This document contains legends for all supplementary tables and the contents for Supplementary Tables 9 and 10; the remaining supplementary tables are provided as Microsoft Excel Worksheets. Supplementary Fig. 2 Differences between spaced and compact promoters. (a) Fractional activation for dose response profiles in Fig. 2a. Fractional activation was determined by dividing each data point by the maximum reporter expression induced by the ZFa on a given reporter. In several conditions, notably ZF1x6-S, excess ZFa (above 50 ng plasmid) resulted in a decrease in reporter expression. In this case, we hypothesize that unbound ZFa competes with bound ZFa for endogenous cofactors required for transcription. (b) Representative flow cytometry histograms for experiments with spaced reporters in Fig. 2a. Data were gated on single, transfected cells ( Supplementary Fig. 15). Reporter expression increases with ZFa dose and number of binding sites. (c) Representative flow cytometry histograms from experiments with compact reporters in Fig. 2a. Data were gated on single, transfected cells. Reporter expression increases with ZFa dose and number of binding sites. Compared to the case of spaced promoters (panel b), cases with compact promoters exhibit a greater fraction of cells that are distinguishably ON, i.e., expressing more EYFP than cells without ZFa. (d) Investigating different minimal promoters with COMET. ZF1a dose responses were conducted using ZF1x6-C promoters with either the YB_TATA, CMV, or SV40 minimal promoters. The SV40 minimal promoter produced low levels of gene expression, while the YB_TATA minimal promoter conferred a maximal gene expression level similar to that of the CMV minimal promoter. Although the CMV minimal promoter was more responsive at lower levels of ZFa expression, this promoter also had higher leaky gene expression (in the absence of ZFa) than did the YB_TATA minimal promoter (quantified in Supplementary Table 10 with fitted parameters). Thus, the YB_TATA minimal promoter resulted in higher fold inductions than did the CMV minimal promoter (approximately 220-fold for YB_TATA, as compared to 60-fold for CMV_min) without sacrificing maximal gene expression. (e) Investigating maximal inducible EYFP expression. Cells were transfected with ZF1a plasmid and with reporter plasmid containing a ZF1x6-S (left) or ZF1x6-C (right) promoter. The ZFa plasmid and reporter plasmid were maintained at a ratio of 1:2 (ZFa:reporter) as the doses were scaled. On the x-axis, a value of 1 denotes a condition with 100 ng of ZF1a plasmid and 200 ng of reporter plasmid. In previous experiments, reporter expression typically plateaued at the level indicated by plasmid doses corresponding to 1 on the x-axis and could not be increased by the addition of more ZFa plasmid (Supplementary Fig. 2a). However, doubling the amount of both ZFa plasmid and reporter plasmid led to twice the reporter expression, which indicates the amount of plasmid was the limiting factor in gene expression as opposed to a downstream step such as tr...

Engineering mammalian cells to carry out sophisticated and customizable genetic programs requires a toolkit of multiple orthogonal and well-characterized transcription factors (TFs). To address this need, we developed the COmposable Mammalian Elements of Transcription (COMET)-an ensemble of TFs and promoters that enable the design and tuning of gene expression to an extent not previously possible.COMET currently comprises 44 activating and 12 inhibitory zinc-finger TFs and 83 cognate promoters, combined in a framework that readily accommodates new parts. This system can tune gene expression over three orders of magnitude, provides chemically inducible control of TF activity, and enables singlelayer Boolean logic. We also develop a mathematical model that provides mechanistic insights into COMET performance characteristics. Altogether, COMET enables the design and construction of customizable genetic programs in mammalian cells. for useful discussion on the computational model. We thank Ahmad Khalil (Boston University) for sharing plasmids encoding the ZFa from his 2012 study 15 . created reagents, designed and performed experiments, and analyzed the data. J.J.M developed the computational models and code.

Synthetic receptors are powerful tools for engineering mammalian cell-based devices. These biosensors enable cell-based therapies to perform complex tasks such as regulating therapeutic gene expression in response to sensing physiological cues. Although multiple synthetic receptor systems now exist, many aspects of receptor performance are poorly understood. In general, it would be useful to understand how receptor design choices influence performance characteristics. In this study, we examined the modular extracellular sensor architecture (MESA) and systematically evaluated previously unexamined design choices, yielding substantially improved receptors. A key finding that might extend to other receptor systems is that the choice of transmembrane domain (TMD) is important for generating high-performing receptors. To provide mechanistic insights, we adopted and employed a Förster resonance energy transfer (FRET)-based assay to elucidate how TMDs affect receptor complex formation and connected these observations to functional performance. To build further insight into these phenomena, we developed a library of new MESA receptors that sense an expanded set of ligands. Based upon these explorations, we conclude that TMDs affect signaling primarily by modulating intracellular domain geometry. Finally, to guide the design of future receptors, we propose general principles for linking design choices to biophysical mechanisms and performance characteristics.

Genetically engineering cells to perform customizable functions is an emerging frontier with numerous technological and translational applications. However, it remains challenging to systematically engineer mammalian cells to execute complex functions. To address this need, we developed a method enabling accurate genetic program design using high-performing genetic parts and predictive computational models. We built multifunctional proteins integrating both transcriptional and posttranslational control, validated models for describing these mechanisms, implemented digital and analog processing, and effectively linked genetic circuits with sensors for multi-input evaluations. The functional modularity and compositional versatility of these parts enable one to satisfy a given design objective via multiple synonymous programs. Our approach empowers bioengineers to predictively design mammalian cellular functions that perform as expected even at high levels of biological complexity.

Genetically engineering cells to perform customizable functions is an emerging frontier with numerous technological and translational applications. However, it remains challenging to systematically engineer mammalian cells to execute complex functions. To address this need, we developed a method enabling accurate genetic program design using high-performing genetic parts and predictive computational models. We built multi-functional proteins integrating both transcriptional and post-translational control, validated models for describing these mechanisms, implemented digital and analog processing, and effectively linked genetic circuits with sensors for multi-input evaluations. The functional modularity and compositional versatility of these parts enable one to satisfy a given design objective via multiple synonymous programs. Our approach empowers bioengineers to predictively design mammalian cellular functions that perform as expected even at high levels of biological complexity.