Skip to main content
SpringerLink
Log in

Modular Assembly of Synthetic Secondary Chromosomes

  • Protocol
  • First Online:
Bacterial Chromatin

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1837))

Abstract

The development of novel DNA assembly methods in recent years has paved the way for the construction of synthetic replicons to be used for basic research and biotechnological applications. Questions of how chromosomes need to be constructed to maintain the genetic information can now be answered by a learning-by-building approach. Here, we describe an efficient pipeline for the design and assembly of synthetic, secondary chromosomes in Escherichia coli based on the popular Modular Cloning system (MoClo).

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

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 109.00
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. Gibson DG, Glass JI, Lartigue C, Noskov VN, Chuang R-Y, 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 Z-Q, Segall-Shapiro TH, Calvey CH, Parmar PP, Hutchison CA, Smith HO, Venter JC (2010) Creation of a bacterial cell controlled by a chemically synthesized genome. Science 329:52–56. https://doi.org/10.1126/science.1190719

    Article  PubMed  CAS  Google Scholar 

  2. Annaluru N, Muller H, Mitchell LA, Ramalingam S, Stracquadanio G, Richardson SM, Dymond JS, Kuang Z, Scheifele LZ, Cooper EM, Cai Y, Zeller K, Agmon N, Han JS, Hadjithomas M, Tullman J, Caravelli K, Cirelli K, Guo Z, London V, Yeluru A, Murugan S, Kandavelou K, Agier N, Fischer G, Yang K, Martin JA, Bilgel M, Bohutskyi P, Boulier KM, Capaldo BJ, Chang J, Charoen K, Choi WJ, Deng P, DiCarlo JE, Doong J, Dunn J, Feinberg JI, Fernandez C, Floria CE, Gladowski D, Hadidi P, Ishizuka I, Jabbari J, Lau CYL, Lee PA, Li S, Lin D, Linder ME, Ling J, Liu J, Liu J, London M, Ma H, Mao J, McDade JE, McMillan A, Moore AM, Oh WC, Ouyang Y, Patel R, Paul M, Paulsen LC, Qiu J, Rhee A, Rubashkin MG, Soh IY, Sotuyo NE, Srinivas V, Suarez A, Wong A, Wong R, Xie WR, Xu Y, Yu AT, Koszul R, Bader JS, Boeke JD, Chandrasegaran S (2014) Total synthesis of a functional designer eukaryotic chromosome. Science 344:55–58. https://doi.org/10.1126/science.1249252

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  3. Schindler D, Waldminghaus T (2015) Synthetic chromosomes. FEMS Microbiol Rev 39:871–891. https://doi.org/10.1093/femsre/fuv030

    Article  PubMed  CAS  Google Scholar 

  4. Touzain F, Petit MA, Schbath S, El Karoui M (2011) DNA motifs that sculpt the bacterial chromosome. Nat Rev Microbiol 9:15–26. https://doi.org/10.1038/nrmicro2477

    Article  PubMed  CAS  Google Scholar 

  5. Milbredt S, Farmani N, Sobetzko P, Waldminghaus T (2016) DNA replication in engineered escherichia coli genomes with extra replication origins. ACS Synth Biol 5:1167–1176. https://doi.org/10.1021/acssynbio.6b00064

    Article  PubMed  CAS  Google Scholar 

  6. Hutchison CA 3rd, Chuang RY, Noskov VN, Assad-Garcia N, Deerinck TJ, Ellisman MH, Gill J, Kannan K, Karas BJ, Ma L, Pelletier JF, Qi ZQ, Richter RA, Strychalski EA, Sun L, Suzuki Y, Tsvetanova B, Wise KS, Smith HO, Glass JI, Merryman C, Gibson DG, Venter JC (2016) Design and synthesis of a minimal bacterial genome. Science 351:aad6253. https://doi.org/10.1126/science.aad6253

    Article  PubMed  CAS  Google Scholar 

  7. Yu W, Han F, Gao Z, Vega JM, Birchler JA (2007) Construction and behavior of engineered minichromosomes in maize. Proc Natl Acad Sci U A 104:8924–8929. https://doi.org/10.1073/pnas.0700932104

    Article  CAS  Google Scholar 

  8. Birchler JA (2015) Promises and pitfalls of synthetic chromosomes in plants. Trends Biotechnol 33:189–194. https://doi.org/10.1016/j.tibtech.2014.12.010

