gibson assembly efficiency

Each fragment is made up of commonly used constitutive promoters of varying strengths and RBSs. (b) Efficiency of 3-fragment assembly by SENAX. 1116, 235244. V.L.D. Natl. The fully amplified 807bp 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. 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. Nonetheless, using the 12bp and 10bp homology arm sizes still yielded fluorescent colonies, thereby showing that the SENAX method works even with smaller homology arm sizes. Samples with no enzyme protein were used as the control. 5). Desalted oligonucleotide primers are suitable for PCR for SENAX. Hence, 10bp homology can be considered the lower limit for SENAX design. For sequence homology-based methods, one approach is to standardize the overlapping regions that are basically independent of the sequence of DNA parts. Gibson Assembly Tips for More Efficient Reactions - SGI-DNA Get the most important science stories of the day, free in your inbox. This enzyme does not attack the single stranded DNA since the hydrolysis is specific for base-paired nucleotides in this enzyme24. Gibson Assembly Master Mix | NEB The correct plasmid was introduced into E. coli BL21 for protein expression. The resulting duplex DNA solution was then kept at 20C and was used for subsequent multiple different DNA assembly constructs. 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. S3). 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 inserted XthA and junctions were verified by nucleotide sequencing to confirm that the cloning was in frame. XthA is also commercially available for research use, such as for the preparation of single-stranded DNA for dideoxy sequencing or site direct mutagenesis. S2). 67(1), 88101. S9). 65(C), 201211. The efficiency of co-transformation cloning is however low and the Gibson assembly reagents are expensive. Article This has proven to be an efficient and effective method for the assembly of plasmids, and molecular biologists now use this method extensively. Do note that the two commercial kits were used according to the manufacturer recommended protocol and optimal conditions (e.g., at a temperature of 50C for at least 15min). If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. Although the XthA product is commercially available for molecular biology studies (e.g., NEB M0206 L), the storage buffer contains some chemicals that might affect DNA assembly. Watanabe, K., Tominaga, K., Kitamura, M. & Kato, J. I. Yang, Y. et al. Twenty nanograms of each fragment was used, and the reaction was performed at 37C for 15min. Scientific Reports (Sci Rep) Google Scholar. PubMed [26] Exonuclease III of Escherichia Coli K-12, an AP endonuclease. 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. Hence, we performed the 3-fragment assembly of construct B using different competent cells, including DH5-alpha and 10Beta (NEB), for the transformation step. This temperature range would also be more compatible with a high-throughput automation system. XthA is known as a multi-functional enzyme, and its homologues are involved in the DNA repair system in various bacterial species. Nucleic Acids Res. Top right, a plot showing the efficiency of assembly along with an increasing number of fragments involved. 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 18bp. This depends on the original concentration of short-fragment stock synthesized by the company (IDT). Under a new group order, Alstom will supply the metropolitan areas of Toulouse, Brest and Besanon in France with a total of 22 Citadis trams. Assembly and transformation in just under two hours Flexible sequence design (scar-less cloning) No PCR clean-up step required High transformation efficiencies for inserts up to 20 kb To this end, we studied the efficiency of the mix with only XthA in assembling 3 typical medium-size fragmentsan RSF origin of replication, a kanamycin resistance cassette, and a GFP reporter gene, which were PCR-prepared to have 18bp overlapping regions to each other. Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. EcoSal Plus https://doi.org/10.1128/ecosalplus.4.4.7 (2011). https://doi.org/10.1266/ggs.15-00068 (2016). 