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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.

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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.


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Single-input gates. A) Tetracycline responsive YES gates. B) β-estradiol sensing NOT gates. In both cases, a single operator guarantees higher signal separation whereas two operators permit to achieve a stronger repression. Fluorescence levels of the open gates (indicated with their yeast strain name–see Additional file1) correspond to blue columns. Gates’ schemes are drawn with SBOL[36] icons (all the symbols used throughout the paper are shown in Additional file1: Figure S1).
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Figure 2: Single-input gates. A) Tetracycline responsive YES gates. B) β-estradiol sensing NOT gates. In both cases, a single operator guarantees higher signal separation whereas two operators permit to achieve a stronger repression. Fluorescence levels of the open gates (indicated with their yeast strain name–see Additional file1) correspond to blue columns. Gates’ schemes are drawn with SBOL[36] icons (all the symbols used throughout the paper are shown in Additional file1: Figure S1).

Mentions: Both configurations of the tetracycline-based YES gate show a high signal separation (4601 AU with a single tet operator, 3640 AU with two tet operators–see Figure2A) that permits to unequivocally distinguish between 0 and 1 output signals (no overlap of the error bars) and place the 0/1 threshold at 4000 AU (YES tetOp case) and 2500 AU (YES tetOp2). The single tet operator configuration, however, cannot achieve a strong repression. This causes a rather low 1 to 0 gain (ρ = 3.42) that is almost doubled in the tetOp2 design where, as expected[35], repression is much stronger. Finally, both variants have φ around 1. Therefore, we detected a high affinity between tetracycline and TetR and a moderate affinity between TetR and its operator binding site.


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

Marchisio MA - J Biol Eng (2014)

Single-input gates. A) Tetracycline responsive YES gates. B) β-estradiol sensing NOT gates. In both cases, a single operator guarantees higher signal separation whereas two operators permit to achieve a stronger repression. Fluorescence levels of the open gates (indicated with their yeast strain name–see Additional file1) correspond to blue columns. Gates’ schemes are drawn with SBOL[36] icons (all the symbols used throughout the paper are shown in Additional file1: Figure S1).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Single-input gates. A) Tetracycline responsive YES gates. B) β-estradiol sensing NOT gates. In both cases, a single operator guarantees higher signal separation whereas two operators permit to achieve a stronger repression. Fluorescence levels of the open gates (indicated with their yeast strain name–see Additional file1) correspond to blue columns. Gates’ schemes are drawn with SBOL[36] icons (all the symbols used throughout the paper are shown in Additional file1: Figure S1).
Mentions: Both configurations of the tetracycline-based YES gate show a high signal separation (4601 AU with a single tet operator, 3640 AU with two tet operators–see Figure2A) that permits to unequivocally distinguish between 0 and 1 output signals (no overlap of the error bars) and place the 0/1 threshold at 4000 AU (YES tetOp case) and 2500 AU (YES tetOp2). The single tet operator configuration, however, cannot achieve a strong repression. This causes a rather low 1 to 0 gain (ρ = 3.42) that is almost doubled in the tetOp2 design where, as expected[35], repression is much stronger. Finally, both variants have φ around 1. Therefore, we detected a high affinity between tetracycline and TetR and a moderate affinity between TetR and its operator binding site.

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