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In silico design and in vivo implementation of yeast gene Boolean gates.

Marchisio MA - J Biol Eng (2014)

Bottom Line: Moreover, model-driven considerations permitted us to pinpoint a strategy for re-designing gates when a better digital performance is required.Our library of well-characterized Boolean gates is the basis for the assembly of more complex gene digital circuits.As a proof of concepts, we engineered two 2-input OR gates, designed by our software, by combining YES and NOT gates present in our library.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biosystems Science and Engineering (D-BSSE), ETH Zurich, Mattenstrasse 26, Basel 4058, Switzerland. marchisio@hit.edu.cn.

ABSTRACT
In our previous computational work, we showed that gene digital circuits can be automatically designed in an electronic fashion. This demands, first, a conversion of the truth table into Boolean formulas with the Karnaugh map method and, then, the translation of the Boolean formulas into circuit schemes organized into layers of Boolean gates and Pools of signal carriers. In our framework, gene digital circuits that take up to three different input signals (chemicals) arise from the composition of three kinds of basic Boolean gates, namely YES, NOT, and AND. Here we present a library of YES, NOT, and AND gates realized via plasmidic DNA integration into the yeast genome. Boolean behavior is reproduced via the transcriptional control of a synthetic bipartite promoter that contains sequences of the yeast VPH1 and minimal CYC1 promoters together with operator binding sites for bacterial (i.e. orthogonal) repressor proteins. Moreover, model-driven considerations permitted us to pinpoint a strategy for re-designing gates when a better digital performance is required. Our library of well-characterized Boolean gates is the basis for the assembly of more complex gene digital circuits. As a proof of concepts, we engineered two 2-input OR gates, designed by our software, by combining YES and NOT gates present in our library.

No MeSH data available.


IPTG responsive YES gates. A) Single integration. B) lacOp2 double integration. The initial non-working YES gate based on two lac operators was rescued via a double integration of the transcription unit producing Citrine.
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Figure 3: IPTG responsive YES gates. A) Single integration. B) lacOp2 double integration. The initial non-working YES gate based on two lac operators was rescued via a double integration of the transcription unit producing Citrine.

Mentions: We found a similar trend in the IPTG-LacI system: a single lac operator (YES lacOp) gives a higher signal separation (4930 AU) whereas two lac operators permit to switch off fluorescence almost completely (see Figure3A). However, the YES lacOp2 configuration presents a drawback: the bipartite promoter cannot be re-activated with 40 mM IPTG (higher concentrations were also of no use–data not shown). Indeed, the 1 output corresponds to only about one quarter of the open gate fluorescence and the signal separation (471 AU) is not high enough to determine a precise threshold between 0 and 1 fluorescent signals. According to our digital circuit computational analysis in[12], σ can be improved by increasing the transcription initiation rate of the promoter that leads to reporter protein production. Here, instead of re-engineering the lacOp2-containing bipartite promoter, we simply double integrated (DI) the transcription unit hosting it in order to mimic an increase of transcription initiation rate. As it is shown in Figure3B, this procedure permitted to boost the 1 output (though still far from the YES lacOp2-DI open gate fluorescence that amounts to 4800.5 AU) without a significant increment in the 0 one. This allowed fixing an unequivocal 0/1 threshold at 500 AU.


In silico design and in vivo implementation of yeast gene Boolean gates.

Marchisio MA - J Biol Eng (2014)

IPTG responsive YES gates. A) Single integration. B) lacOp2 double integration. The initial non-working YES gate based on two lac operators was rescued via a double integration of the transcription unit producing Citrine.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3926364&req=5

Figure 3: IPTG responsive YES gates. A) Single integration. B) lacOp2 double integration. The initial non-working YES gate based on two lac operators was rescued via a double integration of the transcription unit producing Citrine.
Mentions: We found a similar trend in the IPTG-LacI system: a single lac operator (YES lacOp) gives a higher signal separation (4930 AU) whereas two lac operators permit to switch off fluorescence almost completely (see Figure3A). However, the YES lacOp2 configuration presents a drawback: the bipartite promoter cannot be re-activated with 40 mM IPTG (higher concentrations were also of no use–data not shown). Indeed, the 1 output corresponds to only about one quarter of the open gate fluorescence and the signal separation (471 AU) is not high enough to determine a precise threshold between 0 and 1 fluorescent signals. According to our digital circuit computational analysis in[12], σ can be improved by increasing the transcription initiation rate of the promoter that leads to reporter protein production. Here, instead of re-engineering the lacOp2-containing bipartite promoter, we simply double integrated (DI) the transcription unit hosting it in order to mimic an increase of transcription initiation rate. As it is shown in Figure3B, this procedure permitted to boost the 1 output (though still far from the YES lacOp2-DI open gate fluorescence that amounts to 4800.5 AU) without a significant increment in the 0 one. This allowed fixing an unequivocal 0/1 threshold at 500 AU.

Bottom Line: Moreover, model-driven considerations permitted us to pinpoint a strategy for re-designing gates when a better digital performance is required.Our library of well-characterized Boolean gates is the basis for the assembly of more complex gene digital circuits.As a proof of concepts, we engineered two 2-input OR gates, designed by our software, by combining YES and NOT gates present in our library.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biosystems Science and Engineering (D-BSSE), ETH Zurich, Mattenstrasse 26, Basel 4058, Switzerland. marchisio@hit.edu.cn.

ABSTRACT
In our previous computational work, we showed that gene digital circuits can be automatically designed in an electronic fashion. This demands, first, a conversion of the truth table into Boolean formulas with the Karnaugh map method and, then, the translation of the Boolean formulas into circuit schemes organized into layers of Boolean gates and Pools of signal carriers. In our framework, gene digital circuits that take up to three different input signals (chemicals) arise from the composition of three kinds of basic Boolean gates, namely YES, NOT, and AND. Here we present a library of YES, NOT, and AND gates realized via plasmidic DNA integration into the yeast genome. Boolean behavior is reproduced via the transcriptional control of a synthetic bipartite promoter that contains sequences of the yeast VPH1 and minimal CYC1 promoters together with operator binding sites for bacterial (i.e. orthogonal) repressor proteins. Moreover, model-driven considerations permitted us to pinpoint a strategy for re-designing gates when a better digital performance is required. Our library of well-characterized Boolean gates is the basis for the assembly of more complex gene digital circuits. As a proof of concepts, we engineered two 2-input OR gates, designed by our software, by combining YES and NOT gates present in our library.

No MeSH data available.