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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.


YES lacOp2-multiple integration analysis. Experimental data (exp) and computational results (cpu) show a good agreement concerning Citrine single and double integration both in the close (IPTG = 0 and IPTG = 1) and the open YES gate. Citrine double integration is equivalent to an increase of the transcription initiation rate (k2) of pLacOp2 from 0.1 to 0.218s-1.
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Figure 6: YES lacOp2-multiple integration analysis. Experimental data (exp) and computational results (cpu) show a good agreement concerning Citrine single and double integration both in the close (IPTG = 0 and IPTG = 1) and the open YES gate. Citrine double integration is equivalent to an increase of the transcription initiation rate (k2) of pLacOp2 from 0.1 to 0.218s-1.

Mentions: We had to include in our model LacI cooperative interactions in order to mimic YES lacOp2 gate fluorescence levels. In our computational framework we had to assign to the two lac operators different affinities towards LacI molecules. The weaker operator (i.e. the one close to the TSS box) is characterized by the same binding rate constant as in the pLacOp case (αw = α). The stronger operator shows, in contrast, an almost 10 fold higher affinity (αs = 7.1 107M-1s-1). When the operator close to the TATA box is taken by LacI, αw is increased the to αs value. Moreover, as a consequence of the stronger bond to the DNA, we had to drastically lower the value of γ (more than 130 folds) used for pLacOp. With this choice of parameter values, a double concentration of pLacOp2 in the closed gate configuration gives results fairly near to the experimental data (see Figure6). Moreover, we calculated that a double integration has the same effect as an increase of pLacOp2 transcription rate from 0.1 to 0.218s-1. This proves that multiple gene integration is a valid (and easier to achieve) alternative to promoter re-engineering.


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

Marchisio MA - J Biol Eng (2014)

YES lacOp2-multiple integration analysis. Experimental data (exp) and computational results (cpu) show a good agreement concerning Citrine single and double integration both in the close (IPTG = 0 and IPTG = 1) and the open YES gate. Citrine double integration is equivalent to an increase of the transcription initiation rate (k2) of pLacOp2 from 0.1 to 0.218s-1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: YES lacOp2-multiple integration analysis. Experimental data (exp) and computational results (cpu) show a good agreement concerning Citrine single and double integration both in the close (IPTG = 0 and IPTG = 1) and the open YES gate. Citrine double integration is equivalent to an increase of the transcription initiation rate (k2) of pLacOp2 from 0.1 to 0.218s-1.
Mentions: We had to include in our model LacI cooperative interactions in order to mimic YES lacOp2 gate fluorescence levels. In our computational framework we had to assign to the two lac operators different affinities towards LacI molecules. The weaker operator (i.e. the one close to the TSS box) is characterized by the same binding rate constant as in the pLacOp case (αw = α). The stronger operator shows, in contrast, an almost 10 fold higher affinity (αs = 7.1 107M-1s-1). When the operator close to the TATA box is taken by LacI, αw is increased the to αs value. Moreover, as a consequence of the stronger bond to the DNA, we had to drastically lower the value of γ (more than 130 folds) used for pLacOp. With this choice of parameter values, a double concentration of pLacOp2 in the closed gate configuration gives results fairly near to the experimental data (see Figure6). Moreover, we calculated that a double integration has the same effect as an increase of pLacOp2 transcription rate from 0.1 to 0.218s-1. This proves that multiple gene integration is a valid (and easier to achieve) alternative to promoter re-engineering.

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.