Design and Assembly of Large Synthetic DNA Constructs

Aleksandr E. Miklos1, Randall A. Hughes1, Andrew D. Ellington1

1 The University of Texas at Austin, Applied Research Laboratories, Department of Chemistry and Biochemistry, Center for Systems and Synthetic Biology, Austin, Texas
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 3.23
DOI:  10.1002/0471142727.mb0323s99
Online Posting Date:  July, 2012
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Abstract

The availability of custom synthetic gene‐length DNA products removes numerous bottlenecks in research efforts, making gene synthesis an increasingly common commercial service. However, the assembly of synthetic oligonucleotides into large, custom DNA constructs is not especially difficult, and performing “in‐house” gene synthesis has time and cost advantages. This unit will treat both the concerns of design and physical assembly in gene synthesis, including how to design DNA sequences for synthesis and the design of overlapping oligonucleotide schemes to ensure facile assembly into the final product. Assembly is accomplished using a reliable series of PCR reactions, with a troubleshooting assembly protocol included, which not only assembles difficult sequences but allows identification of the source of a failure down to a pair of oligonucleotides. Curr. Protoc. Mol. Biol. 99:3.23.1‐3.23.18. © 2012 by John Wiley & Sons, Inc.

Keywords: gene synthesis; synthetic genes; synthetic DNA; synthetic biology; protein expression; gene assembly; oligonucleotides; oligonucleotide assembly; oligonucleotide synthesis; inside‐out nucleation; PCR

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Sequence Design for Protein‐Encoding Genes
  • Basic Protocol 2: Design of a Gene Assembly Scheme
  • Basic Protocol 3: Two‐Step Assembly of Overlapping Oligonucleotides Using Thermodynamically Balanced Inside‐Out PCR and Overlap Extension PCR to Generate a Long, Synthetic DNA Sequence
  • Basic Protocol 4: Troubleshooting by Sequential‐TBIO Assembly of Primary Fragments
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Sequence Design for Protein‐Encoding Genes

  Materials
  • Protein sequence for desired gene
  • Computer with Internet access
  • Sequence‐editing software: e.g., Gene Design (http://genedesign.thruhere.net/gd/) or CircDesigNA (http://cssb.utexas.edu/circdesigna/)

Basic Protocol 2: Design of a Gene Assembly Scheme

  Materials
  • Sequence to be synthesized
  • Biological sequence‐editing software (e.g., BioEdit; Hall, )

Basic Protocol 3: Two‐Step Assembly of Overlapping Oligonucleotides Using Thermodynamically Balanced Inside‐Out PCR and Overlap Extension PCR to Generate a Long, Synthetic DNA Sequence

  Materials
  • Oligonucleotides to be assembled (diluted to 1 µM)
  • Molecular biology–grade water (nuclease‐free)
  • Assembly reaction master mix (see recipe)
  • Analytical‐grade agarose
  • 6× DNA loading dye (see recipe)
  • 100‐bp dsDNA ladder
  • 1‐kb dsDNA ladder
  • Suitable buffer for agarose gel electrophoresis (e.g., TAE, TBE)
  • 200‐µl thin‐walled nuclease‐free PCR tubes (or microplates)
  • PCR thermal cycler
  • Agarose gel electrophoresis equipment (gel box, casting rig, power supply; unit 2.5)

Basic Protocol 4: Troubleshooting by Sequential‐TBIO Assembly of Primary Fragments

  Materials
  • Oligonucleotides to be assembled (1 µM)
  • Molecular biology‐grade water (nuclease‐free)
  • Assembly reaction master mix (see recipe)
  • Analytical‐grade agarose
  • 6× DNA loading dye (see recipe)
  • 100‐bp dsDNA ladder
  • Suitable buffer for agarose gel electrophoresis (e.g., TAE, TBE)
  • 200‐µl thin‐walled nuclease‐free PCR tubes (or microplates)
  • Thermal cycler
  • Agarose gel electrophoresis equipment (gel box, casting rig, power supply)
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Figures

