Mechanistic Modeling of a Rewritable Recombinase Addressable Data Module

Bowyer, Jack; Zhao, Jia; Subsoontorn, Pakpoom; Wong, Wilson; Rosser, Susan J.; Bates, Declan G.

doi:10.1109/tbcas.2016.2526668

Cited by 10 publications

(15 citation statements)

References 60 publications

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“…Thus, the likelihood of a productive collision beween activated att L and att R would also be low, resulting in little or no LxR recombination observed. Future studies will focus on testing this mechanism quantitatively, as done for other systems (39–41). …”

Section: Discussionmentioning

confidence: 99%

Coiled-coil interactions mediate serine integrase directionality

Gupta

Sharp

Yuan

et al. 2017

Nucleic Acids Research

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Serine integrases are bacteriophage enzymes that carry out site-specific integration and excision of their viral genomes. The integration reaction is highly directional; recombination between the phage attachment site attP and the host attachment site attB to form the hybrid sites attL and attR is essentially irreversible. In a recent model, extended coiled-coil (CC) domains in the integrase subunits are proposed to interact in a way that favors the attPxattB reaction but inhibits the attLxattR reaction. Here, we show for the Listeria innocua integrase (LI Int) system that the CC domain promotes self-interaction in isolated Int and when Int is bound to attachment sites. Three independent crystal structures of the CC domain reveal the molecular nature of the CC dimer interface. Alanine substitutions of key residues in the interface support the functional significance of the structural model and indicate that the same interaction is responsible for promoting integration and for inhibiting excision. An updated model of a LI Int•attL complex that incorporates the high resolution CC dimer structure provides insights that help to explain the unusual CC dimer structure and potential sources of stability in Int•attL and Int•attR complexes. Together, the data provide a molecular basis for understanding serine integrase directionality.

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Section: Discussionmentioning

confidence: 99%

Coiled-coil interactions mediate serine integrase directionality

Gupta

Sharp

Yuan

et al. 2017

Nucleic Acids Research

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“…Although dCas9 allows for programmable interconnections, its response function is leaky leading to signal degradation when layered 18 . Site-specific recombinases have been employed in genetic circuits as a means to reduce leak 35 36 37 , but there are a limited number of such enzymes restricting the scalability of this approach. Here, we address these issues, advancing the art of engineering living digital circuits by focusing on two main engineering goals.…”

mentioning

confidence: 99%

Digital logic circuits in yeast with CRISPR-dCas9 NOR gates

et al. 2017

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Natural genetic circuits enable cells to make sophisticated digital decisions. Building equally complex synthetic circuits in eukaryotes remains difficult, however, because commonly used components leak transcriptionally, do not arbitrarily interconnect or do not have digital responses. Here, we designed dCas9-Mxi1-based NOR gates in Saccharomyces cerevisiae that allow arbitrary connectivity and large genetic circuits. Because we used the chromatin remodeller Mxi1, our gates showed minimal leak and digital responses. We built a combinatorial library of NOR gates that directly convert guide RNA (gRNA) inputs into gRNA outputs, enabling the gates to be ‘wired' together. We constructed logic circuits with up to seven gRNAs, including repression cascades with up to seven layers. Modelling predicted the NOR gates have effectively zero transcriptional leak explaining the limited signal degradation in the circuits. Our approach enabled the largest, eukaryotic gene circuits to date and will form the basis for large, synthetic, cellular decision-making systems.

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“…This dynamical behaviour defines a truth table for the standard two-input biological logic gate [Table 1(a)]. State transitioning is a unidirectional process unless excisionase-mediated reset functions are incorporated; however, this will invariably compromise the dynamical behaviour of the logic gate or toggle switch in question [9,19].…”

Section: Biological Boolean Logicmentioning

confidence: 99%

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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Mechanistic Modeling of a Rewritable Recombinase Addressable Data Module

Cited by 10 publications

References 60 publications

Coiled-coil interactions mediate serine integrase directionality

Coiled-coil interactions mediate serine integrase directionality

Digital logic circuits in yeast with CRISPR-dCas9 NOR gates

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

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