Skip to main content
SpringerLink
Log in

ACEMBL Tool-Kits for High-Throughput Multigene Delivery and Expression in Prokaryotic and Eukaryotic Hosts

  • Chapter
  • First Online:
Advanced Technologies for Protein Complex Production and Characterization

Part of the book series: Advances in Experimental Medicine and Biology ((AEMB,volume 896))

Abstract

Multicomponent biological systems perform a wide variety of functions and are crucially important for a broad range of critical health and disease states. A multitude of applications in contemporary molecular and synthetic biology rely on efficient, robust and flexible methods to assemble multicomponent DNA circuits as a prerequisite to recapitulate such biological systems in vitro and in vivo. Numerous functionalities need to be combined to allow for the controlled realization of information encoded in a defined DNA circuit. Much of biological function in cells is catalyzed by multiprotein machines typically made up of many subunits. Provision of these multiprotein complexes in the test-tube is a vital prerequisite to study their structure and function, to understand biology and to develop intervention strategies to correct malfunction in disease states. ACEMBL is a technology concept that specifically addresses the requirements of multicomponent DNA assembly into multigene constructs, for gene delivery and the production of multiprotein complexes in high-throughput. ACEMBL is applicable to prokaryotic and eukaryotic expression hosts, to accelerate basic and applied research and development. The ACEMBL concept, reagents, protocols and its potential are reviewed in this contribution.

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 149.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 199.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 199.99
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. Schelshorn D, Ljubicic S, Berger I, Fitzgerald DJ (2015) Synthetic biology in biopharmaceutical production. Eur Biopharm Rev 12(228):3995–3997

    Google Scholar 

  2. Tirabassi R (2014) Foundations of molecular cloning – past, present and future. http://www.neb-online.fr/pdfs/Article_MolecularCloning2014_NEBFR.pdf

  3. Luria SE, Human ML (1952) A nonhereditary, host-induced variation of bacterial viruses. J Bacteriol 64:557–569

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Bartani G, Weigle JJ (1953) Host controlled variation in bacterial viruses. J Bacteriol 65:113–121

    Google Scholar 

  5. Linn S, Arber W (1968) Host specificity of DNA produced by Escherichia coli, X. In vitro restriction of phage fd replicative form. Proc Natl Acad Sci U S A 59:1300–1306

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Smith HO, Wilcox KW (1970) A restriction enzyme from Hemophilus influenzae. I. Purification and general properties. J Mol Biol 51:379–391

    Article  CAS  PubMed  Google Scholar 

  7. Kellenberger G, Zichichi ML, Weigle JJ (1961) Exchange of DNA in the recombination of bacteriophage lambda. Proc Natl Acad Sci U S A 47:869–878

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Meselson M, Weigle JJ (1961) Chromosome brekage accompanying genetic recombination in bacteriophage. Proc Natl Acad Sci U S A 47:857–868

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Bode VC, Kaiser AD (1965) Changes in the structure and activity of lambda DNA in a superinfected immune bacterium. J Mol Biol 14:399–417

    Article  CAS  PubMed  Google Scholar 

  10. Cozzarelli NR, Melechen NE, Jovin TM, Kornberg A (1967) Polynucleotide cellulose as a substrate for a polynucleotide ligase induced by phage T4. Biochem Biophys Res Commun 28:578–586

    Article  CAS  PubMed  Google Scholar 

  11. Gefter ML, Becker A, Hurwitz J (1967) The enzymatic repair of DNA. I. Formation of circular lambda-DNA. Proc Natl Acad Sci U S A 58:240–247

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Gellert M (1967) Formation of covalent circles of lambda DNA by E. coli extracts. Proc Natl Acad Sci U S A 57:148–155

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Olivera BM, Lehman IR (1967) Linkage of polynucleotides through phosphodiester bonds by an enzyme from Escherichia coli. Proc Natl Acad Sci U S A 57:1426–1433

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Weiss B, Richardson CC (1967) Enzymatic breakage and joining of deoxyribonucleic acid, I. Repair of single-strand breaks in DNA by an enzyme system from Escherichia coli infected with T4 bacteriophage. Proc Natl Acad Sci U S A 57:1021–1028