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 (0100ng) for each 10 L reaction. Tests of the 70bp fragment with the ho1 template were successful, achieving a high number of colonies (36). Conley, E. C., Saunders, V. A., Jackson, V. & Saunders, J. R. Mechanism of intramolecular recyclizatlon and deletion formation following transformation of Escherichia coli with linearized plasmid DNA. (2006). Virus Res. 4b). Assembly mixes of the 3-fragment assembly reaction after 15min with different XthA amounts were verified by agarose electrophoresis (below). Gibson, D. G. et al. In some experiments, the cultures were incubated at different temperatures for optimization purposes. Alstom teams are especially proud to win the contract for Line 18 of the le-de-France network. Efficiency of assembly decreases as the number or length of fragments increases. 10(1), 3294. https://doi.org/10.1038/s41467-019-11263-0 (2019). Promoters/Catalog/Andersonparts.igem.org. 3b) and naringenin-producing gene cluster (Fig. GeneArt Gibson assembly EX kits are ideal for assembling multiple inserts. However, there were only a few colonies without fluorescence when the temperature was 32C, while the number of nonfluorescent colonies gradually increased when the temperature was reduced to 30C and lower. Alstom was selected by Socit du Grand Paris, in agreement with le-de-France Mobilits. Biol. This suggests that the DNA assembly activity does not depend on specific components of Stellar E. coli. On the other hand, in vitro assembly methods have been widely employed for routine DNA construction, as they are more stable and have higher efficiency and accuracy. Find out what industry suppliers are up to and read in-depth editorials. Taken together, SENAX can achieve high accuracy at reasonable efficiency for short-fragment assembly, and the minimum length of the short fragment that can be assembled directly into a template is 70bp. In the last step, the buffer containing the purified protein fraction was changed to 50mM TrisHCl pH 7.5. Five short fragments of different lengths (200bp, 150bp, 100bp, 88bp, 70bp) were designed (Table S2). All authors have no other conflicts of interest to declare. A. Line 18 is a 35-kilometre automatic metro line that will include 14km of overhead lines. We also investigated the effect of temperature on SENAX efficiency. (b) A naringenin-producing plasmid (10.5kb) was separated by PCR into several linear fragments (34567) with 18bp homology arms. PubMed However, this requires the use of long primers (usually 50100bp or more), resulting in a higher cost of DNA synthesis. However, the efficiency was lower when the concentrated Stellar cell extract supplemented with XthA was used. With the 2.8kb backbone plasmid, both Gibson and In-Fusion methods generated a number of fluorescent colonies for the assembly of the 200bp and 150bp short fragments. Shorter than 10min of incubation greatly decreased the efficiency, as approximately 2 times less activity was observed. marie.bah-monteilliet@veolia.com. Gibson Assembly - Snapgene 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. The results show that SENAX is much more effective in assembling fragments shorter than 100bp into backbones of varied sizes compared to Gibson and In-Fusion. Meanwhile, the nicked DNA substrate is known to be a weak substrate for XthA compared to other exonucleases20. This could be due to chemicals present in the storage buffer by the manufacturer. Nucleic Acids Res. The In-Fusion method uses a polymerase with exonuclease activity to accomplish the reaction22,23. Approximately 1g was loaded onto SDSPAGE (Trisglycine 10% polyacrylamide gel). Gibson Assembly Protocol (E5510) | NEB 5). PubMed Central 4b). Introduction to Gibson Assembly | NEB 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 amplicon was then treated with the corresponding restriction enzyme to release the 55 overhang fragment (XbaI-BamHI) and 53 overhang fragment (XbaI-KpnI) (Fig. These constructs were used for multifragment assembly and short-fragment assembly tests. email. I've noticed the efficiency of Gibson Assembly can be pretty dependent on the purity of my fragments, the length/tm of my overlaps, and the proximity of the ends to either regions with. Constructs A, B, C, D (2.8kb); E (4.0kb); F (5.0kb); and G (6.3kb) were used for the test (see Fig. The efficiency achieved by SENAX is comparable to that achieved by commonly used DNA assembly methods (Gibson and In-Fusion) while requiring shorter homology arms and lower reaction temperatures. 83(9), 42364250. PubMedGoogle Scholar. Nucleic Acids Res. DNA Cloning Tips-Build Clones with DNA Fragments using Gibson Assembly Systematic identification of synthetic lethal mutations with reduced-genome Escherichia coli: Synthetic genetic interactions among yoaA, xthA and holC related to survival from MMS exposure. N.R. S4). Google Scholar. S8b) and using 16bp homology arms for short-fragment assembly. 