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1.
Int J Mol Sci ; 21(19)2020 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-32998303

RESUMO

Some years inspire more hindsight reflection and future-gazing than others. This is even more so in 2020 with its evocation of perfect vision and the landmark ring to it. However, no futurist can reliably predict what the world will look like the next time that a year's first two digits will match the second two digits-a numerical pattern that only occurs once in a century. As we leap into a new decade, amid uncertainties triggered by unforeseen global events-such as the outbreak of a worldwide pandemic, the accompanying economic hardship, and intensifying geopolitical tensions-it is important to note the blistering pace of 21st century technological developments indicate that while hindsight might be 20/20, foresight is 50/50. The history of science shows us that imaginative ideas, research excellence, and collaborative innovation can, for example, significantly contribute to the economic, cultural, social, and environmental recovery of a post-COVID-19 world. This article reflects on a history of yeast research to indicate the potential that arises from advances in science, and how this can contribute to the ongoing recovery and development of human society. Future breakthroughs in synthetic genomics are likely to unlock new avenues of impactful discoveries and solutions to some of the world's greatest challenges.


Assuntos
Surtos de Doenças/prevenção & controle , Engenharia Genética/métodos , Genoma Fúngico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Biologia Sintética/métodos , Saccharomyces cerevisiae/classificação
2.
Nat Commun ; 11(1): 4440, 2020 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-32895374

RESUMO

Traditionally engineered genetic circuits have almost exclusively used naturally occurring transcriptional repressors. Recently, non-natural transcription factors (repressors) have been engineered and employed in synthetic biology with great success. However, transcriptional anti-repressors have largely been absent with regard to the regulation of genes in engineered genetic circuits. Here, we present a workflow for engineering systems of non-natural anti-repressors. In this study, we create 41 inducible anti-repressors. This collection of transcription factors respond to two distinct ligands, fructose (anti-FruR) or D-ribose (anti-RbsR); and were complemented by 14 additional engineered anti-repressors that respond to the ligand isopropyl ß-d-1-thiogalactopyranoside (anti-LacI). In turn, we use this collection of anti-repressors and complementary genetic architectures to confer logical control over gene expression. Here, we achieved all NOT oriented logical controls (i.e., NOT, NOR, NAND, and XNOR). The engineered transcription factors and corresponding series, parallel, and series-parallel genetic architectures represent a nascent anti-repressor based transcriptional programming structure.


Assuntos
Bioengenharia/métodos , Repressores Lac/antagonistas & inibidores , Proteínas de Escherichia coli/metabolismo , Expressão Gênica/fisiologia , Redes Reguladoras de Genes , Repressores Lac/síntese química , Ligantes , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/síntese química , Biologia Sintética/métodos , Fatores de Transcrição/síntese química , Fatores de Transcrição/metabolismo
3.
Nat Commun ; 11(1): 4879, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32978379

RESUMO

Synthetic biology allows us to bioengineer cells to synthesize novel valuable molecules such as renewable biofuels or anticancer drugs. However, traditional synthetic biology approaches involve ad-hoc engineering practices, which lead to long development times. Here, we present the Automated Recommendation Tool (ART), a tool that leverages machine learning and probabilistic modeling techniques to guide synthetic biology in a systematic fashion, without the need for a full mechanistic understanding of the biological system. Using sampling-based optimization, ART provides a set of recommended strains to be built in the next engineering cycle, alongside probabilistic predictions of their production levels. We demonstrate the capabilities of ART on simulated data sets, as well as experimental data from real metabolic engineering projects producing renewable biofuels, hoppy flavored beer without hops, fatty acids, and tryptophan. Finally, we discuss the limitations of this approach, and the practical consequences of the underlying assumptions failing.


Assuntos
Aprendizado de Máquina , Engenharia Metabólica/métodos , Biologia Sintética/métodos , Teorema de Bayes , Cerveja , Biocombustíveis , Dodecanol/metabolismo , Escherichia coli/metabolismo , Ácidos Graxos/metabolismo , Saccharomyces cerevisiae/metabolismo
4.
Nat Commun ; 11(1): 4758, 2020 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-32958811

RESUMO

Genetic programs operating in a history-dependent fashion are ubiquitous in nature and govern sophisticated processes such as development and differentiation. The ability to systematically and predictably encode such programs would advance the engineering of synthetic organisms and ecosystems with rich signal processing abilities. Here we implement robust, scalable history-dependent programs by distributing the computational labor across a cellular population. Our design is based on standardized recombinase-driven DNA scaffolds expressing different genes according to the order of occurrence of inputs. These multicellular computing systems are highly modular, do not require cell-cell communication channels, and any program can be built by differential composition of strains containing well-characterized logic scaffolds. We developed automated workflows that researchers can use to streamline program design and optimization. We anticipate that the history-dependent programs presented here will support many applications using cellular populations for material engineering, biomanufacturing and healthcare.


