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Successful completion of a semi-automated enzyme-free cloning method

  • Published:
Journal of Structural and Functional Genomics

Abstract

Nowadays, in scientific fields such as Structural Biology or Vaccinology, there is an increasing need of fast, effective and reproducible gene cloning and expression processes. Consequently, the implementation of robotic platforms enabling the automation of protocols is becoming a pressing demand. The main goal of our study was to set up a robotic platform devoted to the high-throughput automation of the polymerase incomplete primer extension cloning method, and to evaluate its efficiency compared to that achieved manually, by selecting a set of bacterial genes that were processed either in the automated platform (330) or manually (94). Here we show that we successfully set up a platform able to complete, with high efficiency, a wide range of molecular biology and biochemical steps. 329 gene targets (99 %) were effectively amplified using the automated procedure and 286 (87 %) of these PCR products were successfully cloned in expression vectors, with cloning success rates being higher for the automated protocols respect to the manual procedure (93.6 and 74.5 %, respectively).

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References

  1. Boettner M, Prinz B, Holz C, Stahl U, Lang C (2002) High-throughput screening for expression of heterologous proteins in the yeast Pichia pastoris. J Biotechnol 99(1):51–62

    Article  CAS  PubMed  Google Scholar 

  2. Dieckman L, Gu M, Stols L, Donnelly MI, Collart FR (2002) High throughput methods for gene cloning and expression. Protein Expr Purif 25(1):1–7

    Article  CAS  PubMed  Google Scholar 

  3. Joachimiak A (2009) High-throughput crystallography for structural genomics. Curr Opin Struct Biol 19(5):573–584

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Terwilliger TC (2004) Structures and technology for biologists. Nat Struct Mol Biol 11(4):296–297

    Article  CAS  PubMed  Google Scholar 

  5. Khandurina J, Legg E, Wang X, Guttman A (2002) Automated agarose gel electrophoresis of dsDNA fragments on a commercial DNA sequencer. Biotechniques 33(5):1008–1014

    CAS  PubMed  Google Scholar 

  6. Hughes RS, Riedmuller SB, Mertens J, Li X-L, Bischoff K, Cotta M, Farrely P (2005) Development of a liquid handler component for a plasmid-based functional proteomic robotic workcell. J Lab Autom 10:287

    Article  CAS  Google Scholar 

  7. Luan CH, Qiu S, Finley JB, Carson M, Gray RJ, Huang W, Johnson D, Tsao J, Reboul J, Vaglio P, Hill DE, Vidal M, Delucas LJ, Luo M (2004) High-throughput expression of C. elegans proteins. Genome Res 14(10B):2102–2110

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Sievert V, Ergin A, Bussow K (2008) High throughput cloning with restriction enzymes. Methods Mol Biol 426:163–173

    Article  CAS  PubMed  Google Scholar 

  9. Kwon K, Peterson SN (2014) High-throughput cloning for biophysical applications. Methods Mol Biol 1140:61–74

    Article  CAS  PubMed  Google Scholar 

  10. Masignani V, Rappuoli R, Pizza M (2002) Reverse vaccinology: a genome-based approach for vaccine development. Expert Opin Biol Ther 2(8):895–905

    Article  CAS  PubMed  Google Scholar 

  11. Rappuoli R (2000) Reverse vaccinology. Curr Opin Microbiol 3(5):445–450

    Article  CAS  PubMed  Google Scholar 

  12. Delany I, Rappuoli R, De Gregorio E (2014) Vaccines for the 21st century. EMBO Mol Med 6(6):708–720

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Dormitzer PR, Grandi G, Rappuoli R (2012) Structural vaccinology starts to deliver. Nat Rev Microbiol 10(12):807–813

    Article  CAS  PubMed  Google Scholar 

  14. Loomis RJ, Johnson PR (2015) Emerging vaccine technologies. Vaccines 3(2):429–447

    Article  PubMed  PubMed Central  Google Scholar 

  15. Koehn J, Hunt I (2009) High-Throughput Protein Production (HTPP): a review of enabling technologies to expedite protein production. Methods Mol Biol 498:1–18

    Article  CAS  PubMed  Google Scholar 

  16. Chen Y, Qiu S, Luan CH, Luo M (2008) A high throughput platform for eukaryotic genes. Methods Mol Biol 426:209–220

    Article  CAS  PubMed  Google Scholar 

  17. Aoki M, Matsuda T, Tomo Y, Miyata Y, Inoue M, Kigawa T, Yokoyama S (2009) Automated system for high-throughput protein production using the dialysis cell-free method. Protein Expr Purif 68(2):128–136

    Article  CAS  PubMed  Google Scholar 

  18. Sparkes R, Behm DG (2010) Training adaptations associated with an 8-week instability resistance training program with recreationally active individuals. J Strength Cond Res 24(7):1931–1941

    Article  PubMed  Google Scholar 

  19. Kong F, Yuan L, Zheng YF, Chen W (2012) Automatic liquid handling for life science: a critical review of the current state of the art. J Lab Autom 17(3):169–185

    Article  CAS  PubMed  Google Scholar 

  20. Lorenz MGO (2004) Liquid-handling robotic workstations for functional genomics. JALA 9:262–267

    Google Scholar 

  21. Hughes SR, Butt TR, Bartolett S, Riedmuller SB, Farrelly P (2011) Design and construction of a first-generation high-throughput integrated robotic molecular biology platform for bioenergy applications. J Lab Autom 16(4):292–307

    Article  CAS  PubMed  Google Scholar 

  22. Klock HE, White A, Koesema E, Lesley SA (2005) Methods and results for semi-automated cloning using integrated robotics. J Struct Funct Genomics 6(2–3):89–94

    Article  CAS  PubMed  Google Scholar 

  23. Walhout AJ, Temple GF, Brasch MA, Hartley JL, Lorson MA, van den Heuvel S, Vidal M (2000) GATEWAY recombinational cloning: application to the cloning of large numbers of open reading frames or ORFeomes. Methods Enzymol 328:575–592

    Article  CAS  PubMed  Google Scholar 

  24. The Editors BioTechniques (2012) The rise of high throughput. BioTech 53(1):25

  25. Klock HE, Koesema EJ, Knuth MW, Lesley SA (2008) Combining the polymerase incomplete primer extension method for cloning and mutagenesis with microscreening to accelerate structural genomics efforts. Proteins 71(2):982–994

    Article  CAS  PubMed  Google Scholar 

  26. Shubhakar A, Reiding KR, Gardner RA, Spencer DI, Fernandes DL, Wuhrer M (2015) High-throughput analysis and automation for glycomics studies. Chromatographia 78(5–6):321–333

    Article  CAS  PubMed  Google Scholar 

  27. Holmberg RC, Gindlesperger A, Stokes T, Brady D, Thakore N, Belgrader P, Cooney CG, Chandler DP (2013) High-throughput, automated extraction of DNA and RNA from clinical samples using TruTip technology on common liquid handling robots. J Vis Exp 76:e50356

    PubMed  Google Scholar 

  28. Seib KL, Zhao X, Rappuoli R (2012) Developing vaccines in the era of genomics: a decade of reverse vaccinology. Clin Microbiol Infect 18(Suppl 5):109–116

    Article  CAS  PubMed  Google Scholar 

  29. Delany I, Rappuoli R, Seib KL (2013) Vaccines, reverse vaccinology, and bacterial pathogenesis. Cold Spring Harb Perspect Med 3(5):a012476

    Article  PubMed  PubMed Central  Google Scholar 

  30. Chung CT, Niemela SL, Miller RH (1989) One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proc Natl Acad Sci USA 86(7):2172–2175

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25(17):3389–3402

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Rice P, Longden I, Bleasby A (2000) EMBOSS: the European molecular biology open software suite. Trends in Genetics: TIG 16(6):276–277

    Article  CAS  PubMed  Google Scholar 

  33. Klock HE, Lesley SA (2009) The Polymerase Incomplete Primer Extension (PIPE) method applied to high-throughput cloning and site-directed mutagenesis. Methods Mol Biol 498:91–103

    Article  CAS  PubMed  Google Scholar 

  34. Camilo CM, Polikarpov I (2014) High-throughput cloning, expression and purification of glycoside hydrolases using Ligation-Independent Cloning (LIC). Protein Expr Purif 99:35–42

    Article  CAS  PubMed  Google Scholar 

  35. Acton TB, Gunsalus KC, Xiao R, Ma LC, Aramini J, Baran MC, Chiang YW, Climent T, Cooper B, Denissova NG, Douglas SM, Everett JK, Ho CK, Macapagal D, Rajan PK, Shastry R, Shih LY, Swapna GV, Wilson M, Wu M, Gerstein M, Inouye M, Hunt JF, Montelione GT (2005) Robotic cloning and protein production platform of the northeast structural genomics consortium. Methods Enzymol 394:210–243