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. All of the short fragments consist of spacer S1 at the 5 terminus, a promoter, and an RBS. https://doi.org/10.1371/journal.pone.0115318 (2014). For the 6-fragment assembly, we obtained several colonies on the plate. 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. Gibson Assembly constructs may be prepared using SGIDNA Gibson Assembly HiFi 1Step and Ultra kits or by the automated cloning instrument, the BioXp 3200 system. By subscribing you agree to our Privacy Policy. Article XthA is known as a multifunctional DNA repair enzyme that has important biological roles in DNA metabolism (phosphatase, exonuclease, AP endonuclease, RNase H activities)8,9. It will supply a maximum of 37 trainsets, with an initial firm order made for 15. S5h). Department of Biomedical Engineering, National University of Singapore, Singapore, Singapore, Viet Linh Dao,Sheena Chan,Jingyun Zhang,Russell Kai Jie Ngo&Chueh Loo Poh, Synthetic Biology for Clinical and Technological Innovation, National University of Singapore, Singapore, Singapore, You can also search for this author in While a number of enzymes could be responsible for SLiCE assembly and in vivo recombination activity in E. coli20, recent reports revealed the important role of XthA and its homologues in DNA repair in many species, including E. coli, and XthA is required for in vivo DNA cloning using E. coli13. supervised the study. Motohashi, K. A simple and efficient seamless DNA cloning method using SLiCE from Escherichia coli laboratory strains and its application to SLiP site-directed mutagenesis. For example, using current methods, researchers would need to repeatedly synthesize new long primers (over 100bp) to include the short fragment in advance. 35(1), 143151. Biol. This is a common observation, as reported for other assembly methods2. We love trains. In brief, 4.5 L of the Gibson Assembly mixture (containing 601-based . Article This result indicates that having only XthA was sufficient for DNA assembly. We tested the cloning of the blunt end and sticky end (3-prime overhang and 5-prime overhang inserts) using SENAX (Fig. S5Table S3). CAF has been contracted to manufacture 18 Coradia Polyvalent regional trains for the south of France and the nation of Senegal. Optimal Quantities NEB recommends a total of 0.02-0.5 pmols of DNA fragments when 1 or 2 fragments are being assembled into a vector and 0.2-1.0 pmoles of DNA fragments when 4-6 fragments are being assembled. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. This contract also points to the renewed confidence of our customers, le-de-France Mobilits and Socit du Grand Paris.. Therefore, when building larger molecules, it is recommended to keep the number of fragments as small as possible. The transformants were screened on antibiotic screening plates, and the extracted plasmids from several positive colonies were sent for sequencing (1st-BASE, Axil ScientificSanger sequencing service) to confirm the match to the designed constructs. To produce E. coli Stellar XthA for the study, XthA was cloned and expressed in E. coli BL21 (Fig. 3-fragment assembly with different amounts of Mg2+supplemented in the reaction. A similar situation could be assumed for the exonuclease enzyme used in In-Fusion technology. S10). 2a, SENAX effectively catalyzed the assembly of 3, 4, or 5 fragments. 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. In our study, the spacer (18bp) was designed to facilitate the easy reuse of the bioparts. The obtained mixture was heated to 95C for 5min and lowered to 4C at 0.1C/sec to allow annealing. The pathway of recombining short homologous ends in Escherichia coli revealed by the genetic study. PDF A Simple Enhancement for Gibson Isothermal Assembly - bioRxiv Metzger, W. & Heumann, H. Footprinting with exonuclease III. The percentage of fluorescent colonies was reduced by more than 70% compared with the experiment using a 15min incubation time. Constructing arrays of nucleosome positioning sequences using Gibson https://doi.org/10.1007/978-1-62703-764-8_16 (2014). Correspondence to Among the tested short fragment sizes, 88bp appeared to be a good candidate size to harbour bioparts such as promoters and RBSs, which are routinely used for fine-tuning gene expression. Nat. The DNA bands found from 1.5 to 2.0kb probably represented the linear assembled product, in which only 2 DNA fragments were concatenated. Both methods were not effective with fragments shorter than 100bp, particularly with the 9.0kb backbone. To confirm the sequences, three colonies among these fluorescent colonies were examined using sequencing.
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