Skip to main content

Engineering Protein-Based Parts for Genetic Devices in Mammalian Cells

  • Protocol
  • First Online:
Synthetic Gene Circuits

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2229))

Abstract

Synthetic biology has been advancing cellular and molecular biology studies through the design of synthetic circuits capable to examine diverse endogenously or exogenously driven regulatory pathways. While early genetic devices were engineered to be insulated from intracellular crosstalk, more recently the need of achieving dynamic control of cellular behavior has led to the development of smart interfaces that connect signal information (sensor) to desired output activation (actuator). Sensor-actuator circuits can respond to diverse inputs, including small molecules, exogenous and endogenous mRNA, noncoding RNA (i.e., miRNA), and proteins to regulate downstream events, transcriptionally, posttranscriptionally, and translationally. These devices require attentive engineering to either create complex chimeric proteins or modify protein structures to be amenable to the specific circuits’ architecture and/or purpose.

In this chapter, we describe how to implement two different protein-based devices in mammalian cells: (1) a modular platform that sense and respond to disease-associated proteins and (2) a protein-based system that allows simultaneous regulation of RNA translation and protein activity, via RNA-protein and newly engineered protein–protein interactions.

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

Access this chapter

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 139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 219.99
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

Similar content being viewed by others

References

  1. Ausländer D, Eggerschwiler B, Kemmer C, Geering B, Ausländer S, Fussenegger M (2014) A designer cell-based histamine-specific human allergy profiler. Nat Commun 5:4408

    Article  Google Scholar 

  2. di Bernardo D, Marucci L, Menolascina F, Siciliano V (2012) Predicting synthetic gene networks. Methods Mol Biol 813:57–81

    Article  Google Scholar 

  3. Tigges M, Marquez-Lago TT, Stelling J, Fussenegger M (2009) A tunable synthetic mammalian oscillator. Nature 457:309–312

    Article  CAS  Google Scholar 

  4. Siciliano V, Garzilli I, Fracassi C, Criscuolo S, Ventre S, di Bernardo D (2013) MiRNAs confer phenotypic robustness to gene networks by suppressing biological noise. Nat Commun 4:2364

    Article  Google Scholar 

  5. Kipniss NH, Dingal PCDP, Abbott TR, Gao Y, Wang H, Dominguez AA, Labanieh L, Qi LS (2017) Engineering cell sensing and responses using a GPCR-coupled CRISPR-Cas system. Nat Commun 8:2212

    Article  Google Scholar 

  6. Siciliano V, DiAndreth B, Monel B, Beal J, Huh J, Clayton KL, Wroblewska L, McKeon A, Walker BD, Weiss R (2018) Engineering modular intracellular protein sensor-actuator devices. Nat Commun 9:1881

    Article  Google Scholar 

  7. Scheller L, Strittmatter T, Fuchs D, Bojar D, Fussenegger M (2018) Generalized extracellular molecule sensor platform for programming cellular behavior. Nat Chem Biol 14:723–729

    Article  CAS  Google Scholar 

  8. Courbet A, Endy D, Renard E, Molina F, Bonnet J (2015) Detection of pathological biomarkers in human clinical samples via amplifying genetic switches and logic gates. Sci Transl Med 7:289ra83

    Article  Google Scholar 

  9. Sedlmayer F, Fussenegger M (2017) Synthetic biology: a probiotic probe for inflammation. Nat Biomed Eng 1:0097

    Article  CAS  Google Scholar 

  10. Schwarz KA, Daringer NM, Dolberg TB, Leonard JN (2016) Rewiring human cellular input–output using modular extracellular sensors. Nat Chem Biol 13:202

    Article  Google Scholar 

  11. McNamara MA, Nair SK, Holl EK (2015) RNA-based vaccines in cancer immunotherapy. J Immunol Res 2015:794528

    Article  Google Scholar 

  12. Sahin U, Karikó K, Türeci Ö (2014) mRNA-based therapeutics — developing a new class of drugs. Nat Rev Drug Discov 13:759–780

    Article  CAS  Google Scholar 

  13. Cella F, Wroblewska L, Weiss R, Siciliano V (2018) Engineering protein-protein devices for multilayered regulation of mRNA translation using orthogonal proteases in mammalian cells. Nat Commun 9:1–9

    Article  CAS  Google Scholar 

  14. Culler SJ, Hoff KG, Smolke CD (2010) Reprogramming cellular behavior with RNA controllers responsive to endogenous proteins. Science 330:1251–1255

    Article  CAS  Google Scholar 

  15. Wroblewska L, Kitada T, Endo K, Siciliano V, Stillo B, Saito H, Weiss R (2015) Mammalian synthetic circuits with RNA binding proteins for RNA-only delivery. Nat Biotechnol 33:839–841

    Article  CAS  Google Scholar 

  16. PyMOL | pymol.org. https://pymol.org/2/. Accessed 30 Oct 2019

  17. Waterhouse A, Bertoni M, Bienert S, Studer G, Tauriello G, Gumienny R, Heer FT, de Beer TAP, Rempfer C, Bordoli L, Lepore R, Schwede T (2018) SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res 46:W296–W303

    Article  CAS  Google Scholar 

  18. Engler C, Marillonnet S (2014) Golden Gate cloning. Methods Mol Biol 1116:119–131

    Article  CAS  Google Scholar 

  19. Beal J, Wagner TE, Kitada T, Azizgolshani O, Parker JM, Densmore D, Weiss R (2015) Model-driven engineering of gene expression from RNA replicons. ACS Synth Biol 4:48–56

    Article  CAS  Google Scholar 

  20. Beal J, Weiss R, Yaman F, Davidsohn N, Adler A (2012) A method for fast, high-precision characterization of synthetic biology devices. MIT CSAIL Tech Report 2012-008

    Google Scholar 

  21. Moore T, Zhang Y, Fenley MO, Li H (2004) Molecular basis of box C/D RNA-protein interactions; cocrystal structure of archaeal L7Ae and a box C/D RNA. Structure 12:807–818

    Article  CAS  Google Scholar 

  22. Caliendo F, Dukhinova M, Siciliano V (2019) Engineered Cell-Based Therapeutics: Synthetic Biology Meets Immunology. Front. Bioeng. Biotechnol. 7:43

    Google Scholar 

  23. Cella F, Siciliano V (2019) Protein-based parts and devices that respond to intracellular and extracellular signals in mammalian cells. Curr. Opin. Chem. Biol. 52:47–53

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Velia Siciliano .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2021 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Bonfá, G., Cella, F., Siciliano, V. (2021). Engineering Protein-Based Parts for Genetic Devices in Mammalian Cells. In: Menolascina, F. (eds) Synthetic Gene Circuits . Methods in Molecular Biology, vol 2229. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1032-9_16

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-1032-9_16

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1031-2

  • Online ISBN: 978-1-0716-1032-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics