ABSTRACT
Bacillus subtilis employs five purine riboswitches for the control of purine de novo synthesis and transport at the transcription level. All of them are formed by a structurally conserved aptamer, and a variable expression platform harboring a rho-independent transcription terminator. In this study, we characterized all five purine riboswitches under the context of active gene expression processes both in vitro and in vivo. We identified transcription pause sites located in the expression platform upstream of the terminator of each riboswitch. Moreover, we defined a correlation between in vitro transcription readthrough and in vivo gene expression. Our in vitro assay demonstrated that the riboswitches operate in the micromolar range of concentration for the cognate metabolite. Our in vivo assay showed the dynamics of the control of gene expression by each riboswitch. This study deepens the knowledge of the regulatory mechanism of purine riboswitches.
Subject(s)
Riboswitch , Bacillus subtilis/genetics , Bacillus subtilis/metabolism , Purines/metabolism , Riboswitch/geneticsABSTRACT
Modular and tuneable genetic tools for Metabolic Engineering fuels the development of chassis for the efficient production of biocompounds at industrial scale. We have constructed an autoinduction device for gene expression in Bacillus subtilis based on the LuxR/I quorum sensing system [1]. Here, we present raw and processed data regarding to B. subtilis growth measured as OD600, performed in three different scales: microcultivation on 96-well plates (200 µL), test tubes (12 mL), and Erlenmeyer flasks (50 mL). We also present raw and processed data on gene expression measured as GFP fluorescence (485/535 nm), luminescence and riboflavin production. Measurements were performed on a microplate reader Tecan 200 PRO (iControl software) and on spectrophotometer (Thermo Fisher Scientific GENESYS 10S UV-Vis). Processed data are presented as product/OD600, maximum and minimum promoter activity, fold of induction, and the induction OD600.
ABSTRACT
Intense synthesis of proteins and chemicals in engineered microbes impose metabolic burden, frequently leading to reduced growth and heterogeneous cell population. Thus, the correct balance between growth and production is important. Such balance can be engineered through dynamic control of pathways, but few broadly applicable tools are available to achieve this. We present an autonomous control of gene expression mediated by quorum sensing in Bacillus subtilis, able to self-monitor and induce expression without human supervision. Two variations of the induction module and seven of the response module were engineered generating a range of induction folds and strengths for gene expression control. Our strongest response promoter is 2.5 and 3.2 times stronger than the well-characterized promoters PsrfA and Pveg, respectively. We applied our strongest autoinduction device for the production of the vitamin B2. This study presents a toolbox of autoinduction modules for B. subtilis that is modular and tunable.
Subject(s)
Bacillus subtilis , Gene Expression Regulation, Bacterial , Metabolic Engineering , Quorum Sensing , Bacillus subtilis/genetics , Bacillus subtilis/metabolismABSTRACT
AIM: This work aimed to produce a membrane based on fluconazole-loaded natural rubber latex (NRL), and study their interaction, drug release and antifungal susceptibility against Candida albicans. MATERIALS & METHODS: Fluconazole-loaded NRL membrane was obtained by casting method. RESULTS: The Fourier Transform Infrared Spectroscopy showed no modifications either in NRL or fluconazole after the incorporation. Mechanical test presented low Young's modulus and high strain, indicating the membranes have sufficient elasticity for biomedical application. The bio-membrane was able to release the drug and inhibit the growth of C. albicans as demonstrated by disk diffusion and macrodilution assays. CONCLUSION: The biomembrane was able to release fluconazole and inhibit the growth of C. albicans, representing a promising biomaterial for skin application.
