation [2–4]. Next, we incorporated these findings into a suite of webtools that design assembly reactions using the experimental data. This design enables examination of every overhang sequence context in the same assembly reaction. However, it should be noted that we did not compare assembly yield between different Type IIS restriction enzymes. This is especially true for overhang sequences that are prone to mismatch pairing. For four-base overhangs, there are 120 distinct overhangs after eliminating complementary and palindromic overhangs. Materials T4 DNA Ligase ( NEB #M0202 ) https://doi.org/10.1371/journal.pone.0238592.g005. Thus, while it remains to be determined whether selecting only the highest efficiency pairs would enhance the assembly yield or decrease the time necessary to complete the reaction, it does not seem to be a major factor compared to DNA quality or assembly fidelity. Each datapoint represents a single overhang sequence. Methodology, No, Is the Subject Area "Polymerase chain reaction" applicable to this article? Finally, the new backbone with SapI sites, the 4CL with SapI sites, and two synthesized oligos, Prefix-Promoter-LacO-RBS-ZIF268-spacer and terminator-suffix will go through a Golden Gate Assembly with SapI, forming the final 4CL assembly. On average, 71% of the observed transformants harbored correctly assembled products. here. Basically, a type II restriction enzyme is used to cut the DNA. Unlike conventional private equity firms, we operate as a private holding company and recapitalize, restructure, and ultimately build meaningful businesses in partnership with management over an indefinite time horizon. These design principles can inconveniently limit the design of complex assemblies, as the ideal breakpoints in a DNA sequence of interest may call for violation of these rules. Similarly, the efficiency was not always well correlated with the identity of the nucleotide bases adjacent to the ligation site; for example, the 5′-TCCG/5′-CGGA pair is assembled significantly more efficiently than the 5′-TGTG/5′-CACA pair (Fig 2B and 2C). It makes use of type IIS restriction enzymes, such as BsaI, BsmBI, BbsI, SapI, etc., that have the peculiarity of having a recognition site outside their cutting site. (B) For each sequenced assembly product, the overhang pair identity was extracted. For simple, visual assay results, the SARS-CoV-2 Rapid Colorimetric LAMP Assay Kit includes a color-changing pH indicator for detection of SARS-CoV-2 nucleic acid amplification. In addition, we again found that the prediction of efficient overhang combinations is non-trivial based on the sequence composition of the overhang. Contributed equally to this work with: Importantly, GetSet does not use pre-calculated results and instead identifies de novo high-fidelity overhang sets using a stochastic search algorithm. Assembly fragments were purified using the Monarch DNA Cleanup Kit using a 1:1 ratio of sample: binding buffer, and the concentration was determined using the Agilent Bioanalyzer 2100. No, Is the Subject Area "Enzymes" applicable to this article? Methodology, These tools can be found at the following link: https://www.neb.com/research/nebeta-tools, and have also been integrated into a large suite of assembly webtools available at: https://goldengate.neb.com. Full results from each experiment are supplied in the supporting data files (S1–S5 Tables). Ada sebuah teori alternatif menyatakan bahwa itu dibangun di bagian selanjutnya pada abad ke-7 oleh pengrajin … The range and distribution of assembly efficiencies for the Watson-Crick pairs were similar regardless of the restriction enzyme used (Fig 2A). Thus, we recommend DNA stock solutions be purified and accurately quantified for all assembly reactions to maximize assembly efficiency, in addition to selecting junction sets designed to minimize erroneous ligation events. Methodology, To facilitate the design of large, multiple fragment assembly reactions, we systematically examined digestion and ligation of every overhang sequence combination under typical Golden Gate assembly reaction conditions using T4 DNA ligase and BsaI-HFv2, BsmBI-v2, Esp3I, BbsI-HF or SapI. Importantly, transformants harboring correctly assembled constructs turn blue after incubation on media containing IPTG and X-Gal, while transformants harboring constructs with assembly errors form white colonies. To design Golden Gate assembly reactions using our sequencing data, we developed a suite of tools to parse the data in several different ways. Visualization, Chemically competent E. coli strain T7 Express (NEB) lacks a functional lacZ gene, full genotype: fhuA2 lacZ::T7 gene1 [lon] ompT gal sulA11 R(mcr-73::miniTn10—TetS)2 [dcm] R(zgb-210::Tn10—TetS) endA1 Δ(mcrC-mrr)114::IS10. Mismatch frequencies for assembly reactions with T4 DNA ligase and BsaI-HFv2 (blue), BsmBI-v2 (orange), Esp3I (gray), or BbsI-HF (yellow) were grouped according to nucleotide mispair (A:A, A:C, A:G, C:C, C:T, G:G, G:T, T:T). The overhangs are written in a 5′ to 