Assuntos
Modelos Genéticos , Biologia Sintética/métodos , Fenômenos Fisiológicos Celulares/genética , DNA/genética , DNA/metabolismo , Lógica , Recombinases/genética , Recombinases/metabolismo , Software , Fluxo de Trabalho
5.
Nat Commun ; 11(1): 4043, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792475

RESUMO

Genetically fusing protein domains to Cas9 has yielded several transformative technologies; however, the genetic modifications are limited to natural polypeptide chains at the Cas9 termini, which excludes a diverse array of molecules useful for gene editing. Here, we report chemical modifications that allow site-specific and multiple-site conjugation of a wide assortment of molecules on both the termini and internal sites of Cas9, creating a platform for endowing Cas9 with diverse functions. Using this platform, Cas9 can be modified to more precisely incorporate exogenously supplied single-stranded oligonucleotide donor (ssODN) at the DNA break site. We demonstrate that the multiple-site conjugation of ssODN to Cas9 significantly increases the efficiency of precision genome editing, and such a platform is compatible with ssODNs of diverse lengths. By leveraging the conjugation platform, we successfully engineer INS-1E, a ß-cell line, to repurpose the insulin secretion machinery, which enables the glucose-dependent secretion of protective immunomodulatory factor interleukin-10.


Assuntos
Proteína 9 Associada à CRISPR/química , Proteína 9 Associada à CRISPR/metabolismo , Engenharia Celular/métodos , Biologia Sintética/métodos , Proteína 9 Associada à CRISPR/genética , Linhagem Celular , Edição de Genes , Humanos , Sistemas de Infusão de Insulina
6.
Nat Commun ; 11(1): 4050, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792485

RESUMO

Regulatory networks describe the hierarchical relationship between transcription factors, associated proteins, and their target genes. Regulatory networks respond to environmental and genetic perturbations by reprogramming cellular metabolism. Here we design, construct, and map a comprehensive regulatory network library containing 110,120 specific mutations in 82 regulators expected to perturb metabolism. We screen the library for different targeted phenotypes, and identify mutants that confer strong resistance to various inhibitors, and/or enhanced production of target compounds. These improvements are identified in a single round of selection, showing that the regulatory network library is universally applicable and is convenient and effective for engineering targeted phenotypes. The facile construction and mapping of the regulatory network library provides a path for developing a more detailed understanding of global regulation in E. coli, with potential for adaptation and use in less-understood organisms, expanding toolkits for future strain engineering, synthetic biology, and broader efforts.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Edição de Genes/métodos , Engenharia Metabólica/métodos , Biologia Sintética/métodos , Redes Reguladoras de Genes/genética , Redes Reguladoras de Genes/fisiologia
7.
Nat Commun ; 11(1): 4344, 2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32859906

RESUMO

Self-propagating drive systems are capable of causing non-Mendelian inheritance. Here, we report a drive system in yeast referred to as a chromosome drive that eliminates the target chromosome via CRISPR-Cas9, enabling the transmission of the desired chromosome. Our results show that the entire Saccharomyces cerevisiae chromosome can be eliminated efficiently through only one double-strand break around the centromere via CRISPR-Cas9. As a proof-of-concept experiment of this CRISPR-Cas9 chromosome drive system, the synthetic yeast chromosome X is completely eliminated, and the counterpart wild-type chromosome X harboring a green fluorescent protein gene or the components of a synthetic violacein pathway are duplicated by sexual reproduction. We also demonstrate the use of chromosome drive to preferentially transmit complex genetic traits in yeast. Chromosome drive enables entire chromosome elimination and biased inheritance on a chromosomal scale, facilitating genomic engineering and chromosome-scale genetic mapping, and extending applications of self-propagating drives.


