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Tunable synthetic phenotypic diversification on Waddington's landscape through autonomous signaling.

ABSTRACT: Phenotypic diversification of cells is crucial for developmental and regenerative processes in multicellular organisms. The diversification concept is described as the motion of marbles rolling down Waddington's landscape, in which the number of stable states changes as development proceeds. In contrast to this simple concept, the complexity of natural biomolecular processes prevents comprehension of their design principles. We have constructed, in Escherichia coli, a synthetic circuit with just four genes, which programs cells to autonomously diversify as the motion on the landscape through cell-cell communication. The circuit design was based on the combination of a bistable toggle switch with an intercellular signaling system. The cells with the circuit diversified into two distinct cell states, "high" and "low," in vivo and in silico, when all of the cells started from the low state. The synthetic diversification was affected by not only the shape of the landscape determined by the circuit design, which includes the synthesis rate of the signaling molecule, but also the number of cells in the experiments. This cell-number dependency is reminiscent of the "community effect": The fates of developing cells are determined by their number. Our synthetic circuit could be a model system for studying diversification and differentiation in higher organisms. Prospectively, further integrations of our circuit with different cellular functions will provide unique tools for directing cell fates on the population level in tissue engineering.

SUBMITTER: Sekine R

PROVIDER: S-EPMC3207703 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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Tunable synthetic phenotypic diversification on Waddington's landscape through autonomous signaling.

Sekine Ryoji R Yamamura Masayuki M Ayukawa Shotaro S Ishimatsu Kana K Akama Satoru S Takinoue Masahiro M Hagiya Masami M Kiga Daisuke D

Proceedings of the National Academy of Sciences of the United States of America 20111024 44

Phenotypic diversification of cells is crucial for developmental and regenerative processes in multicellular organisms. The diversification concept is described as the motion of marbles rolling down Waddington's landscape, in which the number of stable states changes as development proceeds. In contrast to this simple concept, the complexity of natural biomolecular processes prevents comprehension of their design principles. We have constructed, in Escherichia coli, a synthetic circuit with just ...[more]

PMID: 22025684

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Project description:Antibodies possess properties that make them valuable as therapeutics, diagnostics, and basic research tools. However, antibody chemical reactivity and covalent antigen binding are constrained, or even prevented, by the narrow range of chemistries encoded in canonical amino acids. In this work, we investigate strategies for leveraging an expanded range of chemical functionality using yeast displayed antibodies containing noncanonical amino acids (ncAAs) in or near antibody complementarity determining regions (CDRs). To enable systematic characterization of the effects of ncAA incorporation on antibody function, we first investigated whether diversification of a single antibody loop would support the isolation of binding clones against immunoglobulins from three species. We constructed and screened a billion-member library containing canonical amino acid diversity and loop length diversity only within the third complementarity determining region of the heavy chain (CDR-H3). Isolated clones exhibited moderate affinities (double- to triple-digit nanomolar affinities) and, in several cases, single-species specificity, confirming that antibody specificity can be mediated by a single CDR. This constrained diversity enabled the utilization of additional CDRs for the installation of chemically reactive and photo-cross-linkable ncAAs. Binding studies of ncAA-substituted antibodies revealed that ncAA incorporation is reasonably well tolerated, with observed changes in affinity occurring as a function of ncAA side chain identity, substitution site, and the ncAA incorporation machinery used. Multiple azide-containing ncAAs supported copper-catalyzed azide-alkyne cycloaddition (CuAAC) and strain-promoted azide-alkyne cycloaddition (SPAAC) without the abrogation of binding function. Similarly, several alkyne substitutions facilitated CuAAC without the apparent disruption of binding. Finally, antibodies substituted with a photo-cross-linkable ncAA were evaluated for ultraviolet-mediated cross-linking on the yeast surface. Competition-based assays revealed position-dependent covalent linkages, strongly suggesting successful cross-linking. Key findings regarding CuAAC reactions and photo-cross-linking on the yeast surface were confirmed using soluble forms of ncAA-substituted clones. The consistency of findings on the yeast surface and in solution suggest that chemical diversification can be incorporated into yeast display screening approaches. Taken together, our results highlight the power of integrating the use of yeast display and ncAAs in search of proteins with "chemically augmented" binding functions. This includes strategies for systematically introducing small molecule functionality within binding protein structures and evaluating protein-based covalent target binding. The efficient preparation and chemical diversification of antibodies on the yeast surface open up new possibilities for discovering "drug-like" protein leads in high throughput.

Dataset Information

Tunable synthetic phenotypic diversification on Waddington's landscape through autonomous signaling.

Publications

Tunable synthetic phenotypic diversification on Waddington's landscape through autonomous signaling.

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