Skip to main content
Book cover

Mammalian Synthetic Promoters pp 263–273Cite as

Design of Synthetic Promoters for Gene Circuits in Mammalian Cells

Part of the Methods in Molecular Biology book series (MIMB,volume 1651)

Abstract

Synthetic biology, the synthesis of engineering and biology, has rapidly matured and has dramatically increased the complexity of artificial gene circuits in recent years. The deployment of intricate synthetic gene circuits in mammalian cells requires the establishment of very precise and orthogonal control of transgene expression. In this chapter, we describe methods of modulating the expression of transgenes at the transcriptional level. Using cAMP-response element-binding protein (CREB)-dependent promoters as examples, a tool for the precise tuning of gene expression by using different core promoters and by varying the binding affinity of transcription factor operator sites is described.

Key words

  • Cyclic AMP responsive elements
  • Inducible expression
  • Minimal promoters
  • Promoter design
  • Synthetic promoters

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Benner SA, Sismour AM (2005) Synthetic biology. Nat Rev Genet 6(7):533–543

    CrossRef  CAS  Google Scholar 

  2. Schukur L, Geering B, Charpin-El Hamri G, Fussenegger M (2015) Implantable synthetic cytokine converter cells with AND-gate logic treat experimental psoriasis. Sci Transl Med 7(318):318ra201

    CrossRef  Google Scholar 

  3. Saxena P, Charpin-El Hamri G, Folcher M, Zulewski H, Fussenegger M (2016) Synthetic gene network restoring endogenous pituitary-thyroid feedback control in experimental Graves’ disease. Proc Natl Acad Sci U S A 113(5):1244–1249

    CrossRef  CAS  Google Scholar 

  4. Kramer BP, Fischer C, Fussenegger M (2004) BioLogic gates enable logical transcription control in mammalian cells. Biotechnol Bioeng 87(4):478–484

    CrossRef  CAS  Google Scholar 

  5. Auslander S, Auslander D, Muller M, Wieland M, Fussenegger M (2012) Programmable single-cell mammalian biocomputers. Nature 487(7405):123–127

    CrossRef  Google Scholar 

  6. Bojar D, Fussenegger M (2016) The best of both worlds: reaping the benefits from mammalian and bacterial therapeutic circuits. Curr Opin Chem Biol 34:11–19

    CrossRef  CAS  Google Scholar 

  7. Saxena P, Heng BC, Bai P, Folcher M, Zulewski H, Fussenegger M (2016) A programmable synthetic lineage-control network that differentiates human IPSCs into glucose-sensitive insulin-secreting beta-like cells. Nat Commun 7:11247

    CrossRef  CAS  Google Scholar 

  8. Lienert F, Lohmueller JJ, Garg A, Silver PA (2014) Synthetic biology in mammalian cells: next generation research tools and therapeutics. Nat Rev Mol Cell Biol 15(2):95–107

    CrossRef  CAS  Google Scholar 

  9. Weake VM, Workman JL (2010) Inducible gene expression: diverse regulatory mechanisms. Nat Rev Genet 11(6):426–437

    CrossRef  CAS  Google Scholar 

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

    CrossRef  CAS  Google Scholar 

  11. Gossen M, Bujard H (1992) Tight control of gene expression in mammalian cells by tetracycline -responsive promoters. Proc Natl Acad Sci U S A 89(12):5547–5551

    Google Scholar 

  12. Gitzinger M, Kemmer C, El-Baba MD, Weber W, Fussenegger M (2009) Controlling transgene expression in subcutaneous implants using a skin lotion containing the apple metabolite phloretin. Proc Natl Acad Sci U S A 106(26):10638–10643

    CrossRef  CAS  Google Scholar 

  13. Tornoe J, Kusk P, Johansen TE, Jensen PR (2002) Generation of a synthetic mammalian promoter library by modification of sequences spacing transcription factor binding sites . Gene 297(1–2):21–32

    CrossRef  Google Scholar 

  14. Cheng JK, Alper HS (2016) Transcriptomics-guided design of synthetic promoters for a mammalian system. ACS Synth Biol 5(12):1455–1465

    Google Scholar 

  15. Sassone-Corsi P (2012) The cyclic AMP pathway. Cold Spring Harb Perspect Biol 4(12). doi:10.1101/cshperspect.a011148

    CrossRef  Google Scholar 

  16. Clapham DE (2007) Calcium signaling. Cell 131(6):1047–1058

    CrossRef  CAS  Google Scholar 

  17. Auslander D, Auslander S, Charpin-El Hamri G, Sedlmayer F, Muller M, Frey O, Hierlemann A, Stelling J, Fussenegger M (2014) A synthetic multifunctional mammalian pH sensor and CO2 transgene-control device. Mol Cell 55(3):397–408

    CrossRef  CAS  Google Scholar 

  18. Kemmer C, Fluri DA, Witschi U, Passeraub A, Gutzwiller A, Fussenegger M (2011) A designer network coordinating bovine artificial insemination by ovulation-triggered release of implanted sperms. J Control Release 150(1):23–29

    CrossRef  CAS  Google Scholar 

  19. Lewis DE, Adhya S (2002) In vitro repression of the gal promoters by GalR and HU depends on the proper helical phasing of the two operators. J Biol Chem 277(4):2498–2504

    Google Scholar 

  20. Chi T, Lieberman P, Ellwood K, Carey M (1995) A general mechanism for transcriptional synergy by eukaryotic activators. Nature 377(6546):254–257

    CrossRef  CAS  Google Scholar 

  21. Nikolajczyk BS, Nelsen B, Sen R (1996) Precise alignment of sites required for mu enhancer activation in B cells. Mol Cell Biol 16(8):4544–4554

    CrossRef  CAS  Google Scholar 

  22. Weber W, Kramer BP, Fux C, Keller B, Fussenegger M (2002) Novel promoter/transactivator configurations for macrolide- and streptogramin-responsive transgene expression in mammalian cells. J Gene Med 4(6):676–686

    CrossRef  CAS  Google Scholar 

  23. Ellwood K, Huang W, Johnson R, Carey M (1999) Multiple layers of cooperativity regulate enhanceosome-responsive RNA polymerase II transcription complex assembly. Mol Cell Biol 19(4):2613–2623

    Google Scholar 

  24. Wang J, Ellwood K, Lehman A, Carey MF, She ZS (1999) A mathematical model for synergistic eukaryotic gene activation. J Mol Biol 286(2):315–325

    CrossRef  CAS  Google Scholar 

  25. Chepurny OG, Holz GG (2007) A novel cyclic adenosine monophosphate responsive luciferase reporter incorporating a nonpalindromic cyclic adenosine monophosphate response element provides optimal performance for use in G protein coupled receptor drug discovery efforts. J Biomol Screen 12(5):740–746

    CrossRef  CAS  Google Scholar 

  26. Schlabach MR, Hu JK, Li M, Elledge SJ (2010) Synthetic design of strong promoters. Proc Natl Acad Sci U S A 107(6):2538–2543

    CrossRef  CAS  Google Scholar 

  27. Ede C, Chen X, Lin MY, Chen YY (2016) Quantitative analyses of core promoters enable precise engineering of regulated gene expression in mammalian cells. ACS Synth Biol 5(5):395–404

    Google Scholar 

  28. Haugwitz M, Nourzaie O, Garachtchenko T, Hu L, Gandlur S, Olsen C, Farmer A, Chaga G, Sagawa H (2008) Multiplexing bioluminescent and fluorescent reporters to monitor live cells. Curr Chem Genomics 1:11–19

    CrossRef  CAS  Google Scholar 

  29. Zur H, Tuller T (2013) New universal rules of eukaryotic translation initiation fidelity. PLoS Comput Biol 9(7):e1003136

    CrossRef  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, Basel, CH-4058, Switzerland

    Pratik Saxena, Daniel Bojar & Martin Fussenegger

  2. Faculty of Science, University of Basel, Mattenstrasse 26, Basel, CH-4058, Switzerland

    Martin Fussenegger

Authors
  1. Pratik Saxena
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Daniel Bojar
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Martin Fussenegger
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Martin Fussenegger .

Editor information

Editors and Affiliations

  1. Department of Biochemical Pharmacology, William Harvey Research Institute, Queen Mary University of London, London, UK

    David Gould

Rights and permissions

Reprints and Permissions

Copyright information

© 2017 Springer Science+Business Media LLC

About this protocol

Cite this protocol

Saxena, P., Bojar, D., Fussenegger, M. (2017). Design of Synthetic Promoters for Gene Circuits in Mammalian Cells. In: Gould, D. (eds) Mammalian Synthetic Promoters. Methods in Molecular Biology, vol 1651. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-7223-4_19

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7223-4_19

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-7221-0

  • Online ISBN: 978-1-4939-7223-4

  • eBook Packages: Springer Protocols