Assuntos
Sistemas CRISPR-Cas , Cromossomos/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Saccharomyces cerevisiae/genética , Centrômero , Indóis , Redes e Vias Metabólicas/genética , Saccharomyces cerevisiae/metabolismo , Biologia Sintética/métodos , Termotolerância/genética , Sequenciamento Completo do Genoma
8.
Nat Commun ; 11(1): 4044, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792536

RESUMO

A growing number of optogenetic tools have been developed to reversibly control binding between two engineered protein domains. In contrast, relatively few tools confer light-switchable binding to a generic target protein of interest. Such a capability would offer substantial advantages, enabling photoswitchable binding to endogenous target proteins in cells or light-based protein purification in vitro. Here, we report the development of opto-nanobodies (OptoNBs), a versatile class of chimeric photoswitchable proteins whose binding to proteins of interest can be enhanced or inhibited upon blue light illumination. We find that OptoNBs are suitable for a range of applications including reversibly binding to endogenous intracellular targets, modulating signaling pathway activity, and controlling binding to purified protein targets in vitro. This work represents a step towards programmable photoswitchable regulation of a wide variety of target proteins.


Assuntos
Optogenética/métodos , Biologia Sintética/métodos , Animais , Células HEK293 , Humanos , Camundongos , Células NIH 3T3 , Ligação Proteica , Transporte Proteico/fisiologia , Transdução de Sinais/fisiologia
9.
Nat Commun ; 11(1): 3469, 2020 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-32651386

RESUMO

Insertions and deletions (InDels) are frequently observed in natural protein evolution, yet their potential remains untapped in laboratory evolution. Here we introduce a transposon-based mutagenesis approach (TRIAD) to generate libraries of random variants with short in-frame InDels, and screen TRIAD libraries to evolve a promiscuous arylesterase activity in a phosphotriesterase. The evolution exhibits features that differ from previous point mutagenesis campaigns: while the average activity of TRIAD variants is more compromised, a larger proportion has successfully adapted for the activity. Different functional profiles emerge: (i) both strong and weak trade-off between activities are observed; (ii) trade-off is more severe (20- to 35-fold increased kcat/KM in arylesterase with 60-400-fold decreases in phosphotriesterase activity) and (iii) improvements are present in kcat rather than just in KM, suggesting adaptive solutions. These distinct features make TRIAD an alternative to widely used point mutagenesis, accessing functional innovations and traversing unexplored fitness landscape regions.


Assuntos
Mutação INDEL/genética , Evolução Molecular , Humanos , Mutagênese/genética , Mutagênese/fisiologia , Hidrolases de Triester Fosfórico/genética , Hidrolases de Triester Fosfórico/metabolismo , Biologia Sintética/métodos
10.
Nat Commun ; 11(1): 3659, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32694598

RESUMO

As synthetic biocircuits become more complex, distributing computations within multi-strain microbial consortia becomes increasingly beneficial. However, designing distributed circuits that respond predictably to variation in consortium composition remains a challenge. Here we develop a two-strain gene circuit that senses and responds to which strain is in the majority. This involves a co-repressive system in which each strain produces a signaling molecule that signals the other strain to down-regulate production of its own, orthogonal signaling molecule. This co-repressive consortium links gene expression to ratio of the strains rather than population size. Further, we control the cross-over point for majority via external induction. We elucidate the mechanisms driving these dynamics by developing a mathematical model that captures consortia response as strain fractions and external induction are varied. These results show that simple gene circuits can be used within multicellular synthetic systems to sense and respond to the state of the population.


Assuntos
Engenharia Celular/métodos , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Consórcios Microbianos/genética , Percepção de Quorum/genética , Redes Reguladoras de Genes , Transdução de Sinais/genética , Biologia Sintética/métodos
11.
Proc Natl Acad Sci U S A ; 117(28): 16537-16545, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601191

RESUMO

Engineering biological nitrogen fixation in eukaryotic cells by direct introduction of nif genes requires elegant synthetic biology approaches to ensure that components required for the biosynthesis of active nitrogenase are stable and expressed in the appropriate stoichiometry. Previously, the NifD subunits of nitrogenase MoFe protein from Azotobacter vinelandii and Klebsiella oxytoca were found to be unstable in yeast and plant mitochondria, respectively, presenting a bottleneck to the assembly of active MoFe protein in eukaryotic cells. In this study, we have delineated the region and subsequently a key residue, NifD-R98, from K. oxytoca that confers susceptibility to protease-mediated degradation in mitochondria. The effect observed is pervasive, as R98 is conserved among all NifD proteins analyzed. NifD proteins from four representative diazotrophs, but not their R98 variants, were observed to be unstable in yeast mitochondria. Furthermore, by reconstituting mitochondrial-processing peptidases (MPPs) from yeast, Oryza sativa, Nicotiana tabacum, and Arabidopsis thaliana in Escherichia coli, we demonstrated that MPPs are responsible for cleavage of NifD. These results indicate a pervasive effect on the stability of NifD proteins in mitochondria resulting from cleavage by MPPs. NifD-R98 variants that retained high levels of nitrogenase activity were obtained, with the potential to stably target active MoFe protein to mitochondria. This reconstitution approach could help preevaluate the stability of Nif proteins for plant expression and paves the way for engineering active nitrogenase in plant organelles.


Assuntos
Proteínas de Bactérias/genética , Expressão Gênica , Klebsiella oxytoca/enzimologia , Nitrogenase/genética , Engenharia de Proteínas/métodos , Biologia Sintética/métodos , Proteínas de Bactérias/metabolismo , Klebsiella oxytoca/genética , Mitocôndrias/enzimologia , Mitocôndrias/genética , Nitrogenase/metabolismo , Plantas/genética , Plantas/metabolismo , Processamento de Proteína Pós-Traducional
12.
PLoS One ; 15(7): e0235808, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32722674

RESUMO

One of the central aims of synthetic biology (SB) is to better understand the mechanisms of life by trying to develop and synthesize new forms and perhaps modes of life. While the question of what is life has occupied mankind for centuries, there is a lack of empirical research examining the basic concepts of life scientists within SB themselves refer to and build on. In order to gain insights into these fundamental concepts, we conducted a qualitative interview study with scientists working in the field of SB. The aim was to gain a better understanding of the underlying understandings, principles, and characteristics of (synthetic) life on the one hand, and the entangled consequences for the conducted experiments and studies as well as the pursued scientific approaches. We identified four primarily underlying basic concepts of life which serve as a fundamental framework for current and further scientific research within SB and have implications for research questions, approaches and aims as well as for the evaluation of scientific results.


Assuntos
Biomimética , Biologia Sintética , Biomimética/métodos , Compreensão , Feminino , Humanos , Masculino , Origem da Vida , Pesquisa , Pesquisadores , Biologia Sintética/métodos
13.
Nat Commun ; 11(1): 2739, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32483165

RESUMO

Synthetic biology is a powerful tool to create therapeutics which can be rationally designed to enable unique and combinatorial functionalities. Here we utilize non-pathogenic E coli Nissle as a versatile platform for the development of a living biotherapeutic for the treatment of cancer. The engineered bacterial strain, referred to as SYNB1891, targets STING-activation to phagocytic antigen-presenting cells (APCs) in the tumor and activates complementary innate immune pathways. SYNB1891 treatment results in efficacious antitumor immunity with the formation of immunological memory in murine tumor models and robust activation of human APCs. SYNB1891 is designed to meet manufacturability and regulatory requirements with built in biocontainment features which do not compromise its efficacy. This work provides a roadmap for the development of future therapeutics and demonstrates the transformative potential of synthetic biology for the treatment of human disease when drug development criteria are incorporated into the design process for a living medicine.


Assuntos
Escherichia coli/imunologia , Imunoterapia/métodos , Proteínas de Membrana/imunologia , Neoplasias/terapia , Transdução de Sinais/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Linhagem Celular Tumoral , Escherichia coli/genética , Escherichia coli/metabolismo , Engenharia Genética/métodos , Humanos , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias/genética , Neoplasias/imunologia , Fagócitos/imunologia , Fagócitos/metabolismo , Transdução de Sinais/genética , Biologia Sintética/métodos , Biologia Sintética/tendências
14.
Nat Commun ; 11(1): 2832, 2020 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-32504029

RESUMO

Human amyloids have been shown to interact with viruses and interfere with viral replication. Based on this observation, we employed a synthetic biology approach in which we engineered virus-specific amyloids against influenza A and Zika proteins. Each amyloid shares a homologous aggregation-prone fragment with a specific viral target protein. For influenza we demonstrate that a designer amyloid against PB2 accumulates in influenza A-infected tissue in vivo. Moreover, this amyloid acts specifically against influenza A and its common PB2 polymorphisms, but not influenza B, which lacks the homologous fragment. Our model amyloid demonstrates that the sequence specificity of amyloid interactions has the capacity to tune amyloid-virus interactions while allowing for the flexibility to maintain activity on evolutionary diverging variants.


Assuntos
Amiloide/farmacologia , Antivirais/farmacologia , Genética Reversa/métodos , Biologia Sintética/métodos , Amiloide/genética , Amiloide/uso terapêutico , Animais , Antivirais/uso terapêutico , Modelos Animais de Doenças , Cães , Feminino , Células HEK293 , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Vírus da Influenza A/efeitos dos fármacos , Vírus da Influenza A/genética , Vírus da Influenza A/patogenicidade , Influenza Humana/tratamento farmacológico , Influenza Humana/virologia , Células Madin Darby de Rim Canino , Camundongos , Polimorfismo Genético , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral/efeitos dos fármacos , Zika virus/efeitos dos fármacos , Zika virus/genética , Zika virus/patogenicidade , Infecção por Zika virus/tratamento farmacológico , Infecção por Zika virus/virologia
15.
Nat Commun ; 11(1): 3138, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561745

RESUMO

Synthetic biology has focused on engineering genetic modules that operate orthogonally from the host cells. A synthetic biological module, however, can be designed to reprogram the host proteome, which in turn enhances the function of the synthetic module. Here, we apply this holistic synthetic biology concept to the engineering of cell-free systems by exploiting the crosstalk between metabolic networks in cells, leading to a protein environment more favorable for protein synthesis. Specifically, we show that local modules expressing translation machinery can reprogram the bacterial proteome, changing the expression levels of more than 700 proteins. The resultant feedback generates a cell-free system that can synthesize fluorescent reporters, protein nanocages, and the gene-editing nuclease Cas9, with up to 5-fold higher expression level than classical cell-free systems. Our work demonstrates a holistic approach that integrates synthetic and systems biology concepts to achieve outcomes not possible by only local, orthogonal circuits.


Assuntos
Proteínas de Bactérias/genética , Engenharia Metabólica/métodos , Proteoma/genética , Biologia Sintética/métodos , Proteínas de Bactérias/metabolismo , Sistema Livre de Células/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Redes Reguladoras de Genes , Redes e Vias Metabólicas/genética , Biossíntese de Proteínas/genética , Proteoma/metabolismo
16.
Nat Commun ; 11(1): 3154, 2020 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-32572025

RESUMO

An orthogonal aminoacyl-tRNA synthetase/tRNA pair is a crucial prerequisite for site-specific incorporation of unnatural amino acids. Due to its high codon suppression efficiency and full orthogonality, the pyrrolysyl-tRNA synthetase/pyrrolysyl-tRNA pair is currently the ideal system for genetic code expansion in both eukaryotes and prokaryotes. There is a pressing need to discover or engineer other fully orthogonal translation systems. Here, through rational chimera design by transplanting the key orthogonal components from the pyrrolysine system, we create multiple chimeric tRNA synthetase/chimeric tRNA pairs, including chimera histidine, phenylalanine, and alanine systems. We further show that these engineered chimeric systems are orthogonal and highly efficient with comparable flexibility to the pyrrolysine system. Besides, the chimera phenylalanine system can incorporate a group of phenylalanine, tyrosine, and tryptophan analogues efficiently in both E. coli and mammalian cells. These aromatic amino acids analogous exhibit unique properties and characteristics, including fluorescence, post-translation modification.


Assuntos
Aminoácidos/biossíntese , Código Genético , RNA de Transferência , Biologia Sintética/métodos , Alanina/análogos & derivados , Quimera/genética , Quimera/metabolismo , Escherichia coli , Células HEK293 , Histidina/análogos & derivados , Humanos , Lisina/análogos & derivados , Fenilalanina/análogos & derivados , RNA de Transferência/genética , RNA de Transferência/metabolismo , Triptofano/análogos & derivados , Tirosina/análogos & derivados
17.
PLoS Comput Biol ; 16(6): e1007776, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32555578

RESUMO

We show that logic computational circuits in gene regulatory networks arise from a fibration symmetry breaking in the network structure. From this idea we implement a constructive procedure that reveals a hierarchy of genetic circuits, ubiquitous across species, that are surprising analogues to the emblematic circuits of solid-state electronics: starting from the transistor and progressing to ring oscillators, current-mirror circuits to toggle switches and flip-flops. These canonical variants serve fundamental operations of synchronization and clocks (in their symmetric states) and memory storage (in their broken symmetry states). These conclusions introduce a theoretically principled strategy to search for computational building blocks in biological networks, and present a systematic route to design synthetic biological circuits.