3′ orientation. Standard Taq polymerase buffer is: 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2. PLoS ONE 15(9): Methodology, However, many experimental factors are expected to impact assembly efficiency, as described above. T4 DNA ligase reaction buffer (1X) is: 50 mM Tris–HCl (pH 7.5), 10 mM MgCl2, 1 mM ATP, 10 mM DTT. Your profile has been mapped to an Institution, please sign back for your profile updates to be completed. Finally, both studies were limited to four-base overhangs, and there are currently no resources available to guide assembly design with three-base overhangs, such as those generated by SapI. (1) In addition to allowing users to assemble large protein coding sequences or operons, we also anticipate that this tool could be utilized to quickly generate variants of assembly parts. Two level II plasmids ( T7:His-SUMO-CCaMK and T7:StrepII-SUMO-CYCLOPS ) were … The GetSet tool identified overhang sets containing up to 10 overhang pairs predicted to join >99% accurately, however the predicted fidelity for assemblies with sets containing 11–30 overhangs pairs decreased with each additional overhang added to the set. sgRNA N20 sequences were cloned into the dCas9 plasmid at the sgRNA cloning locus (containing the SNR52 promoter, SapI cloning locus, and sgRNA tail) using Golden Gate cloning , as previously described . https://doi.org/10.1371/journal.pone.0238592.g001. Conceptualization, Conceptualization, (B) For each sequenced assembly product, the overhang pair identity was extracted. Therefore, we designed the SplitSet tool to efficiently design high-fidelity assembly fragments from a desired target DNA sequence. The relative frequency of each overhang pair was determined and was represented as a frequency heat map (log-scaled). To learn more and manage cookies, please refer to our Cookie Statement. Golden Gate assembly reactions (20 μL final volume) with SapI (15 U) and T4 DNA ligase (500 U) were carried out with 3 nM of each PCR assembly fragment in 1X T4 DNA ligase buffer. For this example, we choose the BsmBI-v2 restriction enzyme and 42°C/16°C thermocycling protocol. Writing – review & editing, Roles Blue colonies from both the 13-fragment (SapI + T4 DNA Ligase) and 35-fragment (BsmBI-v2 + T4 DNA Ligase) assembly reactions were subjected to PCR with amplification primers that flank the desired insertion site. I would like to fuse multiple fragments using Golden Gate cloning. Type IIS restriction enzymes cleave outside of their recognition sequence [5]. SapI We are excited to announce that we are in the process of switching all reaction buffers to be BSA-free. It should be noted that other simulation annealing schedules can be used, however, the current optimization strategies already demonstrate an efficient convergence to the near optimum solutions. Writing – original draft, This frequency was slightly higher than the predicted assembly fidelity of 79% and could reflect that some incorrect assembly products cannot be propagated in E. coli under antibiotic selection, such as those that fail to produce circular constructs. However, the scope of this study was limited to approximately 80% of the possible sequence contexts, and the use of only one Type IIS restriction enzyme. For comparison, we also analyzed the fidelity of overhang sets that were selected at random. Add 950 µl of room temperature NEB 10-beta/Stable Outgrowth Medium (. BBa_K2842666 is a BioBrick-compatible standard with improved flexibility that enables the integration of conventional cloning methods into iGEM’s workflow. Click through the PLOS taxonomy to find articles in your field. (B-C) The most and least frequently observed overhang pairs and their relative frequency per 100,000 ligation events are shown. As the field of synthetic biology continues to grow, rapid and robust build phases driven by highly efficient DNA assembly techniques are ever more critical. No, Is the Subject Area "DNA fragment ligation" applicable to this article? We found that results were similar in both cases, however, the simpler implementation required less iterations to arrive at the similar optimum. SapI R0569 New England Biolabs. Each substrate precursor oligonucleotide (20 μM final concentration) was combined with 100 U of Klenow Fragment (3′→5′ exo-), 0.2 U yeast inorganic pyrophosphatase, 1 mM of each dNTP (final concentration), and 1X NEBuffer 2 reaction buffer (final concentration) in a 100 μl reaction volume. It should be noted the resulting overhang set contained several sequence combinations that are not allowed using traditional overhang design standards, including overhang pairs with 100% A/T or G/C content and many with only one base difference from multiple other members of the set (Table 2). The Ligase Fidelity Viewer is used to check assembly fidelity of overhang sets, the GetSet tool is used to design high-fidelity overhang sets, and the SplitSet tool is used to divide up a target DNA sequencing into high-fidelity assembly fragments. Overhangs are listed alphabetically left to right (AAAA, AAAC…TTTG, TTTT) and bottom to top such that the Watson–Crick pairings are shown on the diagonal represented above. Reactions (20 μL final volume) containing T4 DNA ligase (500 U) and BbsI-HF (15 U) were carried out in 1X CutSmart Buffer supplemented with 10 mM DTT and 1 mM ATP. Golden Gate Assembly of 13 fragments using SapI restriction enzyme with T4 DNA Ligase and subsequent transformation into NEB 10-beta Competent E. coli (High Efficiency). These tools, described in more detail below, are freely available on the web at: https://www.neb.com/research/nebeta-tools. Please sign back in to continue your session. 