Skip to main content
SpringerLink
Log in

Back to Basics: Creating Genetic Diversity

  • Chapter
  • First Online:
Directed Enzyme Evolution: Advances and Applications

Abstract

Directed evolution has emerged as a key enabling technology for improving the properties of biomolecules, biochemical pathways, and microorganisms to satisfy a wide range of biotechnological applications, from synthetic biology through to industrial biocatalysis. Laboratory evolution is an iterative process, alternating between creating genetic diversity and selection/screening to identify improved variants. This book chapter focuses on genetic diversity only. We describe and critically review recent advances in the methods for DNA assembly, random mutagenesis, focused mutagenesis, and DNA recombination. We also identify trends in these areas and highlight commercial kits that are developed to streamline and expedite these molecular biology techniques.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Chapter
USD 29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 139.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 179.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 179.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Wong TS, Zhurina D, Schwaneberg U (2006) The diversity challenge in directed protein evolution. Comb Chem High Throughput Screen 9:271–288

    Article  CAS  PubMed  Google Scholar 

  2. Tee KL, Wong TS (2013) Polishing the craft of genetic diversity creation in directed evolution. Biotechnol Adv 31:1707–1721

    Article  CAS  PubMed  Google Scholar 

  3. Eriksen DT, Hsieh PC, Lynn P, Zhao H (2013) Directed evolution of a cellobiose utilization pathway in Saccharomyces cerevisiae by simultaneously engineering multiple proteins. Microb Cell Factories 12:61

    Article  CAS  Google Scholar 

  4. Yuan Y, Zhao H (2013) Directed evolution of a highly efficient cellobiose utilizing pathway in an industrial Saccharomyces cerevisiae strain. Biotechnol Bioeng 110:2874–2881

    Article  CAS  PubMed  Google Scholar 

  5. Gao S, Li Y, Zhang J, Chen H, Ren D, Zhang L, An Y (2014) A modified version of the digestion-ligation cloning method for more efficient molecular cloning. Anal Biochem 453:55–57

    Article  CAS  PubMed  Google Scholar 

  6. Gao S, Zhang J, Miao T, Ma D, Su Y, An Y, Zhang Q (2015) A simple and convenient sticky/blunt-end ligation method for fusion gene construction. Biochem Genet 53:42–48

    Article  CAS  PubMed  Google Scholar 

  7. Hansen NB, Lubeck M, Lubeck PS (2014) Advancing USER cloning into simpleUSER and nicking cloning. J Microbiol Methods 96:42–49

    Article  CAS  PubMed  Google Scholar 

  8. Bitinaite J, Rubino M, Varma KH, Schildkraut I, Vaisvila R, Vaiskunaite R (2007) USER friendly DNA engineering and cloning method by uracil excision. Nucleic Acids Res 35:1992–2002

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Yang J, Zhang Z, Zhang XA, Luo Q (2010) A ligation-independent cloning method using nicking DNA endonuclease. BioTechniques 49:817–821

    Article  CAS  PubMed  Google Scholar 

  10. Sun S, Huang H, Qi YB, Qiu M, Dai ZM (2015) Complementary annealing mediated by exonuclease: a method for seamless cloning and conditioning site-directed mutagenesis. Biotechnol Biotechnol Equip 29:105–110

    Article  CAS  PubMed  Google Scholar 

  11. Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA 3rd, Smith HO (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6:343–345

    Article  CAS  PubMed  Google Scholar 

  12. Kahl LJ, Endy D (2013) A survey of enabling technologies in synthetic biology. J Biol Eng 7:13

    Article  PubMed  PubMed Central  Google Scholar 

  13. Fu C, Donovan WP, Shikapwashya-Hasser O, Ye X, Cole RH (2014) Hot Fusion: an efficient method to clone multiple DNA fragments as well as inverted repeats without ligase. PLoS ONE 9:e115318

    Article  PubMed  PubMed Central  Google Scholar 

  14. Taniguchi N, Nakayama S, Kawakami T, Murakami H (2013) Patch cloning method for multiple site-directed and saturation mutagenesis. BMC Biotechnol 13:91

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Lund AM, Kildegaard HF, Petersen MB, Rank J, Hansen BG, Andersen MR, Mortensen UH (2014) A versatile system for USER cloning-based assembly of expression vectors for mammalian cell engineering. PLoS ONE 9:e96693

    Article  PubMed  PubMed Central  Google Scholar 

  16. Jensen NB, Strucko T, Kildegaard KR, David F, Maury J, Mortensen UH, Forster J, Nielsen J, Borodina I (2014) EasyClone: method for iterative chromosomal integration of multiple genes in Saccharomyces cerevisiae. FEMS Yeast Res 14:238–248

    Article  CAS  PubMed  Google Scholar 

  17. Zhang Z, Yang J, Barford D (2015) Recombinant expression and reconstitution of multiprotein complexes by the USER cloning method in the insect cell-baculovirus expression system. Methods 95:13–25

    Google Scholar 

  18. Storch M, Casini A, Mackrow B, Fleming T, Trewhitt H, Ellis T, Baldwin GS (2015) BASIC: a new biopart assembly standard for idempotent cloning provides accurate, single-tier DNA assembly for synthetic biology. ACS Synth Biol 4:781–787

    Article  CAS  PubMed  Google Scholar 

  19. Beyer HM, Gonschorek P, Samodelov SL, Meier M, Weber W, Zurbriggen MD (2015) AQUA cloning: a versatile and simple enzyme-free cloning approach. PLoS ONE 10:e0137652

    Article  PubMed  PubMed Central  Google Scholar 

  20. Jacobus AP, Gross J (2015) Optimal cloning of PCR fragments by homologous recombination in Escherichia coli. PLoS ONE 10:e0119221

    Article  PubMed  PubMed Central  Google Scholar 

  21. Wang Y, Liu Y, Chen J, Tang MJ, Zhang SL, Wei LN, Li CH, Wei DB (2015) Restriction-ligation-free (RLF) cloning: a high-throughput cloning method by in vivo homologous recombination of PCR products. Genet Mol Res: GMR 14:12306–12315

    Article  CAS  PubMed  Google Scholar 

  22. Zhang Y, Werling U, Edelmann W (2012) SLiCE: a novel bacterial cell extract-based DNA cloning method. Nucleic Acids Res 40:e55

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Okegawa Y, Motohashi K (2015) Evaluation of seamless ligation cloning extract preparation methods from an Escherichia coli laboratory strain. Anal Biochem 486:51–53

    Article  CAS  PubMed  Google Scholar 

  24. Okegawa Y, Motohashi K (2015) A simple and ultra-low cost homemade seamless ligation cloning extract (SLiCE) as an alternative to a commercially available seamless DNA cloning kit. Biochem Biophys Rep 4:148–151

    Google Scholar 

  25. Li MV, Shukla D, Rhodes BH, Lall A, Shu J, Moriarity BS, Largaespada DA (2014) HomeRun Vector Assembly System: a flexible and standardized cloning system for assembly of multi-modular DNA constructs. PLoS ONE 9:e100948

    Article  PubMed  PubMed Central  Google Scholar 

  26. Ma H, Kunes S, Schatz PJ, Botstein D (1987) Plasmid construction by homologous recombination in yeast. Gene 58:201–216

    Article  CAS  PubMed  Google Scholar 

  27. Joska TM, Mashruwala A, Boyd JM, Belden WJ (2014) A universal cloning method based on yeast homologous recombination that is simple, efficient, and versatile. J Microbiol Methods 100:46–51

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Holmberg MA, Gowda NKC, Andreasson C (2014) A versatile bacterial expression vector designed for single-step cloning of multiple DNA fragments using homologous recombination. Protein Expr Purif 98:38–45

    Article  CAS  PubMed  Google Scholar 

  29. Gibson DG, Benders GA, Andrews-Pfannkoch C, Denisova EA, Baden-Tillson H, Zaveri J, Stockwell TB, Brownley A, Thomas DW, Algire MA et al (2008) Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome. Science 319:1215–1220

