Skip to main content
SpringerLink
Log in

Optimization of Synthetic Operons Using Libraries of Post-Transcriptional Regulatory Elements

  • Protocol
  • First Online:
Strain Engineering

Part of the book series: Methods in Molecular Biology ((MIMB,volume 765))

Abstract

Constructing polycistronic operons is an advantageous strategy for coordinating the expression of ­multiple genes in a prokaryotic host. Unfortunately, a basic construct consisting of an inducible promoter and genes cloned in series does not generally lead to optimal results. Here, a combinatorial approach for tuning relative gene expression in operons is presented. The method constructs libraries of post-­transcriptional regulatory elements that can be cloned into the noncoding sequence between genes. Libraries can be screened to identify sequences that optimize expression of metabolic pathways, multisubunit proteins, or other situations where precise stoichiometric ratios of proteins are desired.

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 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
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. Khosla C., and Keasling J. D. (2003) Timeline - Metabolic engineering for drug discovery and development. Nature Reviews Drug Discovery 2, 1019–1025.

    Article  PubMed  CAS  Google Scholar 

  2. Martin V. J. J., Pitera D. J., Withers S. T., Newman J. D., and Keasling J. D. (2003) Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nat. Biotechnol. 21, 796–802.

    Article  PubMed  CAS  Google Scholar 

  3. Baga M., Goransson M., Normark S., and Uhlin B. E. (1988) Processed messenger-RNA with differential stability in the regulation of E. coli pilin gene expression. Cell 52, 197–206.

    Google Scholar 

  4. Nishizaki T., Tsuge K., Itaya M., Doi N., and Yanagawa H. (2007) Metabolic engineering of carotenoid biosynthesis in Escherichia coli by ordered gene assembly in Bacillus subtilis. Appl. Environ. Microbiol. 73, 1355–1361.

    Article  PubMed  CAS  Google Scholar 

  5. Salis H. M., Mirsky E. A., and Voigt C. A. (2009) Automated design of synthetic ribosome binding sites to control protein expression. Nat. Biotechnol. 27, 946-U112.

    Article  PubMed  CAS  Google Scholar 

  6. Kizer L., Pitera D. J., Pfleger B. F., and Keasling J. D. (2008) Application of functional genomics to pathway optimization for increased isoprenoid production. Appl. Environ. Microbiol. 74, 3229–3241.

    Article  PubMed  CAS  Google Scholar 

  7. Guzman L. M., Belin D., Carson M. J., and Beckwith J. (1995) Tight Regulation, Modulation, and High-Level Expression by Vectors Containing the Arabinose PBAD Promoter. J. Bacteriol. 177, 4121–4130.

    PubMed  CAS  Google Scholar 

  8. Carrier T. A., and Keasling J. D. (1999) Library of synthetic 5‘ secondary structures to ­manipulate mRNA stability in Escherichia coli. Biotechnol. Prog. 15, 58–64.

    Article  PubMed  CAS  Google Scholar 

  9. Smolke C. D., Carrier T. A., and Keasling J. D. (2000) Coordinated, differential expression of two genes through directed mRNA cleavage and stabilization by secondary structures. Appl. Environ. Microbiol. 66, 5399–5405.

    Article  PubMed  CAS  Google Scholar 

  10. Jana S., and Deb J. K. (2005) Strategies for efficient production of heterologous proteins in Escherichia coli. Appl. Microbiol. Biotechnol. 67, 289–298.

    Article  PubMed  CAS  Google Scholar 

  11. Hammer K., Mijakovic I., and Jensen P. R. (2006) Synthetic promoter libraries - tuning of gene expression. Trends Biotechnol. 24, 53–55.

    Article  PubMed  CAS  Google Scholar 

  12. Kozak M. (2005) Regulation of translation via mRNA structure in prokaryotes and eukaryotes. Gene 361, 13–37.

    Article  PubMed  CAS  Google Scholar 

  13. Seo S. W., Yang J., and Jung G. Y. (2009) Quantitative Correlation Between mRNA Secondary Structure Around the Region Downstream of the Initiation Codon and Translational Efficiency in Escherichia coli. Biotechnol. Bioeng. 104, 611–616.

    Google Scholar 

  14. Pfleger B. F., Pitera D. J., D Smolke C., and Keasling J. D. (2006) Combinatorial ­engineering of intergenic regions in operons tunes ­expression of multiple genes. Nat. Biotechnol. 24, 1027–1032.