    Article  PubMed  CAS  Google Scholar 

  9. Dasgupta S, Lobner-Olesen A (2004) Host controlled plasmid replication: Escherichia coli minichromosomes. Plasmid 52:151–168. https://doi.org/10.1016/j.plasmid.2004.08.001

    Article  PubMed  CAS  Google Scholar 

  10. Casini A, Storch M, Baldwin GS, Ellis T (2015) Bricks and blueprints: methods and standards for DNA assembly. Nat Rev Mol Cell Biol 16:568–576. https://doi.org/10.1038/nrm4014

    Article  PubMed  CAS  Google Scholar 

  11. Karas BJ, Suzuki Y, Weyman PD (2015) Strategies for cloning and manipulating natural and synthetic chromosomes. Chromosom Res 23:57–68. https://doi.org/10.1007/s10577-014-9455-3

    Article  CAS  Google Scholar 

  12. 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 

  13. Benders GA, Noskov VN, Denisova EA, Lartigue C, Gibson DG, Assad-Garcia N, Chuang RY, Carrera W, Moodie M, Algire MA, Phan Q, Alperovich N, Vashee S, Merryman C, Venter JC, Smith HO, Glass JI, Hutchison CA 3rd (2010) Cloning whole bacterial genomes in yeast. Nucleic Acids Res 38:2558–2569. https://doi.org/10.1093/nar/gkq119

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  14. Richardson SM, Mitchell LA, Stracquadanio G, Yang K, Dymond JS, DiCarlo JE, Lee D, Huang CLV, Chandrasegaran S, Cai Y, Boeke JD, Bader JS (2017) Design of a synthetic yeast genome. Science 355:1040–1044. https://doi.org/10.1126/science.aaf4557

    Article  PubMed  CAS  Google Scholar 

  15. de Kok S, Stanton LH, Slaby T, Durot M, Holmes VF, Patel KG, Platt D, Shapland EB, Serber Z, Dean J, Newman JD, Chandran SS (2014) Rapid and reliable DNA assembly via ligase cycling reaction. ACS Synth Biol 3:97–106. https://doi.org/10.1021/sb4001992

    Article  PubMed  CAS  Google Scholar 

  16. Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S (2011) A modular cloning system for standardized assembly of multigene constructs. PLoS One 6:e16765. https://doi.org/10.1371/journal.pone.0016765

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  17. Werner S, Engler C, Weber E, Gruetzner R, Marillonnet S (2012) Fast track assembly of multigene constructs using golden gate cloning and the MoClo system. Bioeng Bugs 3:38–43. https://doi.org/10.4161/bbug.3.1.18223

    Article  PubMed  Google Scholar 

  18. Lee ME, DeLoache WC, Cervantes B, Dueber JE (2015) A highly characterized yeast toolkit for modular, multipart assembly. ACS Synth Biol 4:975–986. https://doi.org/10.1021/sb500366v

    Article  PubMed  CAS  Google Scholar 

  19. Engler C, Youles M, Gruetzner R, Ehnert TM, Werner S, Jones JD, Patron NJ, Marillonnet S (2014) A golden gate modular cloning toolbox for plants. ACS Synth Biol 3:839–843. https://doi.org/10.1021/sb4001504

    Article  PubMed  CAS  Google Scholar 

  20. Duportet X, Wroblewska L, Guye P, Li Y, Eyquem J, Rieders J, Rimchala T, Batt G, Weiss R (2014) A platform for rapid prototyping of synthetic gene networks in mammalian cells. Nucleic Acids Res 42:13440–13451. https://doi.org/10.1093/nar/gku1082

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  21. Schindler D, Milbredt S, Sperlea T, Waldminghaus T (2016) Design and assembly of DNA sequence libraries for chromosomal insertion in bacteria based on a set of modified MoClo vectors. ACS Synth Biol 5(12):1362–1368. https://doi.org/10.1021/acssynbio.6b00089

    Article  PubMed  CAS  Google Scholar 

  22. Messerschmidt SJ, Kemter FS, Schindler D, Waldminghaus T (2015) Synthetic secondary chromosomes in Escherichia coli based on the replication origin of chromosome II in Vibrio cholerae. Biotechnol J 10:302–314. https://doi.org/10.1002/biot.201400031

