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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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Fine-tuning of circuiting interface between USC and DSC.(A). Schematics of XOR-function gene circuit encoded within the entire microbial consortium. LuxI, a synthase of AHL, works as output of USC. AHL transduces a repressive signal to DSC. (B). Upper panels: experimental results using diluted filtrate from induced USC. Four histograms represent results for 4 different RBS sequences: AAAGAGGAGAAA (BBa_B0034), ATTAAAGTTGAGAAA (Mutant 1), GCTCCATCCCCG (Mutant 2), and GCTCCTCCGATC (Mutant 3), with RBS strength 9-, 108- and 150-fold attenuated, respectively, predicted by RBS Calculator. In each histogram, corresponding inputs are: (left to right) no inducers (blank), arabinose only (Ara), salicylate only (Sal), and both inducers (Ara+Sal). Error bars are calculated as mean ± s. d. Lower panels: phase diagrams of the entire circuit predicted by model using characterization data for individual logic operating cells.
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pone-0057482-g003: Fine-tuning of circuiting interface between USC and DSC.(A). Schematics of XOR-function gene circuit encoded within the entire microbial consortium. LuxI, a synthase of AHL, works as output of USC. AHL transduces a repressive signal to DSC. (B). Upper panels: experimental results using diluted filtrate from induced USC. Four histograms represent results for 4 different RBS sequences: AAAGAGGAGAAA (BBa_B0034), ATTAAAGTTGAGAAA (Mutant 1), GCTCCATCCCCG (Mutant 2), and GCTCCTCCGATC (Mutant 3), with RBS strength 9-, 108- and 150-fold attenuated, respectively, predicted by RBS Calculator. In each histogram, corresponding inputs are: (left to right) no inducers (blank), arabinose only (Ara), salicylate only (Sal), and both inducers (Ara+Sal). Error bars are calculated as mean ± s. d. Lower panels: phase diagrams of the entire circuit predicted by model using characterization data for individual logic operating cells.

Mentions: After characterizing USC and DSC, we need to circuit them together according to our design [Fig. 2(A)]. Through quorum-sensing molecule AHL, USC and DSC can be coupled, where LuxI, LuxR, and promoter Plux_rep together work as a transcription-inhibitory chemical wire for cell-cell signaling [Fig. 3(A)]. Fine-tuning process, however, was necessary. Hoping for rational fine-tuning, we utilized RBS Calculator as the tool. It could predict relative translation strength of a given RBS sequence and in silico design synthetic ribosome binding site (RBS) sequences with requested relative translation strength [30]. For this purpose, we use our initial construction [the RBS sequence was AAAGAGGAGAAA, numbered BBa_B0034 in Fig. 3(B)] as the reference. Filtrate from induced USC was used to culture DSC (filtrate was blended 1∶3 in volume with fresh Luria–Bertani broth, and arabinose and/or salicylate were also supplied as needed), and corresponding florescence of DSC were measured. Results showed that leakage expression of luxI in USC generated excessive AHL to repress Plux_rep in DSC [Fig. 3(B), first panel in the upper row], indicating BBa_B0034 was too strong.


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)

Fine-tuning of circuiting interface between USC and DSC.(A). Schematics of XOR-function gene circuit encoded within the entire microbial consortium. LuxI, a synthase of AHL, works as output of USC. AHL transduces a repressive signal to DSC. (B). Upper panels: experimental results using diluted filtrate from induced USC. Four histograms represent results for 4 different RBS sequences: AAAGAGGAGAAA (BBa_B0034), ATTAAAGTTGAGAAA (Mutant 1), GCTCCATCCCCG (Mutant 2), and GCTCCTCCGATC (Mutant 3), with RBS strength 9-, 108- and 150-fold attenuated, respectively, predicted by RBS Calculator. In each histogram, corresponding inputs are: (left to right) no inducers (blank), arabinose only (Ara), salicylate only (Sal), and both inducers (Ara+Sal). Error bars are calculated as mean ± s. d. Lower panels: phase diagrams of the entire circuit predicted by model using characterization data for individual logic operating cells.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585339&req=5

pone-0057482-g003: Fine-tuning of circuiting interface between USC and DSC.(A). Schematics of XOR-function gene circuit encoded within the entire microbial consortium. LuxI, a synthase of AHL, works as output of USC. AHL transduces a repressive signal to DSC. (B). Upper panels: experimental results using diluted filtrate from induced USC. Four histograms represent results for 4 different RBS sequences: AAAGAGGAGAAA (BBa_B0034), ATTAAAGTTGAGAAA (Mutant 1), GCTCCATCCCCG (Mutant 2), and GCTCCTCCGATC (Mutant 3), with RBS strength 9-, 108- and 150-fold attenuated, respectively, predicted by RBS Calculator. In each histogram, corresponding inputs are: (left to right) no inducers (blank), arabinose only (Ara), salicylate only (Sal), and both inducers (Ara+Sal). Error bars are calculated as mean ± s. d. Lower panels: phase diagrams of the entire circuit predicted by model using characterization data for individual logic operating cells.
Mentions: After characterizing USC and DSC, we need to circuit them together according to our design [Fig. 2(A)]. Through quorum-sensing molecule AHL, USC and DSC can be coupled, where LuxI, LuxR, and promoter Plux_rep together work as a transcription-inhibitory chemical wire for cell-cell signaling [Fig. 3(A)]. Fine-tuning process, however, was necessary. Hoping for rational fine-tuning, we utilized RBS Calculator as the tool. It could predict relative translation strength of a given RBS sequence and in silico design synthetic ribosome binding site (RBS) sequences with requested relative translation strength [30]. For this purpose, we use our initial construction [the RBS sequence was AAAGAGGAGAAA, numbered BBa_B0034 in Fig. 3(B)] as the reference. Filtrate from induced USC was used to culture DSC (filtrate was blended 1∶3 in volume with fresh Luria–Bertani broth, and arabinose and/or salicylate were also supplied as needed), and corresponding florescence of DSC were measured. Results showed that leakage expression of luxI in USC generated excessive AHL to repress Plux_rep in DSC [Fig. 3(B), first panel in the upper row], indicating BBa_B0034 was too strong.

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