ACS Publications. Most Trusted. Most Cited. Most Read

OR SEARCH CITATIONS

My Activity
Publications
CONTENT TYPES

Figure 1Loading Img
Download Hi-Res ImageDownload to MS-PowerPointCite This:ACS Synth. Biol. 2016, 5, 5, 395-404
RETURN TO ISSUEPREVResearch ArticleNEXT

Quantitative Analyses of Core Promoters Enable Precise Engineering of Regulated Gene Expression in Mammalian Cells

View Author Information
Department of Chemical and Biomolecular Engineering, University of California—Los Angeles, Los Angeles, California 90095, United States
*Tel: +1-310-825-2816. E-mail: [email protected]
Cite this: ACS Synth. Biol. 2016, 5, 5, 395–404
Publication Date (Web):February 16, 2016
https://doi.org/10.1021/acssynbio.5b00266
Copyright © 2016 American Chemical Society
RIGHTS & PERMISSIONS

Article Views

4267

Altmetric

-

Citations

51
LEARN ABOUT THESE METRICS
Read OnlinePDF (1 MB)
Get e-Alerts
Supporting Info (1)»
SUBJECTS:

Abstract

Abstract Image

Inducible transcription systems play a crucial role in a wide array of synthetic biology circuits. However, the majority of inducible promoters are constructed from a limited set of tried-and-true promoter parts, which are susceptible to common shortcomings such as high basal expression levels (i.e., leakiness). To expand the toolbox for regulated mammalian gene expression and facilitate the construction of mammalian genetic circuits with precise functionality, we quantitatively characterized a panel of eight core promoters, including sequences with mammalian, viral, and synthetic origins. We demonstrate that this selection of core promoters can provide a wide range of basal gene expression levels and achieve a gradient of fold-inductions spanning 2 orders of magnitude. Furthermore, commonly used parts such as minimal CMV and minimal SV40 promoters were shown to achieve robust gene expression upon induction, but also suffer from high levels of leakiness. In contrast, a synthetic promoter, YB_TATA, was shown to combine low basal expression with high transcription rate in the induced state to achieve significantly higher fold-induction ratios compared to all other promoters tested. These behaviors remain consistent when the promoters are coupled to different genetic outputs and different response elements, as well as across different host-cell types and DNA copy numbers. We apply this quantitative understanding of core promoter properties to the successful engineering of human T cells that respond to antigen stimulation via chimeric antigen receptor signaling specifically under hypoxic environments. Results presented in this study can facilitate the design and calibration of future mammalian synthetic biology systems capable of precisely programmed functionality.

KEYWORDS:

Supporting Information

ARTICLE SECTIONS
Jump To

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssynbio.5b00266.

  • Figure S1: Transfection efficiency of plasmids encoding core promoter panels in HEK 293T cells. Figure S2: Comparison of gene expression vs distance between promoter and start codon in expression plasmids. Figure S3: Population gating strategy for flow cytometry data. Figure S4: sfGFP expression by core promoters in the uninduced state. Figure S5: Gluc output and fold-induction of IL-6-responsive transcription systems in transiently transfected HEK 293T cells. Table S1: List of core promoter sequences. (PDF)

Terms & Conditions

Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Cited By

This article is cited by 51 publications.