    Article  PubMed  CAS  Google Scholar 

  23. Harrison PW, Lower RPJ, Kim NKD, Young JPW (2010) Introducing the bacterial “chromid”: not a chromosome, not a plasmid. Trends Microbiol 18:141–148. https://doi.org/10.1016/j.tim.2009.12.010

    Article  PubMed  CAS  Google Scholar 

  24. Heidelberg JF, Eisen JA, Nelson WC, Clayton RA, Gwinn ML, Dodson RJ, Haft DH, Hickey EK, Peterson JD, Umayam L, Gill SR, Nelson KE, Read TD, Tettelin H, Richardson D, Ermolaeva MD, Vamathevan J, Bass S, Qin H, Dragoi I, Sellers P, McDonald L, Utterback T, Fleishmann RD, Nierman WC, White O, Salzberg SL, Smith HO, Colwell RR, Mekalanos JJ, Venter JC, Fraser CM (2000) DNA sequence of both chromosomes of the cholera pathogen Vibrio cholerae. Nature 406:477–483. https://doi.org/10.1038/35020000

    Article  PubMed  CAS  Google Scholar 

  25. Egan ES, Waldor MK (2003) Distinct replication requirements for the two vibrio cholerae chromosomes. Cell 114:521–530. https://doi.org/10.1016/S0092-8674(03)00611-1

    Article  PubMed  CAS  Google Scholar 

  26. Pal D, Venkova-Canova T, Srivastava P, Chattoraj DK (2005) Multipartite regulation of rctB, the replication initiator gene of vibrio cholerae chromosome II. J Bacteriol 187:7167–7175. https://doi.org/10.1128/JB.187.21.7167-7175.2005

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  27. Messerschmidt SJ, Schindler D, Zumkeller CM, Kemter FS, Schallopp N, Waldminghaus T (2016) Optimization and characterization of the synthetic secondary chromosome synVicII in Escherichia coli. Front Bioeng Biotechnol 4:96. https://doi.org/10.3389/fbioe.2016.00096

    Article  PubMed  PubMed Central  Google Scholar 

  28. Rondon MR, Raffel SJ, Goodman RM, Handelsman J (1999) Toward functional genomics in bacteria: analysis of gene expression in Escherichia coli from a bacterial artificial chromosome library of Bacillus cereus. Proc Natl Acad Sci U S A 96:6451–6455

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Heringa SD, Monroe JD, Herrick JB. A simple, rapid method for extracting large plasmid DNA from Bacteria. Nat Preced. 2007. doi: https://doi.org/10.1038/npre.2007.1249.1

  30. Ferrières L, Hémery G, Nham T, Guérout A-M, Mazel D, Beloin C, Ghigo J-M (2010) Silent mischief: bacteriophage Mu insertions contaminate products of Escherichia coli random mutagenesis performed using suicidal transposon delivery plasmids mobilized by broad-host-range rp4 conjugative machinery. J Bacteriol 192:6418–6427. https://doi.org/10.1128/JB.00621-10

    Article  PubMed  PubMed Central  CAS  Google Scholar 

Download references

Author information

Author notes

  1. Daniel Schindler

    Present address: School of Chemistry, Manchester Institute of Biotechnology, University of Manchester, Manchester, UK

Authors and Affiliations

  1. Chromosome Biology Group, LOEWE Center for Synthetic Microbiology—SYNMIKRO, Philipps-Universität Marburg, Marburg, Germany

    Celine Zumkeller, Daniel Schindler & Torsten Waldminghaus

Authors
  1. Celine Zumkeller
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Daniel Schindler
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Torsten Waldminghaus
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Torsten Waldminghaus .

Editor information

Editors and Affiliations

  1. Leiden Institute of Chemistry and Centre for Microbial Cell Biology, Leiden University, Leiden, The Netherlands

    Remus T. Dame

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Zumkeller, C., Schindler, D., Waldminghaus, T. (2018). Modular Assembly of Synthetic Secondary Chromosomes. In: Dame, R. (eds) Bacterial Chromatin. Methods in Molecular Biology, vol 1837. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8675-0_5

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-8675-0_5

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8674-3

  • Online ISBN: 978-1-4939-8675-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation