A synthetic close-loop controller circuit for the regulation of an extracellular molecule by engineered bacteria

Pasotti, Lorenzo; Bellato, Massimo; Politi, Nicolò; Casanova, Michela; Zucca, Susanna; Angelis, Maria Gabriella Cusella De; Magni, Paolo

doi:10.1109/tbcas.2018.2883350

Cited by 9 publications

(46 citation statements)

References 48 publications

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“…In many works considering a constant LuxR production, not changing throughout the experiments [22,23,[31][32][33][34], H(L) is modeled via a Hill equation and the circuit output, y, can be written as in Equation 5…”

Section: Assumptionsmentioning

confidence: 99%

“…Models were parametrized using plausible values, according to available biological knowledge (e.g., DNA/protein concentrations) and previously published experimental data (e.g., P lux activation curve, resource usage [23,31,37]). A summary of parameters is provided in Table 1 for each model used in this work.…”

Section: Model Parametrizationmentioning

confidence: 99%

“…Circuit output, i.e., RFP synthesis rate per cell (S cell ) at steady-state, was measured for recombinant MG1655-Z1 strain [31] bearing the low-copy plasmid pSB4C5 with X 3 r as insert [23]. In this construct, previously described in [23] and with sequence available as BBa_J107032 code in the Registry of Standard Biological Parts (http://parts.igem.org), luxR is under the control of an anhydrotetracycline (ATc) inducible promoter, which works as gene expression knob in Escherichia coli strains overexpressing TetR, like the one used in this study.…”

Section: In Vivo Experimentsmentioning

confidence: 99%

“…We then analyzed the in vivo data from a previously published experiment, in which the activation curve of a medium copy plasmid-borne lux inducible device was characterized in absence and presence of a second co-transformed plasmid, considered as an additional constant load for the host [31]. Transfer functions were measured in four conditions (with/without load, low/high expression of IPTG-driven LuxR) and a summary of the resulting Hill function parameters is reported in Table 2.…”

Section: Effects Of Cell Loadmentioning

confidence: 99%

“…Differently from Equation (48), the expressions of Q 2 (which can be calculated from Equations (17), (31) and (33)) and y show LuxR level-dependent Hill coefficients. For both Q 2 and y, such features can be observed in the closed-form expressions which can be obtained from M1T (Section 2.2.4).…”

Section: Effects Of Cell Loadmentioning

confidence: 99%

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Mechanistic Models of Inducible Synthetic Circuits for Joint Description of DNA Copy Number, Regulatory Protein Level, and Cell Load

et al. 2019

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Accurate predictive mathematical models are urgently needed in synthetic biology to support the bottom-up design of complex biological systems, minimizing trial-and-error approaches. The majority of models used so far adopt empirical Hill functions to describe activation and repression in exogenously-controlled inducible promoter systems. However, such equations may be poorly predictive in practical situations that are typical in bottom-up design, including changes in promoter copy number, regulatory protein level, and cell load. In this work, we derived novel mechanistic steady-state models of the lux inducible system, used as case study, relying on different assumptions on regulatory protein (LuxR) and cognate promoter (Plux) concentrations, inducer-protein complex formation, and resource usage limitation. We demonstrated that a change in the considered model assumptions can significantly affect circuit output, and preliminary experimental data are in accordance with the simulated activation curves. We finally showed that the models are identifiable a priori (in the analytically tractable cases) and a posteriori, and we determined the specific experiments needed to parametrize them. Although a larger-scale experimental validation is required, in the future the reported models may support synthetic circuits output prediction in practical situations with unprecedented details.

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Section: Assumptionsmentioning

confidence: 99%

Section: Model Parametrizationmentioning

confidence: 99%

Section: In Vivo Experimentsmentioning

confidence: 99%

Section: Effects Of Cell Loadmentioning

confidence: 99%

Section: Effects Of Cell Loadmentioning

confidence: 99%

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Mechanistic Models of Inducible Synthetic Circuits for Joint Description of DNA Copy Number, Regulatory Protein Level, and Cell Load

et al. 2019

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Design and biofabrication of bacterial living materials with robust and multiplexed biosensing capabilities

Usai¹,

Loi²,

Scocozza³

et al. 2023

Materials Today Bio

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Design and Model-Driven Analysis of Synthetic Circuits with the Staphylococcus aureus Dead-Cas9 (sadCas9) as a Programmable Transcriptional Regulator in Bacteria

De Marchi,

Shaposhnikov,

Gobaa

et al. 2024

ACS Synth. Biol.

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Synthetic circuit design is crucial for engineering microbes that process environmental cues and provide biologically relevant outputs. To reliably scale-up circuit complexity, the availability of parts toolkits is central. Streptococcus pyogenes (sp)-derived CRISPR interference/dead-Cas9 (CRISPRi/spdCas9) is widely adopted for implementing programmable regulations in synthetic circuits, and alternative CRISPRi systems will further expand our toolkits of orthogonal components. Here, we showcase the potential of CRISPRi using the engineered dCas9 from Staphylococcus aureus (sadCas9), not previously used in bacterial circuits, that is attractive for its low size and high specificity. We designed a collection of ∼20 increasingly complex circuits and variants in Escherichia coli, including circuits with static function like one-/two-input logic gates (NOT, NAND), circuits with dynamic behavior like incoherent feedforward loops (iFFLs), and applied sadCas9 to fix a T7 polymerase-based cascade. Data demonstrated specific and efficient target repression (100-fold) and qualitatively successful functioning for all circuits. Other advantageous features included low sadCas9-borne cell load and orthogonality with spdCas9. However, different circuit variants showed quantitatively unexpected and previously unreported steady-state responses: the dynamic range, switch point, and slope of NOT/NAND gates changed for different output promoters, and a multiphasic behavior was observed in iFFLs, differing from the expected bell-shaped or sigmoidal curves. Model analysis explained the observed curves by complex interplays among components, due to reporter geneborne cell load and regulator competition. Overall, CRISPRi/sadCas9 successfully expanded the available toolkit for bacterial engineering. Analysis of our circuit collection depicted the impact of generally neglected effects modulating the shape of component dose−response curves, to avoid drawing wrong conclusions on circuit functioning.

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A synthetic close-loop controller circuit for the regulation of an extracellular molecule by engineered bacteria

Cited by 9 publications

References 48 publications

Mechanistic Models of Inducible Synthetic Circuits for Joint Description of DNA Copy Number, Regulatory Protein Level, and Cell Load

Mechanistic Models of Inducible Synthetic Circuits for Joint Description of DNA Copy Number, Regulatory Protein Level, and Cell Load

Design and biofabrication of bacterial living materials with robust and multiplexed biosensing capabilities

Design and Model-Driven Analysis of Synthetic Circuits with the Staphylococcus aureus Dead-Cas9 (sadCas9) as a Programmable Transcriptional Regulator in Bacteria

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