  1. Tingxi Guo, Dacheng Ma, Timothy K. Lu. Sense-and-Respond Payload Delivery Using a Novel Antigen-Inducible Promoter Improves Suboptimal CAR-T Activation. ACS Synthetic Biology 2022, 11 (4) , 1440-1453. https://doi.org/10.1021/acssynbio.1c00236
  2. Polina Govorkova, Chee Ka Candice Lam, Kevin Truong. Design of Synthetic Mammalian Promoters Using Highly Palindromic Subsequences. ACS Synthetic Biology 2022, 11 (3) , 1096-1105. https://doi.org/10.1021/acssynbio.1c00600
  3. Chee Ka Candice Lam, Kevin Truong. Engineering a Synthesis-Friendly Constitutive Promoter for Mammalian Cell Expression. ACS Synthetic Biology 2020, 9 (10) , 2625-2631. https://doi.org/10.1021/acssynbio.0c00310
  4. Rui Shen, Jin Yin, Jian-Wen Ye, Rui-Juan Xiang, Zhi-Yu Ning, Wu-Zhe Huang, Guo-Qiang Chen. Promoter Engineering for Enhanced P(3HB-co-4HB) Production by Halomonas bluephagenesis. ACS Synthetic Biology 2018, 7 (8) , 1897-1906. https://doi.org/10.1021/acssynbio.8b00102
  5. Sen Yang, Qingtao Liu, Yunfeng Zhang, Guocheng Du, Jian Chen, and Zhen Kang . Construction and Characterization of Broad-Spectrum Promoters for Synthetic Biology. ACS Synthetic Biology 2018, 7 (1) , 287-291. https://doi.org/10.1021/acssynbio.7b00258
  6. Andrea Martella, Mantas Matjusaitis, Jamie Auxillos, Steven M. Pollard, and Yizhi Cai . EMMA: An Extensible Mammalian Modular Assembly Toolkit for the Rapid Design and Production of Diverse Expression Vectors. ACS Synthetic Biology 2017, 6 (7) , 1380-1392. https://doi.org/10.1021/acssynbio.7b00016
  7. Cindy T. Wei, Nicholas A. Popp, Omri Peleg, Rachel L. Powell, Elhanan Borenstein, Dustin J. Maly, Douglas M. Fowler. A chemically controlled Cas9 switch enables temporal modulation of diverse effectors. Nature Chemical Biology 2023, 337 https://doi.org/10.1038/s41589-023-01278-6
  8. Hui-Shan Li, Divya V. Israni, Keith A. Gagnon, Kok Ann Gan, Michael H. Raymond, Jeffry D. Sander, Kole T. Roybal, J. Keith Joung, Wilson W. Wong, Ahmad S. Khalil. Multidimensional control of therapeutic human cell function with synthetic gene circuits. Science 2022, 378 (6625) , 1227-1234. https://doi.org/10.1126/science.ade0156
  9. Ze-Yan Zhang, Yingwen Ding, Ravesanker Ezhilarasan, Tenzin Lhakhang, Qianghu Wang, Jie Yang, Aram S. Modrek, Hua Zhang, Aristotelis Tsirigos, Andrew Futreal, Giulio F. Draetta, Roel G. W. Verhaak, Erik P. Sulman. Lineage-coupled clonal capture identifies clonal evolution mechanisms and vulnerabilities of BRAFV600E inhibition resistance in melanoma. Cell Discovery 2022, 8 (1) https://doi.org/10.1038/s41421-022-00462-7
  10. Tilman L. B. Hölting, Florencia Cidre-Aranaz, Dana Matzek, Bastian Popper, Severin J. Jacobi, Cornelius M. Funk, Florian H. Geyer, Jing Li, Ignazio Piseddu, Bruno L. Cadilha, Stephan Ledderose, Jennifer Zwilling, Shunya Ohmura, David Anz, Annette Künkele, Frederick Klauschen, Thomas G. P. Grünewald, Maximilian M. L. Knott. Neomorphic DNA-binding enables tumor-specific therapeutic gene expression in fusion-addicted childhood sarcoma. Molecular Cancer 2022, 21 (1) https://doi.org/10.1186/s12943-022-01641-6
  11. Ross D. Jones, Yili Qian, Katherine Ilia, Benjamin Wang, Michael T. Laub, Domitilla Del Vecchio, Ron Weiss. Robust and tunable signal processing in mammalian cells via engineered covalent modification cycles. Nature Communications 2022, 13 (1) https://doi.org/10.1038/s41467-022-29338-w
  12. Omri Sharabi, Yariv Greenshpan, Noa Ofir, Aner Ottolenghi, Tamar Levi, Leonid Olender, Zachor Adler-Agmon, Angel Porgador, Roi Gazit. High throughput screen for the improvement of inducible promoters for tumor microenvironment cues. Scientific Reports 2022, 12 (1) https://doi.org/10.1038/s41598-022-11021-1
