DNA Assembly for Plant Biology

Nicola J. Patron1

1 The Earlham Institute, Norwich Research Park, Norwich, Norfolk
Publication Name:  Current Protocols in Plant Biology
Unit Number:   
DOI:  10.1002/cppb.20038
Online Posting Date:  December, 2016
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Parallel DNA assembly methods allow multiple fragments of DNA to be compiled in a desired order in a single reaction. Several methods enable the efficient one‐step assembly of multiple DNA parts into a suitable plasmid acceptor at high efficiency. Type IIS‐mediated assembly offers the specific advantage of a one‐step reaction that does not require proprietary reagents or the amplification and purification of linear DNA fragments. Instead, multiple plasmids housing standardized DNA parts of interest are combined in an enzyme cocktail. To make these standard parts, DNA sequences with defined functions are assigned specific sequence features. This allows parts to be interoperable and reusable. The availability of collections of DNA parts and molecular toolkits that allow the facile assembly of multigene binary constructs and the establishment of standards for the creation of new parts means Type IIS‐mediated assembly has become a powerful technology for modern plant molecular biologists. © 2016 by John Wiley & Sons, Inc.

Keywords: DNA assembly; gene cloning; modular cloning; biological standards

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Assembly of Standard Parts in the Common Syntax into Transcriptional Units
  • Support Protocol 1: Making New Standard Parts
  • Basic Protocol 2: Assembly of Transcriptional Units into Multi‐Gene Constructs Using MoClo
  • Alternate Protocol 1: Assembly of Transcriptional Units into Multigene Constructs Using GoldenBraid
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Assembly of Standard Parts in the Common Syntax into Transcriptional Units

