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A formalized design process for bacterial consortia that perform logic computing.

Ji W, Shi H, Zhang H, Sun R, Xi J, Wen D, Feng J, Chen Y, Qin X, Ma Y, Luo W, Deng L, Lin H, Yu R, Ouyang Q - PLoS ONE (2013)

Bottom Line: Despite of all its benefits, however, there are still problems remaining for large-scaled multicellular gene circuits, for example, how to reliably design and distribute the circuits in microbial consortia with limited number of well-behaved genetic modules and wiring quorum-sensing molecules.The construction and characterization of logic operators is independent of "wiring" and provides predictive information for fine-tuning.This formalized design process provides guidance for the design of microbial consortia that perform distributed biological computation.

View Article: PubMed Central - PubMed

Affiliation: Peking University Team for the International Genetically Engineered Machine Competition (iGEM), Peking University, Beijing, China.

ABSTRACT
The concept of microbial consortia is of great attractiveness in synthetic biology. Despite of all its benefits, however, there are still problems remaining for large-scaled multicellular gene circuits, for example, how to reliably design and distribute the circuits in microbial consortia with limited number of well-behaved genetic modules and wiring quorum-sensing molecules. To manage such problem, here we propose a formalized design process: (i) determine the basic logic units (AND, OR and NOT gates) based on mathematical and biological considerations; (ii) establish rules to search and distribute simplest logic design; (iii) assemble assigned basic logic units in each logic operating cell; and (iv) fine-tune the circuiting interface between logic operators. We in silico analyzed gene circuits with inputs ranging from two to four, comparing our method with the pre-existing ones. Results showed that this formalized design process is more feasible concerning numbers of cells required. Furthermore, as a proof of principle, an Escherichia coli consortium that performs XOR function, a typical complex computing operation, was designed. The construction and characterization of logic operators is independent of "wiring" and provides predictive information for fine-tuning. This formalized design process provides guidance for the design of microbial consortia that perform distributed biological computation.

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The simplest logic of XOR function, its distribution into separate logic operating cells, and characterization of two logic operators.(A). XOR function and its distribution. Left: The simplest logic of XOR gate expressed as the combination of basic logic units, according to the four rules in main text. XOR gate is distributed into two different logic-operating cells, USC and DSC. USC bears a genetic AND gate, with the output signal linked to DSC. DSC processes three inputs; two environmental inputs and an intermediate signal from USC. NOT gate does not belong to either cell, but is realized by transcription-inhibitory “chemical wire”. Such construction satisfies truth table of XOR gate presented in the right panel. (B). Gene circuit to characterize transfer function of USC. Only when both inputs exist, functional T7 polymerase would activate T7 promoter and produce output, GFP. (C). Left: Experimental results for transfer function of USC. Florescence was measured and normalized by cell density. The measured sets are for 10−1, 10−2, 10−3, 10−4, 10−5, 10−6, 10−7 and 10−8 M arabinose, and 10−3, 10−4, 10−5, 10−6, 10−7, 10−8, 10−9 and 10−10 M salicylate. Right: Corresponding simulation prediction. (D). Gene circuit of DSC. Both environmental inputs can drive the expression supD tRNA through corresponding promoters, composing an OR gate. With no AHL, T7ptag would be expressed, and thereby GFP could be produced when either arabinose or salicylate (or both of them) present. (E). Transfer function of DSC, showing combinations of every two inputs. Columns from left to right: arabinose and salicylate, arabinose and AHL, and salicylate and AHL. Upper panels show experimental data compared with corresponding simulation prediction (lower panel). The data are for 10−1, 10−2, 10−3, 10−4, 10−5, 10−6, and 10−7 M arabinose, 10−3, 10−4, 10−5, 10−6, 10−7, 10−8, and 10−9 M salicylate, and 10−5, 10−6, 10−7, 10−8, 10−9, 10−10, and 10−11 M AHL. AHL was artificially supplied to DSC rather than a signal from USC.
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pone-0057482-g002: The simplest logic of XOR function, its distribution into separate logic operating cells, and characterization of two logic operators.(A). XOR function and its distribution. Left: The simplest logic of XOR gate expressed as the combination of basic logic units, according to the four rules in main text. XOR gate is distributed into two different logic-operating cells, USC and DSC. USC bears a genetic AND gate, with the output signal linked to DSC. DSC processes three inputs; two environmental inputs and an intermediate signal from USC. NOT gate does not belong to either cell, but is realized by transcription-inhibitory “chemical wire”. Such construction satisfies truth table of XOR gate presented in the right panel. (B). Gene circuit to characterize transfer function of USC. Only when both inputs exist, functional T7 polymerase would activate T7 promoter and produce output, GFP. (C). Left: Experimental results for transfer function of USC. Florescence was measured and normalized by cell density. The measured sets are for 10−1, 10−2, 10−3, 10−4, 10−5, 10−6, 10−7 and 10−8 M arabinose, and 10−3, 10−4, 10−5, 10−6, 10−7, 10−8, 10−9 and 10−10 M salicylate. Right: Corresponding simulation prediction. (D). Gene circuit of DSC. Both environmental inputs can drive the expression supD tRNA through corresponding promoters, composing an OR gate. With no AHL, T7ptag would be expressed, and thereby GFP could be produced when either arabinose or salicylate (or both of them) present. (E). Transfer function of DSC, showing combinations of every two inputs. Columns from left to right: arabinose and salicylate, arabinose and AHL, and salicylate and AHL. Upper panels show experimental data compared with corresponding simulation prediction (lower panel). The data are for 10−1, 10−2, 10−3, 10−4, 10−5, 10−6, and 10−7 M arabinose, 10−3, 10−4, 10−5, 10−6, 10−7, 10−8, and 10−9 M salicylate, and 10−5, 10−6, 10−7, 10−8, 10−9, 10−10, and 10−11 M AHL. AHL was artificially supplied to DSC rather than a signal from USC.

Mentions: In formulation of two-input one-output functions, we found that they could all be implemented in a single cell without violation of above rules, except for XOR gate and EQUALS gate, where two logic operators (i.e., two logic-operating cells) are needed (see Figure S2). XOR and EQUALS functions have been rarely implemented within a single cell in previous attempts [5]. Therefore, as a proof of concept, we set out to biologically implement XOR function in an E. coli consortium consisting of two types of logic operating cells (the design of EQUALS gate is very similar to that of XOR gate, because their truth tables are mutually complementary). The circuit design of two logic operators is presented in Fig. 2(A), where two collaborating cells are named as Upstream Cell (USC) and Downstream Cell (DSC), respectively.


A formalized design process for bacterial consortia that perform logic computing.

Ji W, Shi H, Zhang H, Sun R, Xi J, Wen D, Feng J, Chen Y, Qin X, Ma Y, Luo W, Deng L, Lin H, Yu R, Ouyang Q - PLoS ONE (2013)

The simplest logic of XOR function, its distribution into separate logic operating cells, and characterization of two logic operators.(A). XOR function and its distribution. Left: The simplest logic of XOR gate expressed as the combination of basic logic units, according to the four rules in main text. XOR gate is distributed into two different logic-operating cells, USC and DSC. USC bears a genetic AND gate, with the output signal linked to DSC. DSC processes three inputs; two environmental inputs and an intermediate signal from USC. NOT gate does not belong to either cell, but is realized by transcription-inhibitory “chemical wire”. Such construction satisfies truth table of XOR gate presented in the right panel. (B). Gene circuit to characterize transfer function of USC. Only when both inputs exist, functional T7 polymerase would activate T7 promoter and produce output, GFP. (C). Left: Experimental results for transfer function of USC. Florescence was measured and normalized by cell density. The measured sets are for 10−1, 10−2, 10−3, 10−4, 10−5, 10−6, 10−7 and 10−8 M arabinose, and 10−3, 10−4, 10−5, 10−6, 10−7, 10−8, 10−9 and 10−10 M salicylate. Right: Corresponding simulation prediction. (D). Gene circuit of DSC. Both environmental inputs can drive the expression supD tRNA through corresponding promoters, composing an OR gate. With no AHL, T7ptag would be expressed, and thereby GFP could be produced when either arabinose or salicylate (or both of them) present. (E). Transfer function of DSC, showing combinations of every two inputs. Columns from left to right: arabinose and salicylate, arabinose and AHL, and salicylate and AHL. Upper panels show experimental data compared with corresponding simulation prediction (lower panel). The data are for 10−1, 10−2, 10−3, 10−4, 10−5, 10−6, and 10−7 M arabinose, 10−3, 10−4, 10−5, 10−6, 10−7, 10−8, and 10−9 M salicylate, and 10−5, 10−6, 10−7, 10−8, 10−9, 10−10, and 10−11 M AHL. AHL was artificially supplied to DSC rather than a signal from USC.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585339&req=5