    Google Scholar 

  15. Sambrook J., Russell D. W. (2001) Molecular cloning : a laboratory manual Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

    Google Scholar 

  16. Studier F. W., Rosenberg A. H., Dunn J. J., and Dubendorff J. W. (1990) Use of T7 ­RNA-polymerase to direct expression of cloned genes. Methods Enzymol. 185, 60–89.

    Article  PubMed  CAS  Google Scholar 

  17. Bartlett J. M. S., and Stirling D., (Eds.) (2003) PCR Protocols, Vol. 226, second ed., Humana Press.

    Google Scholar 

  18. (2010) Ambion’s Appendix - DNA and RNA Molecular Weights and Conversions, Applied Biosystems. http://www.ambion.com/techlib/append/na_mw_tables.html.

  19. Meynial-Salles I., Cervin M. A., and Soucaille P. (2005) New tool for metabolic pathway engineering in Escherichia coli: One-step method to modulate expression of chromosomal genes. Appl. Environ. Microbiol. 71, 2140–2144.

    Article  PubMed  CAS  Google Scholar 

  20. Graham J. E. (2004) Sequence-specific Rho-RNA interactions in transcription termination. Nucleic Acids Res. 32, 3093–3100.

    Article  PubMed  CAS  Google Scholar 

  21. Zuker M. (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31, 3406–3415.

    Article  PubMed  CAS  Google Scholar 

  22. Desmit M. H., and Vanduin J. (1994) Translational initiation on structured messengers: another role for the Shine-Dalgarno interaction. J. Mol. Biol. 235, 173–184.

    Article  CAS  Google Scholar 

  23. Canton B., Labno A., and Endy D. (2008) Refinement and standardization of synthetic biological parts and devices. Nat Biotechnol. 26, 787–793.

    Article  PubMed  CAS  Google Scholar 

  24. Aslanidis C., and Dejong P. J. (1990) Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Res. 18, 6069–6074.

    Article  PubMed  CAS  Google Scholar 

  25. Datsenko K. A., and Wanner B. L. (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U.S.A. 97, 6640–6645.

    Article  PubMed  CAS  Google Scholar 

  26. Stols L., Gu M. Y., Dieckman L., Raffen R., Collart F. R., and Donnelly M. I. (2002) A new vector for high-throughput, ligation-independent cloning encoding a tobacco etch virus protease cleavage site. Protein Expression Purif. 25, 8–15.

    Article  CAS  Google Scholar 

  27. Benders G. A., Noskov V. N., Denisova E. A., Lartigue C., Gibson D. G., Assad-Garcia N., Chuang R. Y., Carrera W., Moodie M., Algire M. A., Phan Q., Alperovich N., Vashee S., Merryman C., Venter J. C., Smith H. O., Glass J. I., and Hutchison C. A. (2010) Cloning whole bacterial genomes in yeast. Nucleic Acids Res. 38, 2558–2569.

    Article  PubMed  CAS  Google Scholar 

  28. Gibson D. G., Young L., Chuang R. Y., Venter J. C., Hutchison C. A., and Smith H. O. (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343-U341.

    Article  PubMed  CAS  Google Scholar 

  29. DeWitt W., and Helinski D. R. (1965) Characterization of colicinogenic factor E1 from a non-induced and a mitomycin C-induced Proteus strain. J. Mol. Biol. 13, 692–703.

    Article  CAS  Google Scholar 

  30. Altingmees M. A., and Short J. M. (1989) pBlueScript II: gene mapping vectors. Nucleic Acids Res. 17, 9494–9494.

    Article  CAS  Google Scholar 

  31. Short J. M., Fernandez J. M., Sorge J. A., and Huse W. D. (1988) Lambda ZAP: a bacteriophage lambda expression vector with in vivo excision properties. Nucleic Acids Res. 16, 7583–7600.

    Article  PubMed  CAS  Google Scholar 

  32. Betlach M., Hershfield V., Chow L., Brown W., Goodman H. M., and Boyer H. W. (1976) Restriction endonuclease analysis of bacterial plasmid controlling EcoRI restriction and modification of DNA. Fed Proc 35, 2037–2043.

    Google Scholar 

  33. Yanischperron C., Vieira J., and Messing J. (1985) Improved M13 phage cloning vectors and host strains - nucleotide-sequences of the M13mp18 and pUC19 vectors. Gene 33, 103–119.