Assuntos
Biologia Computacional/métodos , Redes Reguladoras de Genes , Biologia Sintética/métodos , Algoritmos , Animais , Arabidopsis , Bacillus subtilis , Simulação por Computador , Eletrônica , Escherichia coli , Humanos , Modelos Teóricos , Mycobacterium tuberculosis , Oscilometria , Salmonella
18.
Proc Natl Acad Sci U S A ; 117(27): 15573-15580, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32571944

RESUMO

Cyanobacteriochromes (CBCRs) are small, bistable linear tetrapyrrole (bilin)-binding light sensors which are typically found as modular components in multidomain cyanobacterial signaling proteins. The CBCR family has been categorized into many lineages that roughly correlate with their spectral diversity, but CBCRs possessing a conserved DXCF motif are found in multiple lineages. DXCF CBCRs typically possess two conserved Cys residues: a first Cys that remains ligated to the bilin chromophore and a second Cys found in the DXCF motif. The second Cys often forms a second thioether linkage, providing a mechanism to sense blue and violet light. DXCF CBCRs have been described with blue/green, blue/orange, blue/teal, and green/teal photocycles, and the molecular basis for some of this spectral diversity has been well established. We here characterize AM1_1499g1, an atypical DXCF CBCR that lacks the second cysteine residue and exhibits an orange/green photocycle. Based on prior studies of CBCR spectral tuning, we have successfully engineered seven AM1_1499g1 variants that exhibit robust yellow/teal, green/teal, blue/teal, orange/yellow, yellow/green, green/green, and blue/green photocycles. The remarkable spectral diversity generated by modification of a single CBCR provides a good template for multiplexing synthetic photobiology systems within the same cellular context, thereby bypassing the time-consuming empirical optimization process needed for multiple probes with different protein scaffolds.


Assuntos
Proteínas de Bactérias/metabolismo , Evolução Molecular , Luz , Fotorreceptores Microbianos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/efeitos da radiação , Cor , Cianobactérias/genética , Cianobactérias/metabolismo , Cianobactérias/efeitos da radiação , Mutagênese Sítio-Dirigida , Nostoc/genética , Nostoc/metabolismo , Nostoc/efeitos da radiação , Fotobiologia/métodos , Fotorreceptores Microbianos/efeitos da radiação , Biologia Sintética/métodos , Tetrapirróis/metabolismo
19.
Nat Commun ; 11(1): 2708, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32488070

RESUMO

Although many animals have evolved intrinsic transparency for the purpose of concealment, the development of dynamic, that is, controllable and reversible, transparency for living human cells and tissues has remained elusive to date. Here, by drawing inspiration from the structures and functionalities of adaptive cephalopod skin cells, we design and engineer human cells that contain reconfigurable protein-based photonic architectures and, as a result, possess tunable transparency-changing and light-scattering capabilities. Our findings may lead to the development of unique biophotonic tools for applications in materials science and bioengineering and may also facilitate an improved understanding of a wide range of biological systems.


Assuntos
Engenharia Celular/métodos , Cefalópodes , Óptica e Fotônica , Animais , Técnicas de Cultura de Células , Feminino , Engenharia Genética , Células HEK293 , Humanos , Proteínas/química , Pele , Biologia Sintética/métodos
20.
PLoS One ; 15(4): e0232046, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32352996

RESUMO

Advancements in the field of synthetic biology have been possible due to the development of genetic tools that are able to regulate gene expression. However, the current toolbox of gene regulatory tools for eukaryotic systems have been outpaced by those developed for simple, single-celled systems. Here, we engineered a set of gene regulatory tools by combining self-cleaving ribozymes with various upstream competing sequences that were designed to disrupt ribozyme self-cleavage. As a proof-of-concept, we were able to modulate GFP expression in mammalian cells, and then showed the feasibility of these tools in Drosophila embryos. For each system, the fold-reduction of gene expression was influenced by the location of the self-cleaving ribozyme/upstream competing sequence (i.e. 5' vs. 3' untranslated region) and the competing sequence used. Together, this work provides a set of genetic tools that can be used to tune gene expression across various eukaryotic systems.


Assuntos
Engenharia Genética/métodos , RNA Catalítico/fisiologia , Biologia Sintética/métodos , Animais , Drosophila/genética , Eucariotos/genética , Eucariotos/metabolismo , Células Eucarióticas/metabolismo , Expressão Gênica/genética , Expressão Gênica/fisiologia , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/fisiologia , Conformação de Ácido Nucleico , Estudo de Prova de Conceito , RNA Catalítico/genética , RNA Mensageiro/metabolismo
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