pone-0057482-g002: The simplest logic of XOR function, its distribution into separate logic operating cells, and characterization of two logic operators.(A). XOR function and its distribution. Left: The simplest logic of XOR gate expressed as the combination of basic logic units, according to the four rules in main text. XOR gate is distributed into two different logic-operating cells, USC and DSC. USC bears a genetic AND gate, with the output signal linked to DSC. DSC processes three inputs; two environmental inputs and an intermediate signal from USC. NOT gate does not belong to either cell, but is realized by transcription-inhibitory “chemical wire”. Such construction satisfies truth table of XOR gate presented in the right panel. (B). Gene circuit to characterize transfer function of USC. Only when both inputs exist, functional T7 polymerase would activate T7 promoter and produce output, GFP. (C). Left: Experimental results for transfer function of USC. Florescence was measured and normalized by cell density. The measured sets are for 10−1, 10−2, 10−3, 10−4, 10−5, 10−6, 10−7 and 10−8 M arabinose, and 10−3, 10−4, 10−5, 10−6, 10−7, 10−8, 10−9 and 10−10 M salicylate. Right: Corresponding simulation prediction. (D). Gene circuit of DSC. Both environmental inputs can drive the expression supD tRNA through corresponding promoters, composing an OR gate. With no AHL, T7ptag would be expressed, and thereby GFP could be produced when either arabinose or salicylate (or both of them) present. (E). Transfer function of DSC, showing combinations of every two inputs. Columns from left to right: arabinose and salicylate, arabinose and AHL, and salicylate and AHL. Upper panels show experimental data compared with corresponding simulation prediction (lower panel). The data are for 10−1, 10−2, 10−3, 10−4, 10−5, 10−6, and 10−7 M arabinose, 10−3, 10−4, 10−5, 10−6, 10−7, 10−8, and 10−9 M salicylate, and 10−5, 10−6, 10−7, 10−8, 10−9, 10−10, and 10−11 M AHL. AHL was artificially supplied to DSC rather than a signal from USC.
Mentions: In formulation of two-input one-output functions, we found that they could all be implemented in a single cell without violation of above rules, except for XOR gate and EQUALS gate, where two logic operators (i.e., two logic-operating cells) are needed (see Figure S2). XOR and EQUALS functions have been rarely implemented within a single cell in previous attempts [5]. Therefore, as a proof of concept, we set out to biologically implement XOR function in an E. coli consortium consisting of two types of logic operating cells (the design of EQUALS gate is very similar to that of XOR gate, because their truth tables are mutually complementary). The circuit design of two logic operators is presented in Fig. 2(A), where two collaborating cells are named as Upstream Cell (USC) and Downstream Cell (DSC), respectively.

Bottom Line: Despite of all its benefits, however, there are still problems remaining for large-scaled multicellular gene circuits, for example, how to reliably design and distribute the circuits in microbial consortia with limited number of well-behaved genetic modules and wiring quorum-sensing molecules.The construction and characterization of logic operators is independent of "wiring" and provides predictive information for fine-tuning.This formalized design process provides guidance for the design of microbial consortia that perform distributed biological computation.

View Article: PubMed Central - PubMed

Affiliation: Peking University Team for the International Genetically Engineered Machine Competition (iGEM), Peking University, Beijing, China.

ABSTRACT
The concept of microbial consortia is of great attractiveness in synthetic biology. Despite of all its benefits, however, there are still problems remaining for large-scaled multicellular gene circuits, for example, how to reliably design and distribute the circuits in microbial consortia with limited number of well-behaved genetic modules and wiring quorum-sensing molecules. To manage such problem, here we propose a formalized design process: (i) determine the basic logic units (AND, OR and NOT gates) based on mathematical and biological considerations; (ii) establish rules to search and distribute simplest logic design; (iii) assemble assigned basic logic units in each logic operating cell; and (iv) fine-tune the circuiting interface between logic operators. We in silico analyzed gene circuits with inputs ranging from two to four, comparing our method with the pre-existing ones. Results showed that this formalized design process is more feasible concerning numbers of cells required. Furthermore, as a proof of principle, an Escherichia coli consortium that performs XOR function, a typical complex computing operation, was designed. The construction and characterization of logic operators is independent of "wiring" and provides predictive information for fine-tuning. This formalized design process provides guidance for the design of microbial consortia that perform distributed biological computation.

Show MeSH
Related in: MedlinePlus