    Article  CAS  PubMed  Google Scholar 

  30. Kilaru S, Steinberg G (2015) Yeast recombination-based cloning as an efficient way of constructing vectors for Zymoseptoria tritici. Fungal Genet Biol: FG & B 79:76–83

    Article  CAS  Google Scholar 

  31. Cao P, Wang L, Zhou G, Wang Y, Chen Y (2014) Rapid assembly of multiple DNA fragments through direct transformation of PCR products into E. coli and Lactobacillus. Plasmid 76C:40–46

    Article  Google Scholar 

  32. Jajesniak P, Wong TS (2015) QuickStep-Cloning: a sequence-independent, ligation-free method for rapid construction of recombinant plasmids. J Biol Eng 9

    Google Scholar 

  33. Mathieu J, Alvarez E, Alvarez PJ (2014) Recombination-assisted megaprimer (RAM) cloning. MethodsX 1:23–29

    Article  PubMed  PubMed Central  Google Scholar 

  34. van den Ent F, Lowe J (2006) RF cloning: a restriction-free method for inserting target genes into plasmids. J Biochem Biophys Methods 67:67–74

    Article  PubMed  Google Scholar 

  35. de Kok S, Stanton LH, Slaby T, Durot M, Holmes VF, Patel KG, Platt D, Shapland EB, Serber Z, Dean J et al (2014) Rapid and reliable DNA assembly via ligase cycling reaction. ACS Synth Biol 3:97–106

    Article  PubMed  Google Scholar 

  36. Yuan Y, Andersen E, Zhao H (2016) Flexible and versatile strategy for the construction of large biochemical pathways. ACS Synth Biol 5:46–52

    Article  CAS  PubMed  Google Scholar 

  37. Krishnamurthy VV, Khamo JS, Cho E, Schornak C, Zhang K (2015) Multiplex gene removal by two-step polymerase chain reactions. Anal Biochem 481:7–9

    Article  CAS  PubMed  Google Scholar 

  38. Botstein D, Shortle D (1985) Strategies and applications of in vitro mutagenesis. Science 229:1193–1201

    Article  CAS  PubMed  Google Scholar 

  39. Leung DW, Chen EY, Goeddel DV (1989) Techniques 1:11–15

    Google Scholar 

  40. Holland EG, Buhr DL, Acca FE, Alderman D, Bovat K, Busygina V, Kay BK, Weiner MP, Kiss MM (2013) AXM mutagenesis: an efficient means for the production of libraries for directed evolution of proteins. J Immunol Methods 394:55–61

    Article  CAS  PubMed  Google Scholar 

  41. Kunkel TA (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A 82:488–492

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Holland EG, Acca FE, Belanger KM, Bylo ME, Kay BK, Weiner MP, Kiss MM (2015) In vivo elimination of parental clones in general and site-directed mutagenesis. J Immunol Methods 417:67–75

    Article  CAS  PubMed  Google Scholar 

  43. Jakob F, Lehmann C, Martinez R, Schwaneberg U (2013) Increasing protein production by directed vector backbone evolution. AMB Express 3:39

    Article  PubMed  PubMed Central  Google Scholar 

  44. Baldwin AJ, Busse K, Simm AM, Jones DD (2008) Expanded molecular diversity generation during directed evolution by trinucleotide exchange (TriNEx). Nucleic Acids Res 36:e77

    Article  PubMed  PubMed Central  Google Scholar 

  45. Arpino JA, Baldwin AJ, McGarrity AR, Tippmann EM, Jones DD (2015) In-frame amber stop codon replacement mutagenesis for the directed evolution of proteins containing non-canonical amino acids: identification of residues open to bio-orthogonal modification. PLoS ONE 10:e0127504

    Article  PubMed  PubMed Central  Google Scholar 

  46. Zhao J, Kardashliev T, Joelle Ruff A, Bocola M, Schwaneberg U (2014) Lessons from diversity of directed evolution experiments by an analysis of 3000 mutations. Biotechnol Bioeng 111:2380–2389

    Article  CAS  PubMed  Google Scholar 

  47. Wong TS, Tee KL, Hauer B, Schwaneberg U (2004) Sequence saturation mutagenesis (SeSaM): a novel method for directed evolution. Nucleic Acids Res 32:e26

    Article  PubMed  PubMed Central  Google Scholar 

  48. Hutchison CA 3rd, Phillips S, Edgell MH, Gillam S, Jahnke P, Smith M (1978) Mutagenesis at a specific position in a DNA sequence. J Biol Chem 253:6551–6560

    CAS  PubMed  Google Scholar 

  49. Papworth C, Bauer JC, Braman J, Wright DA (1996) Site-directed mutagenesis in one day with >80% efficiency. Strategies 9:3–4

    Article  Google Scholar 

  50. Liu H, Ye R, Wang YY (2015) Highly efficient one-step PCR-based mutagenesis technique for large plasmids using high-fidelity DNA polymerase. Genet Mol Res: GMR 14:3466–3473

    Article  CAS  PubMed  Google Scholar 

  51. Xia Y, Chu W, Qi Q, Xun L (2015) New insights into the QuikChange process guide the use of Phusion DNA polymerase for site-directed mutagenesis. Nucleic Acids Res 43:e12

    Article  PubMed  Google Scholar 

  52. Wang C, Wang TY, Zhang LY, Gao XJ, Wang XW, Jin CJ (2015) Cut-and-paste-based cloning strategy for large gene site-directed mutagenesis. Genet Mol Res: GMR 14:5585–5591

    Article  CAS  PubMed  Google Scholar 

  53. Zhang Z, Xu K, Xin Y, Zhang Z (2015) An efficient method for multiple site-directed mutagenesis using type IIs restriction enzymes. Anal Biochem 476:26–28

    Article  CAS  PubMed  Google Scholar 

  54. Waneskog M, Bjerling P (2014) Multi-fragment site-directed mutagenic overlap extension polymerase chain reaction as a competitive alternative to the enzymatic assembly method. Anal Biochem 444:32–37

    Article  PubMed  Google Scholar 

  55. Motohashi K (2015) A simple and efficient seamless DNA cloning method using SLiCE from Escherichia coli laboratory strains and its application to SLiP site-directed mutagenesis. BMC Biotechnol 15:47

    Article  PubMed  PubMed Central  Google Scholar 

  56. Belsare KD, Ruff AJ, Martinez R, Shivange AV, Mundhada H, Holtmann D, Schrader J, Schwaneberg U (2014) P-LinK: a method for generating multicomponent cytochrome P450 fusions with variable linker length. BioTechniques 57:13–20

    Article  CAS  PubMed  Google Scholar 

  57. Trehan A, Kielbus M, Czapinski J, Stepulak A, Huhtaniemi I, Rivero-Muller A (2016) REPLACR-mutagenesis, a one-step method for site-directed mutagenesis by recombineering. Sci Rep 6:19121

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Gonzalez-Perez D, Molina-Espeja P, Garcia-Ruiz E, Alcalde M (2014) Mutagenic Organized Recombination Process by Homologous IN vivo Grouping (MORPHING) for directed enzyme evolution. PLoS ONE 9:e90919

    Article  PubMed  PubMed Central  Google Scholar 

  59. Warburton M, Omar Ali H, Liong WC, Othusitse AM, Abdullah Zubir AZ, Maddock S, Wong TS (2015) OneClick: a program for designing focused mutagenesis experiments. AIMS Bioeng 2:126–143

    Article  Google Scholar 

  60. Currin A, Swainston N, Day PJ, Kell DB (2015) Synthetic biology for the directed evolution of protein biocatalysts: navigating sequence space intelligently. Chem Soc Rev 44:1172–1239

    Article  CAS  PubMed  Google Scholar 

  61. Kille S, Acevedo-Rocha CG, Parra LP, Zhang ZG, Opperman DJ, Reetz MT, Acevedo JP (2013) Reducing codon redundancy and screening effort of combinatorial protein libraries created by saturation mutagenesis. ACS Synth Biol 2:83–92

