Limits...
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.


A bipartite promoter. Our bipartite promoter is made of fragments of the yeast VPH1 and minimal CYC1 promoters. The former provides the anchor point for RNA polymerases, the latter contains the TSS (see the Additional file1 for their sequences). One or two operators are placed between the TATA box and the TSS such that repressor proteins can bind the DNA and inhibit transcription. TetOp, lacOp, and lexOp sequences are taken, in the order, from[32-34]. We modified the lacOp sequence with a T at the end–when it was placed close to the TATA box (position p1) or it was the only promoter operator–and with an A at the beginning, when it was inserted close to the TSS (position p2). In this way, the three operators used in this work had the same length (19 nucleotides).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: A bipartite promoter. Our bipartite promoter is made of fragments of the yeast VPH1 and minimal CYC1 promoters. The former provides the anchor point for RNA polymerases, the latter contains the TSS (see the Additional file1 for their sequences). One or two operators are placed between the TATA box and the TSS such that repressor proteins can bind the DNA and inhibit transcription. TetOp, lacOp, and lexOp sequences are taken, in the order, from[32-34]. We modified the lacOp sequence with a T at the end–when it was placed close to the TATA box (position p1) or it was the only promoter operator–and with an A at the beginning, when it was inserted close to the TSS (position p2). In this way, the three operators used in this work had the same length (19 nucleotides).

Mentions: Our basic Boolean gates exploit transcriptional repression. They are based on synthetic promoters where repressor operators are placed between the TATA box and the TSS (Transcription Start Site). In this way, it is possible to recreate in yeast the same competition, for the promoter sequence, that takes place in bacterial cells between RNA polymerases and repressors[23]. Once bound to the promoter, repressor proteins prevent RNA polymerase binding and inhibit transcription. Our synthetic promoters are bipartite[18]: they are made of a segment of the yeast VPH1 promoter (containing the TATA box but excluding the two TSS[24]) and another small fragment of the yeast minimal CYC1 promoter (where the TSS is well defined[25]). In between, we placed the repressor operators (see Figure1). Each operator is 19 base-pair long and, when two operators are used, they are always separated by the same three nucleotides (CGT). Bacterial transcription factors have already been extensively used into eukaryotic cells. In literature one can find, for instance, a tetracycline-inducible promoter in Schizosaccharomyces Pombe[26]; a three-input logic gate in mammalian cells where the system tetracycline-tTA is employed[27]; a promoter regulated by LexA in Saccharomices Cerevisiae[28]; and a modified version of the yeast ADH1 promoter able to bind LacI[29].


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

Marchisio MA - J Biol Eng (2014)

A bipartite promoter. Our bipartite promoter is made of fragments of the yeast VPH1 and minimal CYC1 promoters. The former provides the anchor point for RNA polymerases, the latter contains the TSS (see the Additional file1 for their sequences). One or two operators are placed between the TATA box and the TSS such that repressor proteins can bind the DNA and inhibit transcription. TetOp, lacOp, and lexOp sequences are taken, in the order, from[32-34]. We modified the lacOp sequence with a T at the end–when it was placed close to the TATA box (position p1) or it was the only promoter operator–and with an A at the beginning, when it was inserted close to the TSS (position p2). In this way, the three operators used in this work had the same length (19 nucleotides).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: A bipartite promoter. Our bipartite promoter is made of fragments of the yeast VPH1 and minimal CYC1 promoters. The former provides the anchor point for RNA polymerases, the latter contains the TSS (see the Additional file1 for their sequences). One or two operators are placed between the TATA box and the TSS such that repressor proteins can bind the DNA and inhibit transcription. TetOp, lacOp, and lexOp sequences are taken, in the order, from[32-34]. We modified the lacOp sequence with a T at the end–when it was placed close to the TATA box (position p1) or it was the only promoter operator–and with an A at the beginning, when it was inserted close to the TSS (position p2). In this way, the three operators used in this work had the same length (19 nucleotides).
Mentions: Our basic Boolean gates exploit transcriptional repression. They are based on synthetic promoters where repressor operators are placed between the TATA box and the TSS (Transcription Start Site). In this way, it is possible to recreate in yeast the same competition, for the promoter sequence, that takes place in bacterial cells between RNA polymerases and repressors[23]. Once bound to the promoter, repressor proteins prevent RNA polymerase binding and inhibit transcription. Our synthetic promoters are bipartite[18]: they are made of a segment of the yeast VPH1 promoter (containing the TATA box but excluding the two TSS[24]) and another small fragment of the yeast minimal CYC1 promoter (where the TSS is well defined[25]). In between, we placed the repressor operators (see Figure1). Each operator is 19 base-pair long and, when two operators are used, they are always separated by the same three nucleotides (CGT). Bacterial transcription factors have already been extensively used into eukaryotic cells. In literature one can find, for instance, a tetracycline-inducible promoter in Schizosaccharomyces Pombe[26]; a three-input logic gate in mammalian cells where the system tetracycline-tTA is employed[27]; a promoter regulated by LexA in Saccharomices Cerevisiae[28]; and a modified version of the yeast ADH1 promoter able to bind LacI[29].

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.