Development and experimental validation of a mechanistic model of a recombinase-based temporal logic gate

Bowyer, Jack; Hsiao, Victoria; Bates, Declan G.

doi:10.1109/biocas.2016.7833832

Cited by 3 publications

(6 citation statements)

References 15 publications

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“…The parameter K ON is a combined parameter that is a function of the dissociation constant of integrase dimerization and the dissociation constant of integrase dimer binding to each DNA recognition site. More complex models of DNA recombination exist , and may be used to expand our model in the future. However, at present we have utilized a simple hill function to model recombination due to the small number of free parameters it introduces, and to reduce computational complexity.…”

Section: Methodsmentioning

confidence: 99%

A Synthetic Recombinase-Based Feedback Loop Results in Robust Expression

et al. 2017

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Accurate control of a biological process is essential for many critical functions in biology, from the cell cycle to proteome regulation. To achieve this, negative feedback is frequently employed to provide a highly robust and reliable output. Feedback is found throughout biology and technology, but due to challenges posed by its implementation, it is yet to be widely adopted in synthetic biology. In this paper we design a synthetic feedback network using a class of recombinase proteins called integrases, which can be re-engineered to flip the orientation of DNA segments in a digital manner. This system is highly orthogonal, and demonstrates a strong capability for regulating and reducing the expression variability of genes being transcribed under its control. An excisionase protein provides the negative feedback signal to close the loop in this system, by flipping DNA segments in the reverse direction. Our integrase/excisionase negative feedback system thus provides a modular architecture that can be tuned to suit applications throughout synthetic biology and biomanufacturing that require a highly robust and orthogonally controlled output.

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Section: Methodsmentioning

confidence: 99%

A Synthetic Recombinase-Based Feedback Loop Results in Robust Expression

et al. 2017

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“…By analysing the criteria required to mediate both highly efficient integration and excision in silico, we have identified a set of optimal system inputs. For example, our model exhibits higher efficiency associated with integration compared with excision, and also reveals the operative cycles required to realise the desired digital response [18,19].…”

Section: Engineering Cellular Memory Using Dna Recombinationmentioning

confidence: 99%

“…4. We also include the formation of a dysfunctional dimer by both integrases, which is subject to the same degradation as its functional counterpart; this was shown to significantly improve the fit of model simulations to experimental data in our previous study [18,19]. Deletion gives rise to two distinct genetic products, the remaining linear sequence of the target DNA and the excised circular DNA loop, and hence we account for two disparate S b states, S bl (linear) and S bc (circular).…”

Section: Constructing a Mechanistic Model Of The Temporal Logic Gatementioning

confidence: 99%

“…Sequence of DNA:protein interactions facilitating integration, taken from [18], enables the system to transition from the original DNA state (state 1) to the three genetically differentiated DNA states (state 2, state 3 and state 4). Expression and degradation of integrases A and B are denoted by g bas, ind and d, respectively.…”

Section: Fig 4 Schematic Diagram Of the Mechanistic Two-input Logic mentioning

confidence: 99%

“…S ab I 4B Reaction rates are described by the corresponding numbered k, which are retained from [18,19] where necessary. S denotes DNA with subscripts corresponding to the four distinct states (0, a, bl and ab); I denotes integrase with subscripts corresponding to the number of monomers (1, 2, 3 and 4), TP901-1 or Bxb1 (A or B) and/or dysfunctionality (X).…”

Section: Referencesmentioning

confidence: 99%

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Mechanistic modelling of a recombinase‐based two‐input temporal logic gate

et al. 2017

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Site-specific recombinases (SSRs) mediate efficient manipulation of DNA sequences in vitro and in vivo. In particular, serine integrases have been identified as highly effective tools for facilitating DNA inversion, enabling the design of genetic switches that are capable of turning the expression of a gene of interest on or off in the presence of a SSR protein. The functional scope of such circuitry can be extended to biological Boolean logic operations by incorporating two or more distinct integrase inputs. To date, mathematical modelling investigations have captured the dynamical properties of integrase logic gate systems in a purely qualitative manner, and thus such models are of limited utility as tools in the design of novel circuitry. Here, the authors develop a detailed mechanistic model of a twoinput temporal logic gate circuit that can detect and encode sequences of input events. Their model demonstrates quantitative agreement with time-course data on the dynamics of the temporal logic gate, and is shown to subsequently predict dynamical responses relating to a series of induction separation intervals. The model can also be used to infer functional variations between distinct integrase inputs, and to examine the effect of reversing the roles of each integrase on logic gate output.

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Tuning and functionalization of logic gates for time resolved programming of bacterial populations

Bäcker,

Broux,

Weckx

et al. 2024

Preprint

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In order to increase our command over genetically engineered bacterial populations in bioprocessing and therapy, synthetic regulatory circuitry needs to enable the temporal programming of a number of consecutive functional tasks without external interventions. In this context, we have engineered a genetic circuit encoding an autonomous but chemically tunable timer in Escherichia coli, based on the concept of a transcription factor cascade mediated by the cytoplasmic dilution of repressors. As proof-of-concept, we used this circuit to impose a time-resolved two-staged synthetic pathway composed of a production-followed-by-lysis program, via a single input. Moreover, via a recombinase step, this synchronous timer was further engineered into an asynchronous timer in which the generational distance of differentiating daughter cells spawning off from a stem-cell like mother cell becomes a predictable driver and proxy for timer dynamics. Using this asynchronous timer circuit, a temporally defined population heterogeneity can be programmed in bacterial populations.

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Development and experimental validation of a mechanistic model of a recombinase-based temporal logic gate

Cited by 3 publications

References 15 publications

A Synthetic Recombinase-Based Feedback Loop Results in Robust Expression

A Synthetic Recombinase-Based Feedback Loop Results in Robust Expression

Mechanistic modelling of a recombinase‐based two‐input temporal logic gate

Tuning and functionalization of logic gates for time resolved programming of bacterial populations

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