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Maniatis T (2012) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York. ISBN 978-1-936113-42-2

    Google Scholar 

  16. Mullis KB, Faloona FA (1987) Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol 155:335–350

    Article  CAS  PubMed  Google Scholar 

  17. Li MZ, Elledge SJ (2007) Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. Nat Methods 4:251–256

    Article  CAS  PubMed  Google Scholar 

  18. Nisson PE, Rashtchian A, Watkins PC (1991) Rapid and efficient cloning of Alu-PCR products using uracil DNA glycosylase. PCR Methods Appl 1:120–123

    Article  CAS  PubMed  Google Scholar 

  19. Quan J, Tian J (2009) Circular polymerase extension cloning of complex gene libraries and pathways. PLoS One 4(7):e6441

    Article  PubMed  PubMed Central  Google Scholar 

  20. Zhang Y, Werling U, Edelmann W (2012) SLiCE: a novel bacterial cell extract-based DNA cloning method. Nucleic Acids Res 40(8):e55

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Gibson DG (2014) Programming biological operating systems: genome design, assembly and activation. Nat Methods 11(5):521–526

    Article  CAS  PubMed  Google Scholar 

  22. Weber W, Fussenegger M (2011) Emerging biomedical applications of synthetic biology. Nat Rev Genet 13(1):21–35

    Article  PubMed  Google Scholar 

  23. Ye H, Aubel D, Fussenegger M (2013) Synthetic mammalian gene circuits for biomedical applications. Curr Opin Chem Biol 17(6):910–917

    Article  CAS  PubMed  Google Scholar 

  24. Folcher M, Fussenegger M (2012) Synthetic biology advancing clinical applications. Curr Opin Chem Biol 16(3–4):345–354

    Article  CAS  PubMed  Google Scholar 

  25. Ausländer S, Fussenegger M (2013) From gene switches to mammalian designer cells: present and future prospects. Trends Biotechnol 31(3):155–168

    Article  PubMed  Google Scholar 

  26. Geering B, Fussenegger M (2015) Synthetic immunology: modulating the human immune system. Trends Biotechnol 33(2):65–79

    Article  CAS  PubMed  Google Scholar 

  27. Terwilliger TC, Stuart D, Yokoyama S (2009) Lessons from structural genomics. Annu Rev Biophys 38:371–383

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Almo SC et al (2013) Protein production from the structural genomics perspective: achievements and future needs. Curr Opin Struct Biol 23(3):335–344

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Torella J et al (2014) Unique nucleotide sequence-guided assembly of repetitive DNA parts for synthetic biology applications. Nat Protoc 9:2075–2089

    Article  CAS  PubMed  Google Scholar 

  30. Leinert F et al (2013) Two- and three-input TALE-based and logic computation in embryonic stem cells. Nucleic Acids Res 41:9967–9975

    Article  Google Scholar 

  31. Vijayachandran LS et al (2011) Robots, pipelines, polyproteins: enabling multiprotein expression in prokaryotic and eukaryotic cells. J Struct Biol 175(2):198–208

    Article  CAS  PubMed  Google Scholar 

  32. Trowitzsch S, Palmberger D, Fitzgerald D, Takagi Y, Berger I (2012) MultiBac complexomics. Expert Rev Proteomics 9(4):363–373

    Article  CAS  PubMed  Google Scholar 

  33. Bieniossek C et al (2009) Automated unrestricted multigene recombineering for multiprotein complex production. Nat Methods 6(6):447–450

    Article  CAS  PubMed  Google Scholar 

  34. Bieniossek C, Imasaki T, Takagi Y, Berger I (2012) MultiBac: expanding the research toolbox for multiprotein complexes. Trends Biochem Sci 37(2):49–57

    Article  CAS  PubMed  Google Scholar 

  35. Kriz A et al (2011) A plasmid-based multigene expression system for mammalian cells. Nat Commun 1:e120

    Article  Google Scholar 

  36. Trowitzsch S, Klumpp M, Thoma R, Carralot JP, Berger I (2011) Light it up: highly efficient multigene delivery in mammalian cells. Bioessays 33(12):946–955