14225 Collier Blvd, 4021 7th Ave NW, 4161 7th Ave NW, and 4191 7th Ave NW. We also note mispairing is common in assembly reactions, and the observed assembly outcomes are complex and not trivially reduced to simple trends or rules. This property gives several advantages during cloning: Selection of a Type IIS restriction enzyme is typically guided by compatibility with a modular cloning system or a desired recognition sequence. (A) Schematic of the 13-fragment lac operon cassette test system. We observed all 32 Watson-Crick overhang pairs and >500 distinct mispairs in the assembly products (S5 Table). Using randomly selected non-palindromic overhang pairs, we identified overhang sets containing up to 6–8 overhang pairs anticipated to join with high-fidelity. Taken together, these data further support and emphasize the difficulty of designing complex assemblies by hand. Reactions (20 μL final volume) with T4 DNA ligase and BsaIHF-v2 or BsmBI-v2 were carried out using their respective NEB Golden Gate Enzyme Mixes (2 μL) in 1X T4 DNA ligase buffer. Initially, a random set of n overhangs is generated, and its fidelity is estimated (so). MoClo) as well as the formation of robust new high-fidelity standards. No, Is the Subject Area "Cloning" applicable to this article? Visualization, Golden Gate Assembly, Restriction Enzyme Digestion Properties & Usage Unit Definition One unit is defined as the amount of enzyme required to digest 1 µg of λ DNA in 1 hour at 37°C in a total reaction volume of 50 µl. 1X CutSmart® Buffer Incubate at 37°C . We again noted that assembly was promiscuous, as each overhang usually mispaired with >10 non-complementary partners (S1 Fig). Presumably, this is due to differences in the reaction temperatures and buffer conditions between the two studies. To help guide overhang selection for assemblies with Type IIS restriction enzymes that generate three-base overhangs, we examined Golden Gate assembly with T4 DNA ligase and SapI. Furthermore, these guidelines are laborious to implement when designing assemblies by hand, especially for assemblies of >10 fragments. Suboptimal conditions, such as temperature or buffer conditions where the activity of the restriction enzyme is poor and cutting is inefficient relative to re-ligation, could decrease the assembly yield. We find the choice of a Type IIS restriction enzyme marginally impacts the observed assembly efficiencies for each overhang pair, suggesting that cleavage is robust with the commonly used Type IIS restriction enzymes under typical Golden Gate reaction conditions. Control reactions and additional screening on a subset of blue colonies were carried out as described above (S2 Fig). These data demonstrate that DAD can be used to easily design robust assembly reactions of unprecedented complexity, and we anticipate that utilizing these tools will be helpful to design robust assembly reactions of any size. Lastly, we use DAD to design and carry out the most complex one-pot Golden Gate assembly reactions to date: 13-fragment assembly with three-base overhangs and 35-fragment assembly with four-base overhangs. international site. We tested the predictions of these tools under challenging circumstances, to carry out very large multi-fragment assemblies, and found that the predictions closely matched the observed assembly fidelities. https://doi.org/10.1371/journal.pone.0238592.g002. Copyright: © 2020 Pryor et al. This assay was initially designed to study DNA ligation [29,32]; the modified version was redesigned to more closely mimic the features and reaction conditions of a Golden Gate assembly reaction. 1X CutSmart® Buffer 50 mM Potassium acetate 20 mM Tris-acetate Tambah kegantengan mu dengan strap dari kami! We carried out assembly reactions using BsmBI-v2 and T4 DNA ligase and found that on average 71% of the observed transformants harbored accurately assembled constructs, compared to a theoretical prediction of 65% (Fig 7B and 7C). Selection of the overhang sequences that flank assembly fragments is an important consideration for successful Golden Gate assembly because promiscuous ligation of non-complementary overhang sequences by the DNA ligase can reduce assembly yield and increase the amount of time required to screen for the desired assembly product [29,31]. Yes Visualization, Four replicate experiments were carried out to quantify the number of colony-forming units