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

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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
Copyright © 2016 American Chemical Society

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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.

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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)

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