Gene Synthesis – Enabling Technologies for Synthetic Biology

  • Michael Liss
  • Ralf Wagner


Biotechnology has enabled us to render the adaptation of living natural resources from a top-down approach (breeding) to a bottom-up process (designing). Common modern cloning techniques allow for the rearrangement of genetic building blocks, the removal of cross-species boundaries and minor modifications of the DNA sequence itself. The availability of in silico gene optimization and in vitro gene synthesis from synthetic oligonucleotides has ushered a new era by confering independency of natural templates. The fast development of this technology during the last decade has dramatically advanced the availability of this service to a present level that by now outperforms classical cloning techniques in terms of flexibility, speed and costs. The exponential increase of biological sequence database contents and the growing need for genes designed for industrial applications, rather than natural function, further drives this market. The fast-growing demand for synthetic genes is attended by a rapid improvement of techniques to enable their stable and reliable production. Downscaling reaction volumes, massive parallelization and automation are integral parts in this development. With the emerging field of synthetic biology the requirements for gene synthesis expand particularly in terms of synthesis speed and construct size to allow for the construction of pathway operons or even complete viral and bacterial genomes. This challenges the engineering of novel techniques to assemble and manipulate synthetic DNA building blocks to large molecular entities efficiently to provide the necessary tools for tomorrows biotechnology.


Gene optimization Gene synthesis Synthetic genes Synthetic biology Protein expression Biosafety 


  1. 1.
    Szybalski W, Skalka A (1978) Nobel prizes and restriction enzymes. Gene 4:181–182CrossRefGoogle Scholar
  2. 2.
    Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N (1985) Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230(4732):1350–1354CrossRefGoogle Scholar
  3. 3.
    Agarwal KL, Buchi H, Caruthers MH, Gupta N, Khorana HG, Kleppe K, Kumar A, Ohtsuka E, Rajbhandary UL, Van de Sande JH, Sgaramella V, Weber H, Yamada T (1970) Total synthesis of the gene for an alanine transfer ribonucleic acid from yeast. Nature 227:27–34CrossRefGoogle Scholar
  4. 4.
    Koster H, Blocker H, Frank R, Geussenhainer S, Kaiser W (1975) Total synthesis of a structural gene for the human peptide hormone angiotensin II. Hoppe Seylers Z Physiol Chem 356:1585–1593CrossRefGoogle Scholar
  5. 5.
    Itakura K, Hirose T, Crea R, Riggs AD, Heyneker HL, Bolivar F, Boyer HW (1977) Expression in Escherichia coli of a chemically synthesized gene for the hormone somatostatin. Science 198:1056–1063CrossRefGoogle Scholar
  6. 6.
    Edge MD, Green AR, Heathcliffe GR, Meacock PA, Schuch W, Scanlon DB, Atkinson TC, Newton CR, Markham AF (1981) Total synthesis of a human leukocyte interferon gene. Nature 292:756–762CrossRefGoogle Scholar
  7. 7.
    Barany F, Gelfand DH (1991) Cloning, overexpression and nucleotide sequence of a thermostable DNA ligase-encoding gene. Gene 109:1–11CrossRefGoogle Scholar
  8. 8.
    Young L, Dong Q (2004) Two-step total gene synthesis method. Nucleic Acids Res 32:e59CrossRefGoogle Scholar
  9. 9.
    Mandecki W, Hayden MA, Shallcross MA, Stotland E (1990) A totally synthetic plasmid for general cloning, gene expression and mutagenesis in Escherichia coli. Gene 94:103–107CrossRefGoogle Scholar
  10. 10.
    Stemmer WP, Crameri A, Ha KD, Brennan TM, Heyneker HL (1995) Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. Gene 164:49–53CrossRefGoogle Scholar
  11. 11.
    Cello J, Paul AV, Wimmer E (2002) Chemical synthesis of poliovirus cDNA: generation of infectious virus in the absence of natural template. Science 297:1016–1018CrossRefGoogle Scholar
  12. 12.
    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 USA 101:15573–15578CrossRefGoogle Scholar
  13. 13.
    Menzella HG, Reisinger SJ, Welch M, Kealey JT, Kennedy J, Reid R, Tran CQ, Santi DV (2006) Redesign, synthesis and functional expression of the 6-deoxyerythronolide B polyketide synthase gene cluster. J Ind Microbiol Biotechnol 33:22–28CrossRefGoogle Scholar
  14. 14.
    Gibson DG, Glass JI, Lartigue C, Noskov VN, Chuang RY, Algire MA, Benders GA, Montague MG, Ma L, Moodie MM, Merryman C, Vashee S, Krishnakumar R, Assad-Garcia N, Andrews-Pfannkoch C, Denisova EA, Young L, Qi ZQ, Segall-Shapiro TH, Calvey CH, Parmar PP, Hutchison CA 3rd, Smith HO, Venter JC (2010) Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome. Science 329:52–56CrossRefGoogle Scholar
  15. 15.
    Ikemura T (1981) Correlation between the abundance of Escherichia coli transfer RNAs and the occurrence of the respective codons in its protein genes: a proposal for a synonymous codon choice that is optimal for the E. coli translational system. J Mol Biol 151:389–409CrossRefGoogle Scholar
  16. 16.
    Ikemura T (1985) Codon usage and tRNA content in unicellular and multicellular organisms. Mol Biol Evol 2:13–34Google Scholar
  17. 17.
    Ikemura T (1982) Correlation between the abundance of yeast transfer RNAs and the occurrence of the respective codons in protein genes. Differences in synonymous codon choice patterns of yeast and Escherichia coli with reference to the abundance of isoaccepting transfer RNAs. J Mol Biol 158:573–597CrossRefGoogle Scholar
  18. 18.
    Emilsson V, Naslund AK, Kurland CG (1993) Growth-rate-dependent accumulation of twelve tRNA species in Escherichia coli. J Mol Biol 230:483–491CrossRefGoogle Scholar
  19. 19.
    Raab D, Graf M, Notka F, Schoedl T, Wagner R (2010) The GeneOptimizer Algorithm: Using a sliding window approach to cope with the vast sequence space in multiparameter DNA sequence optimization. Syst Synth Biol 4:215–225CrossRefGoogle Scholar
  20. 20.
    Van den Brulle J, Fischer M, Langmann T, Horn G, Waldmann T, Arnold S, Fuhrmann M, Schatz O, O’Connell T, O’Connell D, Auckenthaler A, Schwer H (2008) A novel solid phase technology for high-throughput gene synthesis. Biotechniques Sep;45(3):340–343Google Scholar
  21. 21.
    Zhou X, Cai S, Hong A, You Q, Yu P, Sheng N, Srivannavit O, Muranjan S, Rouillard JM, Xia Y, Zhang X, Xiang Q, Ganesh R, Zhu Q, Matejko A, Gulari E, Gao X (2004) Microfluidic PicoArray synthesis of oligodeoxynucleotides and simultaneous assembling of multiple DNA sequences. Nucleic Acids Res 32(18):5409–5417. Print 2004Google Scholar
  22. 22.
    Tian J, Gong H, Sheng N, Zhou X, Gulari E, Gao X, Church G (2004) Accurate multiplex gene synthesis from programmable DNA microchips. Nature 432:1050–1054CrossRefGoogle Scholar
  23. 23.
    Greger B, Kemper B (1998) An apyrimidinic site kinks DNA and triggers incision by endonuclease VII of phage T4. Nucleic Acids Res 26(19):4432–4438CrossRefGoogle Scholar
  24. 24.
    Smith J, Modrich P (1997) Removal of polymerase-produced mutant sequences from PCR products. Proc Natl Acad Sci USA 94(13):6847–6850CrossRefGoogle Scholar
  25. 25.
    Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor laboratory, Cold Spring Harbor, NYGoogle Scholar
  26. 26.
    Padgett KA, Sorge JA (1996) Creating seamless junctions independent of restriction sites in PCR cloning. Gene 168(1):31–35CrossRefGoogle Scholar
  27. 27.
    Mullinax RL, Gross EA, Hay BN, Amberg JR, Kubitz MM, Sorge JA (1992) Expression of a heterodimeric Fab antibody protein in one cloning step. Biotechniques 12(6):864–869Google Scholar
  28. 28.
    Dietmaier W, Fabry S, Schmitt R (1993) DISEC-TRISEC: di- and trinucleotide-sticky-end cloning of PCR-amplified DNA. Nucleic Acids Res 21(15):3603–3604CrossRefGoogle Scholar
  29. 29.
    Aslanidis C, de Jong PJ (1990) Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Res 18(20):6069–6074CrossRefGoogle Scholar
  30. 30.
    Gibson DG, Benders GA, Axelrod KC, Zaveri J, Algire MA, Moodie M, Montague MG, Venter JC, Smith HO, Hutchison CA 3rd (2008) One-step assembly in yeast of 25 overlapping DNA fragments to form a complete synthetic Mycoplasma genitalium genome. Proc Natl Acad Sci USA 105(51):20404–20409. Epub 2008 Dec 10Google Scholar
  31. 31.
    Heinemann M, Panke S (2006) Synthetic biology – putting engineering into biology. Bioinformatics 22(22):2790–2799. Epub 2006 Sep 5. ReviewGoogle Scholar
  32. 32.
    Gibson DG, Benders GA, Andrews-Pfannkoch C, Denisova EA, Baden-Tillson H, Zaveri J, Stockwell TB, Brownley A, Thomas DW, Algire MA, Merryman C, Young L, Noskov VN, Glass JI, Venter JC, Hutchison CA 3rd, Smith HO (2008) Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome. Science 319(5867):1215–1220CrossRefGoogle Scholar
  33. 33.
    Fath S, Bauer AP, Liss M, Spriestersbach A, Maertens B, Hahn P, Ludwig C, Schaefer F, Graf M, Wagner R (2011) Multiparameter RNA and codon optimization: a standardized tool to assess and enhance autologous mammalian gene expression. PloS ONE 6:e17596CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Life Technologies Inc. Geneart AGRegensburgGermany
  2. 2.Molecular Microbiology and Gene TherapyUniversity of RegensburgRegensburgGermany

Personalised recommendations