Videos

Literature Cited

   Allert, M., Cox, J.C., and Hellinga, H.W. 2010. Multifactorial determinants of protein expression in prokaryotic open reading frames. J. Mol. Biol. 402:905‐918.
   Angov, E., Hillier, C.J., Kincaid, R.L., and Lyon, J.A. 2008. Heterologous protein expression is enhanced by harmonizing the codon usage frequencies of the target gene with those of the expression host. PLoS One 3:e2189.
   Bikard, D., Julié‐Galau, S., Cambray, G., and Mazel, D. 2010. The synthetic integron: An in vivo genetic shuffling device. Nucleic Acids Res. 38:e153‐e153.
   Burgess‐Brown, N.A., Sharma, S., Sobott, F., Loenarz, C., Oppermann, U., and Gileadi, O. 2008. Codon optimization can improve expression of human genes in Escherichia coli: A multi‐gene study. Protein Expr. Purif. 59:94‐102.
   Caruthers, M.H. 1991. Chemical synthesis of DNA and DNA analogs. Acc. Chem. Res. 24:278‐284.
   Cox, J.C., Lape, J., Sayed, M.A., and Hellinga, H.W. 2007. Protein fabrication automation. Protein Sci. 16:379‐390.
   Crameri, A., Whitehorn, E., Tate, E., Stemmer, W., and Kitts, P. 1996. Improved green fluorescent protein by molecular evolution using. Nat. Biotechnol. 14:315‐319.
   Crook, N.C., Freeman, E.S., and Alper, H.S. 2011. Re‐engineering multicloning sites for function and convenience. Nucleic Acids Res. 39:e92.
   Edwards, S.R. and Wandless, T.J. 2010. Dicistronic regulation of fluorescent proteins in the budding yeast Saccharomyces cerevisiae. Yeast 27:229‐236.
   Gao, X., Yo, P., Keith, A., Ragan, T.J., and Harris, T.K. 2003. Thermodynamically balanced inside‐out (TBIO) PCR‐based gene synthesis: A novel method of primer design for high‐fidelity assembly of longer gene sequences. Nucleic Acids Res. 31:e143.
   Gibson, D.G., Glass, J.I., Lartigue, C., Noskov, V.N., Chuang, R.Y., Algire, M.A., Benders, G.A., Montague, M.G., Ma, L., and Moodie, M.M. 2010. Creation of a bacterial cell controlled by a chemically synthesized genome. Science 329:52.
   Hall, T. 1999. BioEdit: A user‐friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Paper presented at Nucleic Acids Symposium.
   Hartner, F.S., Ruth, C., Langenegger, D., Johnson, S.N., Hyka, P., Lin‐Cereghino, G.P., Lin‐Cereghino, J., Kovar, K., Cregg, J.M., and Glieder, A. 2008. Promoter library designed for fine‐tuned gene expression in Pichia pastoris. Nucleic Acids Res. 36:e76.
   Horton, R.M., Hunt, H.D., Ho, S.N., Pullen, J.K., and Pease, L.R. 1989. Engineering hybrid genes without the use of restriction enzymes: Gene splicing by overlap extension. Gene 77:61‐68.
   Hughes, R.A., Miklos, A.E., and Ellington, A.D. 2011. Gene Synthesis: Methods and Applications. Methods Enzymol. 498:277‐309.
   Kamtekar, S., Schiffer, J.M., Xiong, H., Babik, J.M., and Hecht, M.H. 1993. Protein design by binary patterning of polar and nonpolar amino acids. Science 262:1680.
   Kudla, G., Murray, A.W., Tollervey, D., and Plotkin, J.B. 2009. Coding‐sequence determinants of gene expression in Escherichia coli. Science 324:255.
   Marsic, D., Hughes, R., Byrne‐Steele, M., and Ng, J. 2008. PCR‐based gene synthesis to produce recombinant proteins for crystallization. BMC Biotechnol. 8:44.
   Nagai, T., Ibata, K., Park, E.S., Kubota, M., Mikoshiba, K., and Miyawaki, A. 2002. A variant of yellow fluorescent protein with fast and efficient maturation for cell‐biological applications. Nature Biotechnol. 20:87‐90.
   Patterson, G.H., Knobel, S.M., Sharif, W.D., Kain, S.R., and Piston, D.W. 1997. Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy. Biophys. J. 73:2782‐2790.
   Pédelacq, J.D., Cabantous, S., Tran, T., Terwilliger, T.C., and Waldo, G.S. 2005. Engineering and characterization of a superfolder green fluorescent protein. Nature Biotechnol. 24:79‐88.
   Prasher, D.C., Eckenrode, V.K., Ward, W.W., Prendergast, F.G., and Cormier, M.J. 1992. Primary structure of the Aequorea victoria green‐fluorescent protein. Gene 111:229‐233.
   Richardson, S.M., Wheelan, S.J., Yarrington, R.M., and Boeke, J.D. 2006. GeneDesign: Rapid, automated design of multikilobase synthetic genes. Genome Res. 16:550‐556.
   Rizzo, M.A., Springer, G.H., Granada, B., and Piston, D.W. 2004. An improved cyan fluorescent protein variant useful for FRET. Nature Biotechnol. 22:445‐449.
   Salis, H.M., Mirsky, E.A., and Voigt, C.A. 2009. Automated design of synthetic ribosome binding sites to control protein expression. Nature Biotechnol. 27:946‐950.
   SantaLucia, J. Jr., Allawi, H.T., and Seneviratne, P.A. 1996. Improved nearest‐neighbor parameters for predicting DNA duplex stability. Biochemistry 35:3555‐3562.
   Tsien, R.Y. 1998. The green fluorescent protein. Ann. Rev Biochem. 67:509‐544.
   Wachter, R.M., Elsliger, M.A., Kallio, K., Hanson, G.T., and Remington, S.J. 1998. Structural basis of spectral shifts in the yellow‐emission variants of green fluorescent protein. Structure 6:1267‐1277.
   Welch, M., Govindarajan, S., Ness, J.E., Villalobos, A., Gurney, A., Minshull, J., and Gustafsson, C. 2009. Design parameters to control synthetic gene expression in Escherichia coli. PLoS One 4:e7002.
Key References
   Cox et al., 2007. See above.
  This paper details an automated version of the two‐step gene assembly process described in this unit.
   Gao et al., 2003. See above.
  This paper was the first to report the use of the TBIO oligonucleotide design and assembly method. The oligonucleotide design principles reported in this paper are used in this unit with some modifications.
   Marsic et al., 2008. See above.
  This paper describes the sequential TBIO or seq‐TBIO process for gene synthesis as a robust alternative to the TBIO assembly process.
Internet Resources
   http://genedesign.thruhere.net/gd/
  The Gene Design Website.
   http://www.mbio.ncsu.edu/bioedit/bioedit.html
  BioEdit is a biological sequence alignment editor written for Windows 95/98/NT/2000/XP/7.
   http://www.bioinformatics.org/sms/index.html
  The Sequence Manipulation Suite is a collection of web‐based programs for analyzing and formatting DNA and protein sequences
   http://helixweb.nih.gov/dnaworks/
  DNAWorks is available for automatic oligonucleotide design for PCR‐based gene synthesis.
   http://cssb.utexas.edu/circdesigna/
  CircDesigNA is a utility for the automated design of reactants in DNA reactions.
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