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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.


Related in: MedlinePlus

AND gates responsive to tetracycline and IPTG. A) Single integration. B) Triple integration (AND lacOp-tetOp configuration). Beside the expected increase in signal separation, the three 0 outputs are lower than in the single integration case. This is probably due to an accidental multiple integration of the LacI gene.
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Figure 5: AND gates responsive to tetracycline and IPTG. A) Single integration. B) Triple integration (AND lacOp-tetOp configuration). Beside the expected increase in signal separation, the three 0 outputs are lower than in the single integration case. This is probably due to an accidental multiple integration of the LacI gene.

Mentions: Tet operator positional effects on transcription regulation are even more evident in the "tet AND IPTG" gate (see Figure5A). In this case, only the AND tetOp-lacOp configuration reproduces the AND truth table properly. Here, although the 1 output level is clearly lower than the fluorescence of the open gate (φ = 0.73), σ remains fairly high (2035 AU). In contrast, the AND lacOp-tetOp implementation presents an excess of fluorescence in correspondence to the truth table entry 01 (no tetracycline and 40 mM IPTG). This value is practically indistinguishable from the 1 output level measured for the 11 entry. When this gate is induced with IPTG only (01), the whole promoter regulation is due to the sole TetR whose action, however, is clearly too weak when it binds far from the TATA box. As in the YES lacOp2 case, we managed to engineer a working AND gate with the lacOp-tetOp bipartite promoter configuration via a multiple (triple) integration of the reporter-protein-encoding transcription unit (AND lacOp-tetOp-TI). In this way, the 1 output fluorescence increased highly which permitted to achieve a signal separation (2017.5 AU) comparable to the one measured for the AND tetOp-lacOp configuration (see Figure5B). However, this implementation gives the lowest value for both ρ and φ. Moreover, a general decrease in the 0 outputs is probably imputable to an accidental multiple integration of the LacI gene (see the computational analysis below).


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

Marchisio MA - J Biol Eng (2014)

AND gates responsive to tetracycline and IPTG. A) Single integration. B) Triple integration (AND lacOp-tetOp configuration). Beside the expected increase in signal separation, the three 0 outputs are lower than in the single integration case. This is probably due to an accidental multiple integration of the LacI gene.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: AND gates responsive to tetracycline and IPTG. A) Single integration. B) Triple integration (AND lacOp-tetOp configuration). Beside the expected increase in signal separation, the three 0 outputs are lower than in the single integration case. This is probably due to an accidental multiple integration of the LacI gene.
Mentions: Tet operator positional effects on transcription regulation are even more evident in the "tet AND IPTG" gate (see Figure5A). In this case, only the AND tetOp-lacOp configuration reproduces the AND truth table properly. Here, although the 1 output level is clearly lower than the fluorescence of the open gate (φ = 0.73), σ remains fairly high (2035 AU). In contrast, the AND lacOp-tetOp implementation presents an excess of fluorescence in correspondence to the truth table entry 01 (no tetracycline and 40 mM IPTG). This value is practically indistinguishable from the 1 output level measured for the 11 entry. When this gate is induced with IPTG only (01), the whole promoter regulation is due to the sole TetR whose action, however, is clearly too weak when it binds far from the TATA box. As in the YES lacOp2 case, we managed to engineer a working AND gate with the lacOp-tetOp bipartite promoter configuration via a multiple (triple) integration of the reporter-protein-encoding transcription unit (AND lacOp-tetOp-TI). In this way, the 1 output fluorescence increased highly which permitted to achieve a signal separation (2017.5 AU) comparable to the one measured for the AND tetOp-lacOp configuration (see Figure5B). However, this implementation gives the lowest value for both ρ and φ. Moreover, a general decrease in the 0 outputs is probably imputable to an accidental multiple integration of the LacI gene (see the computational analysis below).

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.


Related in: MedlinePlus