    Article  CAS  PubMed  Google Scholar 

  37. Sari D et al (2015) The MultiBac baculovirus/insect cell expression vector system for producing complex protein biologics. In: Vega C, Fernandez F (eds) Advances in experimental medicine and biology. Springer, New York

    Google Scholar 

  38. Nie Y et al (2009) Getting a grip on complexes. Curr Genomics 10(8):558–572

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Robinson CV, Sali A, Baumeister W (2007) The molecular sociology of the cell. Nature 450(7172):973–982

    Article  CAS  PubMed  Google Scholar 

  40. Romier C et al (2006) Co-expression of protein complexes in prokaryotic and eukaryotic hosts: experimental procedures, database tracking and case studies. Acta Crystallogr D Biol Crystallogr 62(10):1232–1242

    Article  PubMed  Google Scholar 

  41. Busso D et al (2011) Expression of protein complexes using multiple Escherichia coli protein co-expression systems: a benchmarking study. J Struct Biol 175(2):159–170

    Article  CAS  PubMed  Google Scholar 

  42. Diebold ML et al (2011) Deciphering correct strategies for multiprotein complex assembly by co-expression: application to complexes as large as the histone octamer. J Struct Biol 175(2):178–188

    Article  CAS  PubMed  Google Scholar 

  43. Vincentelli R, Romier C (2013) Expression in Escherichia coli: becoming faster and more complex. Curr Opin Struct Biol 23(3):326–334

    Article  CAS  PubMed  Google Scholar 

  44. Haffke M et al (2015) Characterization and production of protein complexes by co-expression in Escherichia coli. Methods Mol Biol 1261:63–89

    Article  CAS  PubMed  Google Scholar 

  45. Abdulrahman W et al (2015) The production of multiprotein complexes in insect cells using the baculovirus expression system. Methods Mol Biol 1261:91–114

    Article  CAS  PubMed  Google Scholar 

  46. Haffke M, Viola C, Nie Y, Berger I (2013) Tandem recombineering by SLIC cloning and Cre-LoxP fusion to generate multigene expression constructs for protein complex research. Methods Mol Biol 1073:131–140

    Article  CAS  PubMed  Google Scholar 

  47. Berger I (2010) New nucleic acid tools for producing multiprotein complexes. WO2010/100278-A2

    Google Scholar 

  48. Guo F, Gopaul DN, van Duyne GD (1997) Structure of Cre recombinase complexed with DNA in a site-specific recombination synapse. Nature 389(6646):40–406

    Article  CAS  PubMed  Google Scholar 

  49. Gopaul DN, Guo F, Van Duyne GD (1998) Structure of the Holliday junction intermediate in Cre-loxP site-specific recombination. EMBO J 17(14):4175–4187

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Metcalf WW, Jiang W, Wanner BL (1994) Use of the rep technique for allele replacement to construct new Escherichia coli hosts for maintenance of R6K gamma origin plasmids at different copy numbers. Gene 138(1–2):1–7

    Article  CAS  PubMed  Google Scholar 

  51. Cre-ACEMBLR software: https://github.com/christianbecke/Cre-ACEMBLER

  52. Becke C, Haffke M, Berger I (2012) Cre-ACEMBLER User Manual. doi:10.13140/2.1.1068.1128

  53. Davis MW. ApE – a plasmid editor. http://www.biology.utah.edu/jorgensen/wayned/ape

  54. Life Technologies Invitrogen Vector NTI. http://www.invitrogen.com

  55. Python programming language. http://python.org/

  56. The GIMP Toolkit. http://www.gtk.org/

  57. PyGTK: GTK+ for Python. http://www.pygtk.org/

  58. Cock PJA et al (2009) Biopython: freely available Python tools for computational molecular biology and bioinformatics. Bioinformatics 25:1422–1423

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Schaffitzel C et al (2006) Structure of the E. coli signal recognition particle bound to a translating ribosome. Nature 444:503–506

    Article  CAS  PubMed  Google Scholar 

  60. Estrozi LF, Boehringer D, Shan SO, Ban N, Schaffitzel C (2011) Cryo-EM structure of the E. coli translating ribosome in complex with SRP and its receptor. Nat Struct Mol Biol 18(1):88–90