harboring correct and incorrect assembly products per 50 μL of E. coli outgrowth plated (0.1 μL of the assembly reaction). https://doi.org/10.1371/journal.pone.0238592.s010. Reaction Conditions. Lastly, we demonstrate how using these tools expands the limits of current assembly systems by carrying out one-pot assemblies of up to 35 DNA fragments. As an example, we used the GetSet tool to expand the standard overhang set used in plant synthetic biology; we found the set size could be increased from 11 overhangs to 20 overhangs with marginally decreasing the predicted assembly fidelity from 81% to 80% (Fig 4B). Briefly, the DNA assembly fragments comprise a cassette of the lac operon that is cloned into a destination vector containing an antibiotic resistance marker. The overhang sequences connecting fragments are selected using broad design guidelines that minimize base pairing between non-complementary overhangs. RESULTS. In addition to uncertainty in the data used to estimate fidelity, other experimental factors such as suboptimal enzyme concentrations, thermocycling conditions, or DNA purity can influence both yield of the final assembly and prevalence of colonies lacking an insert or containing an undesired assembly. For example, this strategy could be used to easily remove internal Type IIS recognition sequences from assembly fragments, or easily generate high diversity libraries with randomized regions at specific sites, simply by setting windows for the junction fusion sites near the areas to be mutagenized. (A) Hairpin DNA substrates containing a Type IIS recognition sequence (orange), randomized nucleotides at the Type IIS restriction site (NNNN), an internal 6-base random barcode (black), and a PacBio SMRTbell adapter sequence (blue) were synthesized. (B) Results of the assembly reactions. At the same time, our construct has a LacZ reporter, which can be used to screen plates for successful colonies. However, as the size of the overhang set increases mismatch ligation becomes more problematic. Libraries were sequenced on a PacBio RSII instrument with at least 2 SMRT cells per library and a 3 h data collection time per cell with ‘stage start’ off. In addition, the assembly efficiency of each Watson-Crick pair was well correlated (Fig 2B and 2C). Don't leave hungry! Conceptualization, This does not alter our adherence to PLOS ONE policies on sharing data and materials. Consensus sequences for each assembly product were generated as described previously [32]. To compare assembly design by DAD with the traditional overhang design standards, we repeated our fidelity predictions for overhang sets generated using the traditional design rules. where Ncorrect is the number of times overhang Oi ligates correctly to its WC pair and vice versa, and Ntotal is the number of times Oi ligates to any overhangs in the set and its WC pair. Users can specify overhang sequences that must be included or excluded from the results. These data support our previous work suggesting that Golden Gate assembly fidelity and bias is predominantly determined at the DNA ligation step [29,32]. Add 2 µl of the assembly reaction; gently mix by flicking the tube 4-5 times. For each assembly, thaw a 50 µl tube of NEB 10-beta competent E. coli cells on ice for 5–10 min. The substrate sequences (S8 Table) include: a 5′ Type IIS recognition sequence, a randomized four-base region, a constant region, an internal six-base randomized region as a control for synthesis bias, and a region corresponding to the SMRTbell sequencing adapter for PacBio sequencing. Place your order before 7:30pm EST for overnight delivery. Writing – review & editing, Affiliation We have also developed a binary vector that uses SapI, a Type IIS enzyme that uses a 7-bp recognition sequence and allows for additional flexibility when performing Golden Gate Cloning. The outgrowth was spread onto prewarmed agar plates (Luria–Bertani broth supplemented with 1 mg/mL dextrose, 1 mg/mL MgCl2, 30 μg/mL Chloramphenicol, 200 μM IPTG and 80 μg/mL X-gal). Blue transformants harbor correct assembly constructs, and white transformants harbor inaccurate assembly products. Writing – review & editing. Conceptualization, https://doi.org/10.1371/journal.pone.0238592, Editor: Ruslan Kalendar, University of Helsinki, FINLAND, Received: April 9, 2020; Accepted: August 19, 2020; Published: September 2, 2020. This website uses cookies to ensure you get the best experience. The final concentration of DNA substrate in each assembly reaction was 100 nM. Discover a faster, simpler path to publishing in a high-quality journal. SapI is the most distinct of the commonly used Type IIS restriction enzymes, as it has a seven base pair recognition sequence and cleaves DNA to generate three-base overhangs with a 5′-phosphate [5]. 345m Golden Gate 347m Kg.Sundang Laut 356m Smk Batu Sapi Sandakan Local Business The assembly products were then re-purified using the Monarch PCR & DNA Cleanup Kit, including a second wash step, and quantified by Agilent Bioanalyzer (DNA 1000).