    Article  CAS  PubMed  Google Scholar 

  36. Stevenson J, Krycer JR, Phan L, Brown AJ (2013) A practical comparison of ligation-independent cloning techniques. PLoS ONE 8(12):e83888

    Article  PubMed  PubMed Central  Google Scholar 

  37. Deller MC, Rupp B (2014) Approaches to automated protein crystal harvesting. Acta Crystallogr F Struct Biol Commun 70(Pt 2):133–155

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Kornienko M, Montalvo A, Carpenter BE, Lenard M, Abeywickrema P, Hall DL, Darke PL, Kuo LC (2005) Protein expression plasmids produced rapidly: streamlining cloning protocols and robotic handling. Assay Drug Dev Technol 3(6):661–674

    Article  CAS  PubMed  Google Scholar 

  39. Kachel V, Sindelar G, Grimm S (2006) High-throughput isolation of ultra-pure plasmid DNA by a robotic system. BMC Biotechnol 6:9

    Article  PubMed  PubMed Central  Google Scholar 

  40. Ben Yehezkel T, Nagar S, Mackrants D, Marx Z, Linshiz G, Shabi U, Shapiro E (2011) Computer-aided high-throughput cloning of bacteria in liquid medium. Biotechniques 50(2):124–127

    Article  CAS  PubMed  Google Scholar 

  41. Keller M, Naue J, Zengerle R, von Stetten F, Schmidt U (2015) Automated forensic animal family identification by nested PCR and melt curve analysis on an off-the-shelf thermocycler augmented with a centrifugal microfluidic disk segment. PLoS ONE 10(7):e0131845

    Article  PubMed  PubMed Central  Google Scholar 

  42. Alzari PM, Berglund H, Berrow NS, Blagova E, Busso D, Cambillau C, Campanacci V, Christodoulou E, Eiler S, Fogg MJ, Folkers G, Geerlof A, Hart D, Haouz A, Herman MD, Macieira S, Nordlund P, Perrakis A, Quevillon-Cheruel S, Tarandeau F, van Tilbeurgh H, Unger T, Luna-Vargas MP, Velarde M, Willmanns M, Owens RJ (2006) Implementation of semi-automated cloning and prokaryotic expression screening: the impact of SPINE. Acta Crystallogr D Biol Crystallogr 62(Pt 10):1103–1113

    Article  PubMed  Google Scholar 

  43. de Jong RN, Daniels MA, Kaptein R, Folkers GE (2006) Enzyme free cloning for high throughput gene cloning and expression. J Struct Funct Genomics 7(3–4):109–118

    Article  CAS  PubMed  Google Scholar 

  44. Sun Y, Nguyen NT, Kwok YC (2008) High-throughput polymerase chain reaction in parallel circular loops using magnetic actuation. Anal Chem 80(15):6127–6130

    Article  CAS  PubMed  Google Scholar 

  45. Cao W, Bean B, Corey S, Coursey JS, Hasson KC, Inoue H, Isano T, Kanderian S, Lane B, Liang H, Murphy B, Owen G, Shinoda N, Zeng S, Knight IT (2015) Automated microfluidic platform for serial polymerase chain reaction and high-resolution melting analysis. J Lab Autom 21:402–411

    Article  PubMed  Google Scholar 

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Acknowledgments

The authors thank Dr. Daniele Maldini and Dr. Giuliano Spezia (Hamilton Robotics) for their precious assistance in setting up and implementing the automated protocols in the workstation.

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Authors and Affiliations

  1. GSK Vaccines, Siena, 53100, Italy

    Stefano Bonacci, Scilla Buccato, Domenico Maione & Roberto Petracca

Authors
  1. Stefano Bonacci
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  2. Scilla Buccato
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  3. Domenico Maione
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  4. Roberto Petracca
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Corresponding author

Correspondence to Stefano Bonacci.

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The authors declare that they have no conflict of interest. The experiments comply with the current laws of the country in which they were performed.

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Bonacci, S., Buccato, S., Maione, D. et al. Successful completion of a semi-automated enzyme-free cloning method. J Struct Funct Genomics 17, 57–66 (2016). https://doi.org/10.1007/s10969-016-9207-z

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  • DOI: https://doi.org/10.1007/s10969-016-9207-z

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