    Google Scholar 

  34. Vieira J., and Messing J. (1982) The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19, 259–268.

    Article  PubMed  CAS  Google Scholar 

  35. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., and Falkow S. (1977) Construction and characterization of new cloning vehicles. II. Multipurpose cloning system. Gene 2, 95–113.

    Google Scholar 

  36. Antoine R., and Locht C. (1992) Isolation and molecular characterization of a novel broad-host-range plasmid from Bordetella bronchiseptica with sequence similarities to plasmids from gram-positive organisms. Mol. Microbiol. 6, 1785–1799.

    Article  PubMed  CAS  Google Scholar 

  37. Kovach M. E., Phillips R. W., Elzer P. H., Roop R. M., and Peterson K. M. (1994) pBBR1MCS: a broad-host-range cloning vector. BioTechniques 16, 800-&.

    Google Scholar 

  38. Cozzarelli N.R, Kelly R. B., and Kornberg A. (1968) A minute circular DNA from Escherichia coli 15. Proc Natl Acad Sci U.S.A. 60, 992–999.

    Article  PubMed  CAS  Google Scholar 

  39. Chang A. C. Y., and Cohen S. N. (1978) Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from p15A cryptic miniplasmid. J. Bacteriol. 134, 1141–1156.

    PubMed  CAS  Google Scholar 

  40. Cohen S. N., and Chang A. C. Y. (1973) Recircularization and autonomous replication of a sheared R-factor DNA segment in Escherichia coli transformants: (plasmid/transformation/antibiotic resistance/DNA). Proc Natl Acad Sci U.S.A. 70, 1293–1297.

    Article  PubMed  CAS  Google Scholar 

  41. Tabor S., and Richardson C. C. (1985) A bacteriophage T7 RNA polymerase promoter ­system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U.S.A. 82, 1074–1078.

    Article  PubMed  CAS  Google Scholar 

  42. Giordano T. J., Deuschle U., Bujard H., and McAllister W. T. (1989) Regulation of coliphage T3 and coliphage T7 RNA polymerases by the lac repressor-operator system. Gene 84, 209–219.

    Article  PubMed  CAS  Google Scholar 

  43. Deboer H. A., Comstock L. J., and Vasser M. (1983) The tac promoter - a functional hybrid derived from the trp and lac promoters. Proc Natl Acad Sci U.S.A. 80, 21–25.

    Article  CAS  Google Scholar 

  44. Brosius J., Erfle M., and Storella J. (1985) Spacing of the −10 and −35 regions in the tac promoter: effect on its in vivo activity. J. Biol. Chem. 260, 3539–3541.

    PubMed  CAS  Google Scholar 

  45. Elvin C. M., Thompson P. R., Argall M. E., Hendry P., Stamford N. P. J., Lilley P. E., and Dixon N. E. (1990) Modified bacteriophage lambda promoter vectors for overproduction of proteins in Escherichia coli. Gene 87, 123–126.

    Article  PubMed  CAS  Google Scholar 

  46. Skerra A. (1994) Use of the tetracycline promoter for the tightly regulated production of a murine antibody fragment in Escherichia coli. Gene 151, 131–135.

    Article  PubMed  CAS  Google Scholar 

  47. Lee S. K., and Keasling J. D. (2005) A propionate-inducible expression system for enteric bacteria. Appl. Environ. Microbiol. 71, 6856–6862.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

Work in the authors’ lab was sponsored by the University of Wisconsin-Madison Graduate School. Daniel Agnew is the recipient­ of an NIH Biotechnology Training Program Graduate Fellowship (NIH 5 T32 GM08349).

Author information

Authors and Affiliations

  1. Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, WI, USA

    Daniel E. Agnew & Brian F. Pfleger

Authors
  1. Daniel E. Agnew
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Brian F. Pfleger
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Brian F. Pfleger .

Editor information

Editors and Affiliations

  1. Nature Technology Corporation, Innovation Drive 4701, Lincoln, 68521, Nebraska, USA

    James A. Williams

Rights and permissions

Reprints and permissions

Copyright information

© 2011 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Agnew, D.E., Pfleger, B.F. (2011). Optimization of Synthetic Operons Using Libraries of Post-Transcriptional Regulatory Elements. In: Williams, J. (eds) Strain Engineering. Methods in Molecular Biology, vol 765. Humana Press. https://doi.org/10.1007/978-1-61779-197-0_7

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-197-0_7

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-196-3

  • Online ISBN: 978-1-61779-197-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation