RESUMEN
The dimerization and high-order oligomerization of transcription factors has endowed them with cooperative regulatory capabilities that play important roles in many cellular functions. However, such advanced regulatory capabilities have not been fully exploited in synthetic biology and genetic engineering. Here, we engineered a C-terminally fused oligomerization domain to improve the cooperativity of transcription factors. First, we found that two of three designed oligomerization domains significantly increased the cooperativity and ultrasensitivity of a transcription factor for the regulated promoter. Then, seven additional transcription factors were used to assess the modularity of the oligomerization domains, and their ultrasensitivity was generally improved, as assessed by their Hill coefficients. Moreover, we also demonstrated that the allosteric capability of the ligand-responsive domain remained intact when fusing with the designed oligomerization domain. As an example application, we showed that the engineered ultrasensitive transcription factor could be used to significantly improve the performance of a "stripe-forming" gene circuit. We envision that the oligomerization modules engineered in this study could act as a powerful tool to rapidly tune the underlying response profiles of synthetic gene circuits and metabolic pathway controllers.
Asunto(s)
Ingeniería de Proteínas/métodos , Proteínas Recombinantes de Fusión/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Regulación Alostérica/efectos de los fármacos , Proteínas Bacterianas/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Proteínas de Escherichia coli/genética , Retroalimentación Fisiológica , Redes Reguladoras de Genes , Isopropil Tiogalactósido/farmacología , Represoras Lac/genética , Ligandos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Regiones Promotoras Genéticas , Dominios Proteicos , Multimerización de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/genética , Factores de Transcripción/químicaRESUMEN
Rational engineering of biological systems is often complicated by the complex but unwanted interactions between cellular components at multiple levels. Here we address this issue at the level of prokaryotic transcription by insulating minimal promoters and operators to prevent their interaction and enable the biophysical modeling of synthetic transcription without free parameters. This approach allows genetic circuit design with extraordinary precision and diversity, and consequently simplifies the design-build-test-learn cycle of circuit engineering to a mix-and-match workflow. As a demonstration, combinatorial promoters encoding NOT-gate functions were designed from scratch with mean errors of <1.5-fold and a success rate of >96% using our insulated transcription elements. Furthermore, four-node transcriptional networks with incoherent feed-forward loops that execute stripe-forming functions were obtained without any trial-and-error work. This insulation-based engineering strategy improves the resolution of genetic circuit technology and provides a simple approach for designing genetic circuits for systems and synthetic biology.Unwanted interactions between cellular components can complicate rational engineering of biological systems. Here the authors design insulated minimal promoters and operators that enable biophysical modeling of bacterial transcription without free parameters for precise circuit design.
Asunto(s)
Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica/genética , Redes Reguladoras de Genes/genética , Elementos Aisladores/genética , Regiones Promotoras Genéticas/genética , Biología Sintética , Ingeniería Genética , PlásmidosRESUMEN
OBJECTIVE: To establish a rapid and convenient method of DNA modification by bisulfite sodium for the detection of DNA methylation. METHODS: Through increasing the bisulfite sodium concentration and the temperature of treatment, cutting down the modification time, besides using glassmilk to adsorb the DNA in the purification and recovery, to improve the methods of DNA modification. Efficiency of cytosine converted to thymine in MAGE-A3 gene and DAP-K gene fragments were analyzed by bisulfite sequencing PCR in order to evaluate the DNA modification effect among the improved method, traditional method and kit method. RESULTS: The operating time of test was shortened to about 3 hours by the improved method; conversion rate of unmethylated cytosine to thymine was over 99%; compared with the traditional method and kit method, there was no significant difference (χ(2) = 0.0564, P > 0.05); the improved method was only for the unmethylated cytosine conversion modification, and there was no significant difference in process of methylated cytosine converted to thymine comparing with the traditional method (χ(2) = 0.0149, P > 0.05). CONCLUSION: The improved method has high efficiency of DNA modification and has no significant effect on excessive modification;meanwhile, it has many advantages such as time-saving and easy to operate etc.
Asunto(s)
Metilación de ADN , ADN/química , Sulfitos/química , Citosina/química , Reacción en Cadena de la Polimerasa , Timina/químicaRESUMEN
Paeoniflorin, the major component of Paeonia lactiflora pall, has previously been reported to prevent thrombosis. Plasminogen activator urokinase (uPA) is a serine protease that markedly facilitates normal thrombosis resolution. Paeoniflorin and uPA have been linked to the mitogenactivated protein kinase (MAPK) signaling pathway. In the current study, the influence of paeoniflorin on the expression of uPA was investigated and the underlying regulatory mechanism was preliminarily determined. The prothrombotic state of the model animals treated with paeoniflorin were assessed by enzymelinked immunosorbent assay (ELISA). Additionally, the cytotoxicity of paeoniflorin on human umbilical vein endothelial cell (HUVEC) cultures was estimated using a methyl thiazolyl tetrazolium assay and the possible pathways involved in the interaction between paeoniflorin and uPA were evaluated using western blot analysis. The ELISA results demonstrated that the levels of 6keto prostaglandin F1a, fibronectin and uPA were significantly upregulated by treatment with paeoniflorin compared with control (P<0.05). By contrast, the expression of fibrinogen, Ddimer and thromboxane B2 were inhibited. With an increase in the concentration of paeoniflorin the cell viability of HUVECs decreased gradually. The results of western blot analysis demonstrated that paeoniflorin increased the phosphorylation of MAPK 14 (p38) and MAPK 8 (JNK). The present study demonstrated that paeoniflorin has the potential to improve the prethrombotic state and recanalize thrombosis by increasing the expression of uPA, which may be mediated via regulation of the p38 and JNK MAPK signaling pathways. However, this treatment effect was dependent on the concentration of paeoniflorin used, an unsuitable concentration of the agent would result in a negative effect on the antithrombosis pathways.