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Von Loeffelholz O et al (2013) Structural basis of signal sequence surveillance and selection by the SRP-SR complex. Nat Struct Mol Biol 20:604–610

    Article  Google Scholar 

  62. Schulze RJ et al (2014) Membrane protein insertion and proton-motive-force-dependent secretion through the bacterial holo-translocon SecYEG-SecDF-YajC-YidC. Proc Natl Acad Sci U S A 111(13):4844–4849

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Komar J, Botte M, Collinson C, Schaffitzel C, Berger I (2015) ACEMBLing a multiprotein transmembrane complex: the functional SecYEG-SecDFYajC-YidC holotranslocon protein secretase/insertase. Methods Enzymol 556:23–49

    Article  PubMed  Google Scholar 

  64. Basters A et al (2014) Molecular characterization of ubiquitin-specific protease 18 reveals substrate specificity for interferon-stimulated gene 15. FEBS J 281(7):1918–1928

    Article  CAS  PubMed  Google Scholar 

  65. Cunna S et al (2011) Genetic disassembly and combinatorial reassembly identify a minimal functional repertoire of type III effectors in Pseudomonas syringae. Proc Natl Acad Sci U S A 108(7):2975–2980

    Article  Google Scholar 

  66. Jakobi AJ, Huizinga EG (2012) A rapid cloning method employing orthogonal end protection. PLoS One 7(6):e37617

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Zheng N, Huang X, Yin B, Wang D, Xie Q (2012) An effective system for detecting protein-protein interaction based on in vivo cleavage by PPV NIa protease. Protein Cell 3(12):921–928

    Article  CAS  PubMed  Google Scholar 

  68. Rode AB, Endoh T, Sugimoto N (2015) Tuning riboswitch-mediated gene regulation by rational control of aptamer ligand binding properties. Angew Chem Int Ed Eng 54(3):905–909

    Article  CAS  Google Scholar 

  69. Ballmer-Hofer K, Andersson AE, Ratcliffe LE, Berger P (2011) Neuropilin-1 promotes VEGFR-2 trafficking through Rab11 vesicles thereby specifying signal output. Blood 118(3):816–826

    Article  CAS  PubMed  Google Scholar 

  70. Gosset G (2005) Improvement of Escherichia coli production strains by modification of the phosphoenolpyruvate:sugar phosphotransferase system. Microb Cell Factories 4:14

    Article  Google Scholar 

  71. Patnaik R (2008) Engineering complex phenotypes in industrial strains. Biotechnol Prog 24:38–47

    Article  CAS  PubMed  Google Scholar 

  72. Chou CP (2007) Engineering cell physiology to enhance recombinant protein production in Escherichia coli. Appl Microbiol Biotechnol 76:521–532

    Article  CAS  PubMed  Google Scholar 

  73. Lee SY, Kim HU, Park JH, Park JM, Kim TY (2009) Metabolic engineering of microorganisms: general strategies and drug production. Drug Discov Today 14:78–88

    Article  CAS  PubMed  Google Scholar 

  74. Chang MCY, Keasling JD (2006) Production of isoprenoid pharmaceuticals by engineered microbes. Nat Chem Biol 2:674–681

    Article  CAS  PubMed  Google Scholar 

  75. Chemler JA, Koffas MAG (2008) Metabolic engineering for plant natural product biosynthesis in microbes. Curr Opin Biotechnol 19:597–605

    Article  CAS  PubMed  Google Scholar 

  76. Lee SK, Chou H, Ham TS, Lee TS, Keasling JD (2008) Metabolic engineering of microorganisms for biofuels production: from bugs to synthetic biology to fuels. Curr Opin Biotechnol 19:556–563

    Article  CAS  PubMed  Google Scholar 

  77. Jarboe LR et al (2010) Metabolic engineering for production of biorenewable fuels and chemicals: contributions of synthetic biology. J Biomed Biotechnol 2010:761042–761060