  Materials
  • Purified DNA of acceptor plasmid in which parts will be assembled
  • Purified plasmid DNA containing DNA parts to be assembled
  • 10× T4 ligase buffer (e.g., New England BioLabs)
  • 200 U/µl T4 DNA ligase (e.g., New England BioLabs)
  • 1 mg/ml bovine serum albumin (BSA; e.g., New England BioLabs)
  • 10 U/µl BsaI (Eco31I; e.g., Thermo Fisher Scientific)
  • Electrocompetent cells (>109 colony forming units [CFU]/µg) or chemically competent cells
  • LB agar plates supplemented with 0.5 mM IPTG, 0.02 mg/ml X‐gal, and appropriate antibiotics (see Elbing and Brent, )
  • LB medium supplemented with appropriate antibiotics (see Elbing and Brent, )
  • Thermal cycler
  • Electroporator (for electrocompetent cells) or 42°C water bath (for chemically competent cells)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
  Annaluru, N., Ramalingam, S., and Chandrasegaran, S. 2015. Rewriting the blueprint of life by synthetic genomics and genome engineering. Genome Biol. 16:125. doi: 10.1186/s13059‐015‐0689‐y.
  Casini, A., Storch, M., Baldwin, G.S., and Ellis, T. 2015. Bricks and blueprints: Methods and standards for DNA assembly. Nat. Rev. Mol. Cell Biol. 16:568‐576. doi: 10.1038/nrm4014.
  Casini, A., Macdonald, J.T., De Jonghe, J., Christodoulou, G., Freemont, P.S., Baldwin, G.S., and Ellis, T. 2014. One‐pot DNA construction for synthetic biology: The Modular Overlap‐Directed Assembly with Linkers (MODAL) strategy. Nucl. Acids Res. 42:1‐13. doi: 10.1093/nar/gkt915.
  de Kok, S., Stanton, L.H., Slaby, T., Durot, M., Holmes, V. F., Patel, K. G., Platt, D., Shapland, E. B., Serber, Z., Dean, J., Newman, J.D., and Chandran, S.S. 2014. Rapid and reliable DNA assembly via ligase cycling reaction. ACS Synth. Biol. 3:97‐106. doi: 10.1021/sb4001992.
  Elbing, K. and Brent, R. 2002. Media preparation and bacteriological tools. Curr. Protoc. Mol. Biol. 59:1.1.1‐1.1.7. doi: 10.1002/0471142727.mb0101s59.
  Ellis, T., Adie, T., and Baldwin, G. S. 2011. DNA assembly for synthetic biology: From parts to pathways and beyond. Integr. Biol. 3:109‐118. doi: 10.1039/c0ib00070a.
  Engler, C., Kandzia, R., and Marillonnet, S. 2008. A one pot, one step, precision cloning method with high throughput capability. PloS One 3:e3647. doi: 10.1371/journal.pone.0003647.
  Engler, C., Youles, M., and Grüetzner, R. 2014. A Golden Gate modular cloning toolbox for plants. ACS Synth. Biol. 3:839‐843. doi: 10.1021/sb4001504.
  Gibson, D., Young, L., Chuang, R., Venter, C., Hutchison III, C.A., and Smith, H. 2009. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6:343‐345. doi: 10.1038/nmeth.1318.
  Hutchison, C.A., Chuang, R.‐Y., Noskov, V.N., Assad‐Garcia, N., Deerinck, T. J., Ellisman, M.H., Gill, J., Kannan, K., Karas, B.J., Ma, L., Pelletier, J.F., Qi, Z.Q., Richter, R.A., Strychalski, E.A., Sun, L., Suzuki, Y., Tsvetanova, B., Wise, K.S., Smith, H.O., Glass, J.I., Merryman, C., Gibson, D.G., and Venter, J.C. 2016. Design and synthesis of a minimal bacterial genome. Science 351:aad6253. doi: 10.1126/science.aad6253.
  Knight, T. 2003. Idempotent vector design for standard assembly of BioBricks. MIT Artificial Intelligence Laboratory, MIT Synthetic Biology Working Group. Available at http://hdl.handle.net/1721.1/21168.
  Lee, L.‐Y. and Gelvin, S.B. 2008. T‐DNA binary vectors and systems. Plant Physiol. 146:325‐332. doi: 10.1104/pp.107.113001.
  Li, M.Z. and Elledge, S.J. 2007. Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. Nat. Methods 4:251‐256. doi: 10.1038/nmeth1010.
  Patron, N.J. 2014. DNA assembly for plant biology: Techniques and tools. Curr. Opin. Plant Biol. 19C:14‐19. doi: 10.1016/j.pbi.2014.02.004.
  Patron, N.J. 2016. Blueprints for green biotech: Development and application of standards for plant synthetic biology. Biochem. Soc. Trans. 44:702‐708. doi: 10.1042/BST20160044.
  Patron, N., Orzaez, D., Marillonnet, S., Warzecha, W., Matthewman, C., Youles, M., Raitskin, O., Leveau, A., Farré, G., Rogers, C., Smith, A., Hibberd, J., Webb, A.A., Locke, J., Schornack, S., Ajioka, J., Baulcombe, D.C., Zipfel, C., Kamoun, S., Jones, J.D., Kuhn, H., Robatzek, S., Van Esse, H.P., Sanders, D., Oldroyd, G., Martin, C., Field, R., O'Connor, S., Fox, S., Wulff, B., Miller, B., Breakspear, A., Radhakrishnan, G., Delaux, P.M., Loqué, D., Granell, A., Tissier, A., Shih, P., Brutnell, T.P., Quick, W.P., Rischer, H., Fraser, P.D., Aharoni, A., Raines, C., South, P.F., Ané, J.M., Hamberger, B.R., Langdale, J., Stougaard, J., Bouwmeester, H., Udvardi, M., Murray, J.A., Ntoukakis, V., Schäfer, P., Denby, K., Edwards, K.J., Osbourn, A., and Haseloff, J. 2015. Standards for plant synthetic biology: A common syntax for exchange of DNA parts. New Phytol. 208:13‐19. doi: 10.1111/nph.13532.
  Quan, J. and Tian, J. 2009. Circular polymerase extension cloning of complex gene libraries and pathways. PloS One 4:e6441. doi: 10.1371/journal.pone.0006441.
  Sarrion‐Perdigones, A., Falconi, E.E., Zandalinas, S.I., Juárez, P., Fernández‐del‐Carmen, A., Granell, A., and Orzaez, D. 2011. GoldenBraid: An iterative cloning system for standardized assembly of reusable genetic modules. PloS One 6:e21622. doi: 10.1371/journal.pone.0021622.
  Sarrion‐Perdigones, A., Vazquez‐Vilar, M., Palací, J., Castelijns, B., Forment, J., Ziarsolo, P., Blanca, J., Granell, A., and Orzaez, D. 2013. GoldenBraid2.0: A comprehensive DNA assembly framework for plant synthetic biology. Plant Physiol. 162:1618‐1631. doi: 10.1104/pp.113.217661.
  Shetty, R.P., Endy, D., and Knight, T.F. 2008. Engineering BioBrick vectors from BioBrick parts. J. Biol. Eng. 2:5. doi: 10.1186/1754‐1611‐2‐5.
  Storch, M., Casini, A., Mackrow, B., Trewhitt, H., Ellis, T., and Baldwin, G.S. 2015. BASIC: A new Biopart Assembly Standard provides accurate, single‐tier DNA assembly for synthetic biology. ACS Synth. Biol. 4:781‐787. doi: 10.1021/sb500356d.
  Weber, E., Engler, C., Gruetzner, R., Werner, S., and Marillonnet, S. 2011. A modular cloning system for standardized assembly of multigene constructs. PloS One 6:e16765. doi: 10.1371/journal.pone.0016765.
  Werner, S., Engler, C., Weber, E., Gruetzner, R., and Marillonnet, S. 2012. Fast track assembly of multigene constructs using Golden Gate cloning and the MoClo system. Bioeng. Bugs 3:38‐43. doi: 10.4161/bbug.3.1.18223.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library