    Article  CAS  PubMed  Google Scholar 

  62. Nov Y, Segev D (2013) Optimal codon randomization via mathematical programming. J Theor Biol 335:147–152

    Article  PubMed  Google Scholar 

  63. Tang L, Gao H, Zhu X, Wang X, Zhou M, Jiang R (2012) Construction of “small-intelligent” focused mutagenesis libraries using well-designed combinatorial degenerate primers. BioTechniques 52:149–158

    CAS  PubMed  Google Scholar 

  64. Acevedo-Rocha CG, Reetz MT, Nov Y (2015) Economical analysis of saturation mutagenesis experiments. Sci Rep 5:10654

    Article  PubMed  PubMed Central  Google Scholar 

  65. Stemmer WP (1994) DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution. Proc Natl Acad Sci U S A 91:10747–10751

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Zhao H, Giver L, Shao Z, Affholter JA, Arnold FH (1998) Molecular evolution by staggered extension process (StEP) in vitro recombination. Nat Biotechnol 16:258–261

    Article  CAS  PubMed  Google Scholar 

  67. Ostermeier M, Shim JH, Benkovic SJ (1999) A combinatorial approach to hybrid enzymes independent of DNA homology. Nat Biotechnol 17:1205–1209

    Article  CAS  PubMed  Google Scholar 

  68. Sieber V, Martinez CA, Arnold FH (2001) Libraries of hybrid proteins from distantly related sequences. Nat Biotechnol 19:456–460

    Article  CAS  PubMed  Google Scholar 

  69. Xu S, Ju J, Misono H, Ohnishi K (2006) Directed evolution of extradiol dioxygenase by a novel in vivo DNA shuffling. Gene 368:126–137

    Article  CAS  PubMed  Google Scholar 

  70. Abecassis V, Pompon D, Truan G (2000) High efficiency family shuffling based on multi-step PCR and in vivo DNA recombination in yeast: statistical and functional analysis of a combinatorial library between human cytochrome P450 1A1 and 1A2. Nucleic Acids Res 28:E88

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Lehtonen SI, Taskinen B, Ojala E, Kukkurainen S, Rahikainen R, Riihimaki TA, Laitinen OH, Kulomaa MS, Hytonen VP (2015) Efficient preparation of shuffled DNA libraries through recombination (Gateway) cloning. Protein Eng Des Select: PEDS 28:23–28

    Article  CAS  Google Scholar 

  72. Mills DR, Peterson RL, Spiegelman S (1967) An extracellular Darwinian experiment with a self-duplicating nucleic acid molecule. Proc Natl Acad Sci U S A 58:217–224

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Joyce GF (2007) Forty years of in vitro evolution. Angew Chem 46:6420–6436

    Article  CAS  Google Scholar 

  74. 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 230:1350–1354

    Article  CAS  PubMed  Google Scholar 

  75. Cline J, Hogrefe H (2000) Randomize gene sequences with new PCR mutagenesis kit. Strategies 13:157–162

    Google Scholar 

  76. Bittker JA, Le BV, Liu DR (2002) Nucleic acid evolution and minimization by nonhomologous random recombination. Nat Biotechnol 20:1024–1029

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. ChELSI Institute and Advanced Biomanufacturing Centre (ABC), Department of Chemical and Biological Engineering, University of Sheffield, Sir Robert Hadfield Building, Mappin Street, Sheffield, S1 3JD, UK

    Kang Lan Tee & Tuck Seng Wong

Authors
  1. Kang Lan Tee
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tuck Seng Wong
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Tuck Seng Wong .

Editor information

Editors and Affiliations

  1. Department of Biocatalysis, Institute of Catalysis and Petrochemistry, CSIC, Madrid, Spain

    Miguel Alcalde

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer International Publishing AG

About this chapter

Cite this chapter

Tee, K.L., Wong, T.S. (2017). Back to Basics: Creating Genetic Diversity. In: Alcalde, M. (eds) Directed Enzyme Evolution: Advances and Applications. Springer, Cham. https://doi.org/10.1007/978-3-319-50413-1_8

Download citation

Publish with us

Policies and ethics

Search

Navigation