Design and evaluation of an incoherent feed-forward loop for an arsenic biosensor based on standard iGEM parts

Barone, Federico; Dorr, Francisco; Marasco, Luciano E.; Mildiner, S; Patop, Inés Lucía; Sosa, Santiago; Vattino, Lucas G.; Vignale, Federico A; Altszyler, Edgar; Basanta, Benjamin; Carlotto, Nicolás; Gasulla, Javier; M, Gimenez; Grande, Alicia; Moreno, Nicolás Nieto; Bonomi, Hernán Ruy; Nadra, Alejandro D.

doi:10.1093/synbio/ysx006

Cited by 14 publications

(17 citation statements)

References 15 publications

(22 reference statements)

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“…There have been a number of synthetic I1-FFLs built using protein regulators (Barone et al, 2017;Cheng, Hirning, Josić, & Bennett, 2017;Entus et al, 2007). Recently, we built a RNA-based I1-FFL that uses N-acyl homoserine lactone to activate expression of a STAR RNA that activates expression of monomeric red fluorescent protein (mRFP) as well as a gRNA and dCas9 that repress mRFP (Chappell et al, 2017).…”

Section: Discussionmentioning

confidence: 99%

“…The I1-FFL is a common network motif in natural bacterial networks (Alon, 2013;Milo et al, 2002;Shen-Orr, Milo, Mangan, & Alon, 2002) and has received much interest due to its ability to produce a pulse of gene expression (Basu, Mehreja, Thiberge, Chen, & Weiss, 2004;Mangan & Alon, 2003) and accelerate the response time (Mangan, Itzkovitz, Zaslaver, & Alon, 2006). I1-FFLs have also been used to implement band-pass filters (Entus, Aufderheide, & Sauro, 2007;Kaplan, Bren, Dekel, & Alon, 2008), fold-change detection (Goentoro, Shoval, Kirschner, & Alon, 2009), biosensing (Barone et al, 2017), and noise buffering (Osella, Bosia, Corá, & Caselle, 2011). An I1-FFL consists of an activator X that activates a gene Z and simultaneously its repressor, Y (Figure 1c).…”

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confidence: 99%

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Distinct timescales of RNA regulators enable the construction of a genetic pulse generator

Westbrook

Tang

Marshall

et al. 2019

Biotech & Bioengineering

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To build complex genetic networks with predictable behaviors, synthetic biologists use libraries of modular parts that can be characterized in isolation and assembled together to create programmable higher‐order functions. Characterization experiments and computational models for gene regulatory parts operating in isolation are routinely used to predict the dynamics of interconnected parts and guide the construction of new synthetic devices. Here, we individually characterize two modes of RNA‐based transcriptional regulation, using small transcription activating RNAs (STARs) and clustered regularly interspaced short palindromic repeats interference (CRISPRi), and show how their distinct regulatory timescales can be used to engineer a composed feedforward loop that creates a pulse of gene expression. We use a cell‐free transcription‐translation system (TXTL) to rapidly characterize the system, and we apply Bayesian inference to extract kinetic parameters for an ordinary differential equation‐based mechanistic model. We then demonstrate in simulation and verify with TXTL experiments that the simultaneous regulation of a single gene target with STARs and CRISPRi leads to a pulse of gene expression. Our results suggest the modularity of the two regulators in an integrated genetic circuit, and we anticipate that construction and modeling frameworks that can leverage this modularity will become increasingly important as synthetic circuits increase in complexity.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Distinct timescales of RNA regulators enable the construction of a genetic pulse generator

Westbrook

Tang

Marshall

et al. 2019

Biotech & Bioengineering

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“…The success of using mathematical modeling to quantify and guide the design of synthetic gene circuits has been reported in various systems from bacteria to mammalian cells [29][30][31][32][33]. Given previous successes with model-guided design with the evaluation of the gene pulse generator and IFFL circuits [25,[34][35][36], here, we develop ordinary differential equation (ODE)-based mathematical models to first explore the capability of an RNA-only circuit functioning as an IFFL circuit for pulse generation and then, to guide the design and experimental realization of an RNA-protein hybrid IFFL circuit with predictable dynamics.…”

Section: Plasmid Construction and E Coli Strains Usedmentioning

confidence: 99%

Design and Evaluation of Synthetic RNA-Based Incoherent Feed-Forward Loop Circuits

et al. 2021

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RNA-based regulators are promising tools for building synthetic biological systems that provide a powerful platform for achieving a complex regulation of transcription and translation. Recently, de novo-designed synthetic RNA regulators, such as the small transcriptional activating RNA (STAR), toehold switch (THS), and three-way junction (3WJ) repressor, have been utilized to construct RNA-based synthetic gene circuits in living cells. In this work, we utilized these regulators to construct type 1 incoherent feed-forward loop (IFFL) circuits in vivo and explored their dynamic behaviors. A combination of a STAR and 3WJ repressor was used to construct an RNA-only IFFL circuit. However, due to the fast kinetics of RNA–RNA interactions, there was no significant timescale difference between the direct activation and the indirect inhibition, that no pulse was observed in the experiments. These findings were confirmed with mechanistic modeling and simulation results for a wider range of conditions. To increase delay in the inhibition pathway, we introduced a protein synthesis process to the circuit and designed an RNA–protein hybrid IFFL circuit using THS and TetR protein. Simulation results indicated that pulse generation could be achieved with this RNA–protein hybrid model, and this was further verified with experimental realization in E. coli. Our findings demonstrate that while RNA-based regulators excel in speed as compared to protein-based regulators, the fast reaction kinetics of RNA-based regulators could also undermine the functionality of a circuit (e.g., lack of significant timescale difference). The agreement between experiments and simulations suggests that the mechanistic modeling can help debug issues and validate the hypothesis in designing a new circuit. Moreover, the applicability of the kinetic parameters extracted from the RNA-only circuit to the RNA–protein hybrid circuit also indicates the modularity of RNA-based regulators when used in a different context. We anticipate the findings of this work to guide the future design of gene circuits that rely heavily on the dynamics of RNA-based regulators, in terms of both modeling and experimental realization.

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“…There have been a number of synthetic I1-FFLs built using protein regulators. 23,26,46 Recently, we built an RNAbased I1-FFL that uses AHL to activate expression of a STAR RNA that activates expression of mRFP as well as a gRNA and dCas9 that repress mRFP. 2 This design relies on an additional RNA cleavage strategy, cascading RNA regulatory events, and slow dCas9 production.…”

Section: Discussionmentioning

confidence: 99%

“…17 The I1-FFL is a common network motif in natural bacterial networks [18][19][20] and has received much interest due to its ability to produce a pulse of gene expression 17,21 and accelerate the response time. 22 I1-FFLs have also been used to implement band-pass filters, 23,24 fold-change detection, 25 biosensing, 26 and noise buffering. 27 An I1-FFL consists of an activator X that activates a gene Z and simultaneously its repressor, Y ( Figure 1C).…”

Section: Introductionmentioning

confidence: 99%

Distinct timescales of RNA regulators enable the construction of a genetic pulse generator

Westbrook

Tang

Marshall

et al. 2018

Preprint

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To build complex genetic networks with predictable behaviours, synthetic biologists use libraries of modular parts that can be characterized in isolation and assembled together to create programmable higher-order functions. Characterization experiments and computational models for gene regulatory parts operating in isolation are routinely employed to predict the dynamics of interconnected parts and guide the construction of new synthetic devices. Here, we individually characterize two modes of RNA-based transcriptional regulation, using small transcription activating RNAs (STARs) and CRISPR interference (CRISPRi), and show how their distinct regulatory timescales can be used to engineer a composed feedforward loop that creates a pulse of gene expression. We use a cell-free transcription-translation system (TXTL) to rapidly characterize the system, and we apply Bayesian inference to extract kinetic parameters for an ODE-based mechanistic model. We then demonstrate in simulation and verify with TXTL experiments that the simultaneous regulation of a single gene target with STARs and CRISPRi leads to a pulse of gene expression. Our results suggest the modularity of the two regulators in an integrated genetic circuit, and we anticipate that construction and modeling frameworks that can leverage this modularity will become increasingly important as synthetic circuits increase in complexity.

show abstract

Design and evaluation of an incoherent feed-forward loop for an arsenic biosensor based on standard iGEM parts

Cited by 14 publications

References 15 publications

Distinct timescales of RNA regulators enable the construction of a genetic pulse generator

Distinct timescales of RNA regulators enable the construction of a genetic pulse generator

Design and Evaluation of Synthetic RNA-Based Incoherent Feed-Forward Loop Circuits

Distinct timescales of RNA regulators enable the construction of a genetic pulse generator

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