    Article  PubMed  PubMed Central  Google Scholar 

  78. Pitera DJ, Paddon CJ, Newman JD, Keasling JD (2007) Balancing a heterologous mevalonate pathway for improved isoprenoid production in Escherichia coli. Metab Eng 9:193–207

    Article  CAS  PubMed  Google Scholar 

  79. Alper H, Miyaoku K, Stephanopoulos G (2005) Construction of lycopene-overproducing E. coli strains by combining systematic and combinatorial gene knockout targets. Nat Biotechnol 23:612–616

    Article  CAS  PubMed  Google Scholar 

  80. Berríos-Rivera SJ, Bennett GN, San KY (2002) Metabolic engineering of Escherichia coli: increase of NADH availability by overexpressing an NAD+-dependent formate dehydrogenase. Metab Eng 4:217–229

    Article  PubMed  Google Scholar 

  81. Park JH, Lee KH, Kim TY, Lee SY (2007) Metabolic engineering of Escherichia coli for the production of L-valine based on transcriptome analysis and in silico gene knockout simulation. Proc Natl Acad Sci U S A 104:7797–7802

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

We thank all members of the Berger laboratory for their support, as well as Lakshmi S. Vijayachandran (Amrita Center for Nanosciences & Molecular Medicine, India), Christoph Bieniossek (Hofmann-La Roche, Basel), Simon Trowitzsch (Goethe University, Frankfurt) and Alexander Craig (S. Karger GmbH, Publishers) for their contribution and helpful discussions. This work was funded by the European Commission (EC) Framework Programme (FP) 7 ComplexINC project (grant number 279039) to DF and IB. YN was a fellow of the Boehringer Ingelheim Fonds (BIF, Germany). MH was recipient of a Kekulé fellowship from the Fonds der Chemischen Industrie (FCI, Germany).

Author information

Authors and Affiliations

  1. European Molecular Biology Laboratory, Grenoble Outstation, 71 avenue des Martyrs, 38042, Grenoble Cedex 9, France

    Yan Nie, Maxime Chaillet, Christian Becke, Matthias Haffke, Martin Pelosse, Christiane Schaffitzel & Imre Berger

  2. Unit of Virus Host-Cell Interactions, University Grenoble Alpes-EMBL-CNRS, UMI 3265, 71 avenue des Martyrs, 38042, Grenoble Cedex 9, France

    Yan Nie, Maxime Chaillet, Matthias Haffke, Martin Pelosse, Christiane Schaffitzel & Imre Berger

  3. Department of Structural Biochemistry, Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227, Dortmund, Germany

    Yan Nie

  4. Department of Biochemistry, Freie Universität Berlin, Takustrasse 6, 14195, Berlin, Germany

    Christian Becke

  5. Novartis Institutes for BioMedical Research, Novartis Campus, 4056, Basel, Switzerland

    Matthias Haffke

  6. Geneva Biotech SARL, Avenue de la Roseraie 64, 1205, Genève, Switzerland

    Daniel Fitzgerald

  7. School of Biochemistry, University of Bristol, Bristol, BS8 1TD, UK

    Ian Collinson, Christiane Schaffitzel & Imre Berger

Authors
  1. Yan Nie
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Maxime Chaillet
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Christian Becke
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Matthias Haffke
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Martin Pelosse
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Daniel Fitzgerald
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Ian Collinson
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Christiane Schaffitzel
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Imre Berger
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Imre Berger .

Editor information

Editors and Affiliations

  1. Center for Biological Research, SpanishNationalResearchCouncil(CIB-CSIC), Madrid, Spain

    M. Cristina Vega

Rights and permissions

Reprints and permissions

Copyright information

© 2016 Springer International Publishing Switzerland

About this chapter

Cite this chapter

Nie, Y. et al. (2016). ACEMBL Tool-Kits for High-Throughput Multigene Delivery and Expression in Prokaryotic and Eukaryotic Hosts. In: Vega, M. (eds) Advanced Technologies for Protein Complex Production and Characterization. Advances in Experimental Medicine and Biology, vol 896. Springer, Cham. https://doi.org/10.1007/978-3-319-27216-0_3

Download citation

Publish with us

Policies and ethics

Search

Navigation