  13. Alan Cabrera, Hailey I. Edelstein, Fokion Glykofrydis, Kasey S. Love, Sebastian Palacios, Josh Tycko, Meng Zhang, Sarah Lensch, Cara E. Shields, Mark Livingston, Ron Weiss, Huimin Zhao, Karmella A. Haynes, Leonardo Morsut, Yvonne Y. Chen, Ahmad S. Khalil, Wilson W. Wong, James J. Collins, Susan J. Rosser, Karen Polizzi, Michael B. Elowitz, Martin Fussenegger, Isaac B. Hilton, Joshua N. Leonard, Lacramioara Bintu, Kate E. Galloway, Tara L. Deans. The sound of silence: Transgene silencing in mammalian cell engineering. Cell Systems 2022, 13 (12) , 950-973. https://doi.org/10.1016/j.cels.2022.11.005
  14. Fokion Glykofrydis, Alistair Elfick. Exploring standards for multicellular mammalian synthetic biology. Trends in Biotechnology 2022, 40 (11) , 1299-1312. https://doi.org/10.1016/j.tibtech.2022.06.001
  15. Yariv Greenshpan, Omri Sharabi, Ksenia M. Yegodayev, Ofra Novoplansky, Moshe Elkabets, Roi Gazit, Angel Porgador. The Contribution of the Minimal Promoter Element to the Activity of Synthetic Promoters Mediating CAR Expression in the Tumor Microenvironment. International Journal of Molecular Sciences 2022, 23 (13) , 7431. https://doi.org/10.3390/ijms23137431
  16. Hannah R. Kempton, Kasey S. Love, Lucie Y. Guo, Lei S. Qi. Scalable biological signal recording in mammalian cells using Cas12a base editors. Nature Chemical Biology 2022, 18 (7) , 742-750. https://doi.org/10.1038/s41589-022-01034-2
  17. Rachael S. Watson-Levings, Glyn D. Palmer, Padraic P. Levings, E. Anthony Dacanay, Christopher H. Evans, Steven C. Ghivizzani. Gene Therapy in Orthopaedics: Progress and Challenges in Pre-Clinical Development and Translation. Frontiers in Bioengineering and Biotechnology 2022, 10 https://doi.org/10.3389/fbioe.2022.901317
  18. Heidi E. Klumpe, Matthew A. Langley, James M. Linton, Christina J. Su, Yaron E. Antebi, Michael B. Elowitz. The context-dependent, combinatorial logic of BMP signaling. Cell Systems 2022, 25 https://doi.org/10.1016/j.cels.2022.03.002
  19. Chee Ka Candice Lam, Kevin Truong. Engineering a synthesis-friendly serum responsive promoter for antibiotic selection of genomic integration in cell-based therapy. Biotechnology Letters 2022, 98 https://doi.org/10.1007/s10529-022-03244-z
  20. Yiqian Wu, Ziliang Huang, Reed Harrison, Longwei Liu, Linshan Zhu, Yinglin Situ, Yingxiao Wang. Engineering CAR T cells for enhanced efficacy and safety. APL Bioengineering 2022, 6 (1) , 011502. https://doi.org/10.1063/5.0073746
  21. Changping Deng, Fabiao Hu, Zhangting Zhao, Yiwen Zhou, Yuping Liu, Tong Zhang, Shihui Li, Wenyun Zheng, Wenliang Zhang, Tianwen Wang, Xingyuan Ma. The Establishment of Quantitatively Regulating Expression Cassette with sgRNA Targeting BIRC5 to Elucidate the Synergistic Pathway of Survivin with P-Glycoprotein in Cancer Multi-Drug Resistance. Frontiers in Cell and Developmental Biology 2022, 9 https://doi.org/10.3389/fcell.2021.797005
  22. Andres Cubillos-Ruiz, Tingxi Guo, Anna Sokolovska, Paul F. Miller, James J. Collins, Timothy K. Lu, Jose M. Lora. Engineering living therapeutics with synthetic biology. Nature Reviews Drug Discovery 2021, 20 (12) , 941-960. https://doi.org/10.1038/s41573-021-00285-3
  23. Kate E. Dray, Hailey I. Edelstein, Kathleen S. Dreyer, Joshua N. Leonard. Control of mammalian cell-based devices with genetic programming. Current Opinion in Systems Biology 2021, 28 , 100372. https://doi.org/10.1016/j.coisb.2021.100372
  24. Masahiro Tominaga, Kenta Nozaki, Daisuke Umeno, Jun Ishii, Akihiko Kondo. Robust and flexible platform for directed evolution of yeast genetic switches. Nature Communications 2021, 12 (1) https://doi.org/10.1038/s41467-021-22134-y
  25. Justina Pajarskienė, Vytautas Kašėta, Kristina Vaikšnoraitė, Virginijus Tunaitis, Augustas Pivoriūnas. MicroRNA-124 acts as a positive regulator of IFN-β signaling in the lipopolysaccharide-stimulated human microglial cells. International Immunopharmacology 2021, 101 , 108262. https://doi.org/10.1016/j.intimp.2021.108262
  26. Ian C. Miller, Ali Zamat, Lee-Kai Sun, Hathaichanok Phuengkham, Adrian M. Harris, Lena Gamboa, Jason Yang, John P. Murad, Saul J. Priceman, Gabriel A. Kwong. Enhanced intratumoural activity of CAR T cells engineered to produce immunomodulators under photothermal control. Nature Biomedical Engineering 2021, 5 (11) , 1348-1359. https://doi.org/10.1038/s41551-021-00781-2
  27. Roberto Di Blasi, Annalise Zouein, Tom Ellis, Francesca Ceroni. Genetic Toolkits to Design and Build Mammalian Synthetic Systems. Trends in Biotechnology 2021, 39 (10) , 1004-1018. https://doi.org/10.1016/j.tibtech.2020.12.007
  28. Andrew P. Cazier, John Blazeck. Advances in promoter engineering: Novel applications and predefined transcriptional control. Biotechnology Journal 2021, 16 (10) , 2100239. https://doi.org/10.1002/biot.202100239
  29. Andrew J. Hou, Laurence C. Chen, Yvonne Y. Chen. Navigating CAR-T cells through the solid-tumour microenvironment. Nature Reviews Drug Discovery 2021, 20 (7) , 531-550. https://doi.org/10.1038/s41573-021-00189-2
  30. Agnieszka Graczyk-Jarzynka. Strategies to improve the safety profile of CAR-T therapy. Postępy Polskiej Medycyny i Farmacji 2021, 8 , 48-60. https://doi.org/10.5604/01.3001.0014.9200
  31. Xiaofan Feng, Mario Andrea Marchisio. Novel S. cerevisiae Hybrid Synthetic Promoters Based on Foreign Core Promoter Sequences. International Journal of Molecular Sciences 2021, 22 (11) , 5704. https://doi.org/10.3390/ijms22115704
  32. Theresa B. Loveless, Joseph H. Grotts, Mason W. Schechter, Elmira Forouzmand, Courtney K. Carlson, Bijan S. Agahi, Guohao Liang, Michelle Ficht, Beide Liu, Xiaohui Xie, Chang C. Liu. Lineage tracing and analog recording in mammalian cells by single-site DNA writing. Nature Chemical Biology 2021, 17 (6) , 739-747. https://doi.org/10.1038/s41589-021-00769-8
  33. Nika Shakiba, Ross D. Jones, Ron Weiss, Domitilla Del Vecchio. Context-aware synthetic biology by controller design: Engineering the mammalian cell. Cell Systems 2021, 12 (6) , 561-592. https://doi.org/10.1016/j.cels.2021.05.011
  34. Stefani Alves Magalhães, Maira Licia Foresti, Vanessa Novaes Barros, Luiz E. Mello. Marmosets have a greater diversity of c‐Fos response after hyperstimulation in distinct cortical regions as compared to rats. Journal of Comparative Neurology 2021, 529 (7) , 1628-1641. https://doi.org/10.1002/cne.25044
  35. Patrick S. Donahue, Joseph W. Draut, Joseph J. Muldoon, Hailey I. Edelstein, Neda Bagheri, Joshua N. Leonard. The COMET toolkit for composing customizable genetic programs in mammalian cells. Nature Communications 2020, 11 (1) https://doi.org/10.1038/s41467-019-14147-5
  36. Tingting Fu, Md. Samiul Islam, Mohsin Ali, Jia Wu, Wubei Dong. Two antimicrobial genes from Aegilops tauschii Cosson identified by the Bacillus subtilis expression system. Scientific Reports 2020, 10 (1) https://doi.org/10.1038/s41598-020-70314-5
  37. Mihe Hong, Justin D. Clubb, Yvonne Y. Chen. Engineering CAR-T Cells for Next-Generation Cancer Therapy. Cancer Cell 2020, 38 (4) , 473-488. https://doi.org/10.1016/j.ccell.2020.07.005
  38. Hannah R. Kempton, Laine E. Goudy, Kasey S. Love, Lei S. Qi. Multiple Input Sensing and Signal Integration Using a Split Cas12a System. Molecular Cell 2020, 78 (1) , 184-191.e3. https://doi.org/10.1016/j.molcel.2020.01.016
  39. Ziliang Huang, Yiqian Wu, Molly E. Allen, Yijia Pan, Phillip Kyriakakis, Shaoying Lu, Ya-Ju Chang, Xin Wang, Shu Chien, Yingxiao Wang. Engineering light-controllable CAR T cells for cancer immunotherapy. Science Advances 2020, 6 (8) https://doi.org/10.1126/sciadv.aay9209
  40. Shota Katayama, Kota Sato, Toru Nakazawa. In vivo and in vitro knockout system labelled using fluorescent protein via microhomology-mediated end joining. Life Science Alliance 2020, 3 (1) , e201900528. https://doi.org/10.26508/lsa.201900528
  41. Laurence C Chen, Yvonne Y Chen. Outsmarting and outmuscling cancer cells with synthetic and systems immunology. Current Opinion in Biotechnology 2019, 60 , 111-118. https://doi.org/10.1016/j.copbio.2019.01.016
  42. Thomas Decoene, Sofie L. De Maeseneire, Marjan De Mey, . Modulating transcription through development of semi-synthetic yeast core promoters. PLOS ONE 2019, 14 (11) , e0224476. https://doi.org/10.1371/journal.pone.0224476
  43. Robert Weinkove, Philip George, Nathaniel Dasyam, Alexander D McLellan. Selecting costimulatory domains for chimeric antigen receptors: functional and clinical considerations. Clinical & Translational Immunology 2019, 8 (5) , e1049. https://doi.org/10.1002/cti2.1049
  44. Richard L. Youngblood, Norman F. Truong, Tatiana Segura, Lonnie D. Shea. It’s All in the Delivery: Designing Hydrogels for Cell and Non-viral Gene Therapies. Molecular Therapy 2018, 26 (9) , 2087-2106. https://doi.org/10.1016/j.ymthe.2018.07.022
  45. Patrick Ho, Yvonne Y. Chen. Synthetic Biology in Immunotherapy and Stem Cell Therapy Engineering. 2018, 349-372. https://doi.org/10.1002/9783527688104.ch17
  46. Patrick Ho, Yvonne Y Chen. Mammalian synthetic biology in the age of genome editing and personalized medicine. Current Opinion in Chemical Biology 2017, 40 , 57-64. https://doi.org/10.1016/j.cbpa.2017.06.003
  47. Cosmas D Arnold, Muhammad A Zabidi, Michaela Pagani, Martina Rath, Katharina Schernhuber, Tomáš Kazmar, Alexander Stark. Genome-wide assessment of sequence-intrinsic enhancer responsiveness at single-base-pair resolution. Nature Biotechnology 2017, 35 (2) , 136-144. https://doi.org/10.1038/nbt.3739
  48. Melina Mathur, Joy S. Xiang, Christina D. Smolke. Mammalian synthetic biology for studying the cell. Journal of Cell Biology 2017, 216 (1) , 73-82. https://doi.org/10.1083/jcb.201611002
  49. Pratik Saxena, Daniel Bojar, Martin Fussenegger. Design of Synthetic Promoters for Gene Circuits in Mammalian Cells. 2017, 263-273. https://doi.org/10.1007/978-1-4939-7223-4_19
  50. Muhammad A. Zabidi, Alexander Stark. Regulatory Enhancer–Core-Promoter Communication via Transcription Factors and Cofactors. Trends in Genetics 2016, 32 (12) , 801-814. https://doi.org/10.1016/j.tig.2016.10.003
  51. Sarah Guiziou, Vincent Sauveplane, Hung-Ju Chang, Caroline Clerté, Nathalie Declerck, Matthieu Jules, Jerome Bonnet. A part toolbox to tune genetic expression in Bacillus subtilis. Nucleic Acids Research 2016, 13 , gkw624. https://doi.org/10.1093/nar/gkw624

Pair your accounts.

Export articles to Mendeley

Get article recommendations from ACS based on references in your Mendeley library.

Pair your accounts.

Export articles to Mendeley

Get article recommendations from ACS based on references in your Mendeley library.

You’ve supercharged your research process with ACS and Mendeley!

STEP 1:
Click to create an ACS ID

Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

MENDELEY PAIRING EXPIRED
Your Mendeley pairing has expired. Please reconnect