Abstract
Simple and reliable DNA assembly methods have become a critical technique in synthetic biology. Here, we present a protocol of the recently developed DATEL (scarless and sequence-independent DNA assembly method using thermostable exonuclease and ligase) method for the construction of genetic circuits and biological pathways from multiple DNA parts in one tube. DATEL is expected to be an applicable choice for both manual and automated high-throughput assembly of DNA fragments, which will greatly facilitate the rapid progress of synthetic biology and metabolic engineering.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Ellis T, Adie T, Baldwin GS (2011) DNA assembly for synthetic biology: from parts to pathways and beyond. Integr Biol (Camb) 3:109–118
Canton B, Labno A, Endy D (2008) Refinement and standardization of synthetic biological parts and devices. Nat Biotechnol 26:787–793
Cohen SN, Chang AC, Boyer HW, Helling RB (1973) Construction of biologically functional bacterial plasmids in vitro. Proc Natl Acad Sci U S A 70:3240–3244
Engler C, Gruetzner R, Kandzia R, Marillonnet S (2009) Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes. PLoS One 4:e5553
Sarrion-Perdigones A, Falconi EE, Zandalinas SI, Juarez P, Fernandez-del-Carmen A, Granell A, Orzaez D (2011) GoldenBraid: an iterative cloning system for standardized assembly of reusable genetic modules. PLoS One 6:e21622
Kodumal SJ, Patel KG, Reid R, Menzella HG, Welch M, Santi DV (2004) Total synthesis of long DNA sequences: synthesis of a contiguous 32-kb polyketide synthase gene cluster. Proc Natl Acad Sci U S A 101:15573–15578
Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S (2011) A modular cloning system for standardized assembly of multigene constructs. PLoS One 6:e16765
Li MZ, Elledge SJ (2007) Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. Nat Methods 4:251–256
Quan JY, Tian JD (2011) Circular polymerase extension cloning for high-throughput cloning of complex and combinatorial DNA libraries. Nat Protoc 6:242–251
Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA, Smith HO (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6:343–345
Sleight SC, Bartley BA, Lieviant JA, Sauro HM (2010) In-fusion BioBrick assembly and re-engineering. Nucleic Acids Res 38:2624–2636
Wang RY, Shi ZY, Guo YY, Chen JC, Chen GQ (2013) DNA fragments assembly based on nicking enzyme system. PLoS One 8:e57943
Kang Z, Zhang J, Jin P, Yang S (2015) Directed evolution combined with synthetic biology strategies expedite semi-rational engineering of genes and genomes. Bioengineered 6:136–140
Smanski MJ, Bhatia S, Zhao D, Park Y, BAW L, Giannoukos G, Ciulla D, Busby M, Calderon J, Nicol R (2014) Functional optimization of gene clusters by combinatorial design and assembly. Nat Biotechnol 32:1241–1249
Gibson DG, Benders GA, Axelrod KC, Zaveri J, Algire MA, Moodie M, Montague MG, Venter JC, Smith HO, Hutchison CA (2008) One-step assembly in yeast of 25 overlapping DNA fragments to form a complete synthetic mycoplasma genitalium genome. Proc Natl Acad Sci U S A 105:20404–20409
Lin QH, Jia B, Mitchell LA, Luo JC, Yang K, Zeller KI, Zhang WQ, Xu ZW, Stracquadanio G, Bader JS, Boeke JD, Yuan YJ (2015) RADOM, an efficient in vivo method for assembling designed DNA fragments up to 10 kb long in Saccharomyces Cerevisiae. ACS Synth Biol 4:213–220
Shao ZY, Zhao H, Zhao HM (2009) DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways. Nucleic Acids Res 37:e16
Zhou JT, Wu RH, Xue XL, Qin ZJ (2016) CasHRA (Cas9-facilitated homologous recombination assembly) method of constructing megabase-sized DNA. Nucleic Acids Res 44:e124
Itaya M, Fujita K, Kuroki A, Tsuge K (2008) Bottom-up genome assembly using the Bacillus Subtilis genome vector. Nat Methods 5:41–43
Jin P, Ding W, Du G, Chen J, Kang Z (2016) DATEL: a scarless and sequence-independent DNA assembly method using thermostable exonucleases and ligase. ACS Synth Biol 5:1028–1032. https://doi.org/10.1021/acssynbio.6b00078
Holland PM, Abramson RD, Watson R, Gelfand DH (1991) Detection of specific polymerase chain reaction product by utilizing the 5′–3′ exonuclease activity of Thermus aquaticus DNA polymerase. Proc Natl Acad Sci U S A 88:7276–7280
Lyamichev V, Brow MAD, Dahlberg JE (1993) Structure-specific endonucleolytic cleavage of nucleic-acids by eubacterial DNA-polymerases. Science 260:778–783
Breslauer KJ, Frank R, Blöcker H, Marky LA (1986) Predicting DNA duplex stability from the base sequence. Proc Natl Acad Sci U S A 83:3746–3750
Acknowledgments
This work was financially supported by the National Natural Science Foundation of China (31670092), the Fundamental Research Funds for the Central Universities (JUSRP51707A), and Program for Changjiang Scholars and Innovative Research Team in University (No. IRT_15R26).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Kang, Z., Ding, W., Jin, P., Du, G., Chen, J. (2018). DNA Assembly with the DATEL Method. In: Braman, J. (eds) Synthetic Biology. Methods in Molecular Biology, vol 1772. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7795-6_24
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7795-6_24
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7794-9
Online ISBN: 978-1-4939-7795-6
eBook Packages: Springer Protocols