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1.
ACS Synth Biol ; 7(2): 339-346, 2018 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-29091420

RESUMO

A gene-directed chemical communication pathway between synthetic protocell signaling transmitters (lipid vesicles) and receivers (proteinosomes) was designed, built and tested using a bottom-up modular approach comprising small molecule transcriptional control, cell-free gene expression, porin-directed efflux, substrate signaling, and enzyme cascade-mediated processing.


Assuntos
Células Artificiais/metabolismo , Transdução de Sinais , Transcrição Gênica , Células Artificiais/química , Sistema Livre de Células/química , Sistema Livre de Células/metabolismo
2.
ACS Synth Biol ; 6(4): 638-647, 2017 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-28100049

RESUMO

Although RNA synthesis can be reliably controlled with different T7 transcriptional promoters during cell-free gene expression with the PURE system, protein synthesis remains largely unaffected. To better control protein levels, we investigated a series of ribosome binding sites (RBSs). Although RBS strength did strongly affect protein synthesis, the RBS sequence could explain less than half of the variability of the data. Protein expression was found to depend on other factors besides the strength of the RBS, including the GC content of the coding sequence. The complexity of protein synthesis in comparison to RNA synthesis was observed by the higher degree of variability associated with protein expression. This variability was also observed in an E. coli cell extract-based system. However, the coefficient of variation was larger with E. coli RNA polymerase than with T7 RNA polymerase, consistent with the increased complexity of E. coli RNA polymerase.


Assuntos
Sistema Livre de Células/metabolismo , RNA/metabolismo , Transcrição Gênica , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Sítios de Ligação , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Fotodegradação , Biossíntese de Proteínas , Proteínas/metabolismo , RNA/química , Dobramento de RNA , Ribossomos/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Proteína Vermelha Fluorescente
3.
J Mol Cell Cardiol ; 89(Pt A): 98-112, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26423156

RESUMO

Long noncoding RNAs (lncRNAs) are emerging as important regulators of developmental pathways. However, their roles in human cardiac precursor cell (CPC) remain unexplored. To characterize the long noncoding transcriptome during human CPC cardiac differentiation, we profiled the lncRNA transcriptome in CPCs isolated from the human fetal heart and identified 570 lncRNAs that were modulated during cardiac differentiation. Many of these were associated with active cardiac enhancer and super enhancers (SE) with their expression being correlated with proximal cardiac genes. One of the most upregulated lncRNAs was a SE-associated lncRNA that was named CARMEN, (CAR)diac (M)esoderm (E)nhancer-associated (N)oncoding RNA. CARMEN exhibits RNA-dependent enhancing activity and is upstream of the cardiac mesoderm-specifying gene regulatory network. Interestingly, CARMEN interacts with SUZ12 and EZH2, two components of the polycomb repressive complex 2 (PRC2). We demonstrate that CARMEN knockdown inhibits cardiac specification and differentiation in cardiac precursor cells independently of MIR-143 and -145 expression, two microRNAs located proximal to the enhancer sequences. Importantly, CARMEN expression was activated during pathological remodeling in the mouse and human hearts, and was necessary for maintaining cardiac identity in differentiated cardiomyocytes. This study demonstrates therefore that CARMEN is a crucial regulator of cardiac cell differentiation and homeostasis.


Assuntos
Padronização Corporal/genética , Diferenciação Celular/genética , Coração/embriologia , Homeostase/genética , RNA Longo não Codificante/metabolismo , Animais , Linhagem da Célula/genética , Elementos Facilitadores Genéticos/genética , Proteína Potenciadora do Homólogo 2 de Zeste , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Miocárdio/patologia , Complexo Repressor Polycomb 2/metabolismo , RNA Longo não Codificante/genética , Células-Tronco/citologia , Transcriptoma/genética
4.
Nat Commun ; 5: 4012, 2014 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-24874202

RESUMO

Previous efforts to control cellular behaviour have largely relied upon various forms of genetic engineering. Once the genetic content of a living cell is modified, the behaviour of that cell typically changes as well. However, other methods of cellular control are possible. All cells sense and respond to their environment. Therefore, artificial, non-living cellular mimics could be engineered to activate or repress already existing natural sensory pathways of living cells through chemical communication. Here we describe the construction of such a system. The artificial cells expand the senses of Escherichia coli by translating a chemical message that E. coli cannot sense on its own to a molecule that activates a natural cellular response. This methodology could open new opportunities in engineering cellular behaviour without exploiting genetically modified organisms.


Assuntos
Células Artificiais/metabolismo , Engenharia Celular/métodos , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas Hemolisinas/metabolismo , Riboswitch/genética , Células Artificiais/efeitos dos fármacos , Vesículas Citoplasmáticas/efeitos dos fármacos , Vesículas Citoplasmáticas/metabolismo , Escherichia coli/efeitos dos fármacos , Proteínas de Escherichia coli/efeitos dos fármacos , Proteínas Hemolisinas/efeitos dos fármacos , Isopropiltiogalactosídeo/metabolismo , Antagonistas de Receptores Purinérgicos P1/farmacologia , Riboswitch/efeitos dos fármacos , Teofilina/farmacologia
5.
ACS Synth Biol ; 3(6): 363-71, 2014 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-24283192

RESUMO

The cell-free transcription-translation of multiple proteins typically exploits genes placed behind strong transcriptional promoters that reside on separate pieces of DNA so that protein levels can be easily controlled by changing DNA template concentration. However, such systems are not amenable to the construction of artificial cells with a synthetic genome. Herein, we evaluated the activity of a series of T7 transcriptional promoters by monitoring the fluorescence arising from a genetically encoded Spinach aptamer. Subsequently the influences of transcriptional promoter strength on fluorescent protein synthesis from one, two, and three gene operons were assessed. It was found that transcriptional promoter strength was more effective at controlling RNA synthesis than protein synthesis in vitro with the PURE system. Conversely, the gene position within the operon strongly influenced protein synthesis but not RNA synthesis.


Assuntos
Ordem dos Genes , Óperon , Regiões Promotoras Genéticas , Biossíntese de Proteínas/genética , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Bacteriófago T7/química , Bacteriófago T7/genética , Sistema Livre de Células/química , DNA de Plantas/química , DNA de Plantas/genética , Regulação da Expressão Gênica , Plasmídeos/química , Plasmídeos/genética , RNA Mensageiro/química , RNA Mensageiro/genética , Análise de Sequência de DNA , Spinacia oleracea/química , Spinacia oleracea/genética , Transcrição Gênica
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