Funding

Ongoing

Young Research Award: Synthetic Biology approaches for deciphering the logic of signal integration in complex bacterial promoters. 03.01.15 - 02.28.20. PI: Prof. Rafael Silva-Rocha

The ability to coordinate expression of thousands of genes in response to different stimuli is an essential feature of any live organism. However, current knowledge on this matter is largely inferred from data on extant systems involving just a few regulatory components. In this context, this project propose the adoption of a genetically tractable experimental object (Escherichia coli) along with the conceptual and material tools of Synthetic Biology for examining the molecular mechanisms (hardware) and the underlying logic program (software) that account for the regulatory behavior of complex biological systems. The principal approach to this end will involve the generation of synthetic promoter libraries assembled adjacent to combinatorial arrays of upstream binding sequences for five known global regulators (IHF, Fis, Crp, NarL and Fnr). The behavior of the resulting promoter libraries will be thoroughly parameterized in single cells and in populations as a whole for quantification of their transcriptional capacity, kinetics and stochastic noise. The resulting data will be employed to feed computational models for identifying general rules that account for the regulation of the thereby generated promoters and predict the outcome of new ones. We expect that the results of this endeavor will contribute not only a fundamental understanding mechanisms of signal integration in prokaryotic promoters, but will also produce rules and methods for designing and building standardized, integrated biological systems to accomplish many particular tasks of biotechnological interest.

Young Research Award: Novel approaches to improve functional screening of biocatalysts in metagenomic libraries. 10.01.15 - 09.30.19. PI: Prof. María Eugenia Guazzaroni

Biotechnology plays a crucial role in the development of biocatalysts for use in industry, agriculture, medicine and energy generation. Currently, there is a growing demand for enzymes with improved catalytic performance or tolerance to process-specific parameters. Metagenomics takes advantage of the wealth of genetic and biochemical diversity present in microbial genomes found in environmental samples and provides a set of new technologies directed towards screening of new catalytic activities with potential biotechnology applications. However, biased and low level of expression of heterologous proteins in Escherichia coli together with the use of non-optimal metagenomic screening strategies generally results in an extremely low success rate for enzymes identification. In this sense, the present proposal aims to perform three main approaches in order to develop new tools to improve target gene recovery. Firstly, two different strategies will be used to overcome the biased expression of foreign proteins in the screenings: (i) Construction of metagenomic libraries both in Pseudomonas putida and E. coli and; (ii) Utilization of the optimized and synthetic pSEVA broad host-range vector for library production. At present, there is no reported work in literature using a vector with similar features in functional metagenomic screenings. On the other hand, a third strategy comprising the engineering of a bacterial host will be developed using a synthetic biology approach. In particular, the strategy aims to improve the recovery rate of enzymes by exploiting the synergistic interaction between the metagenomic enzyme (or other accessory/complementary protein) and an enzyme expressed from the chromosome of the engineered host strain. Together, these approaches would provide a new, high efficient platform for the construction and functional screening of metagenomic libraries.

FAPESP-RCUK BBSRC: Loving the poison: Molecular basis for metabolism and toxicity of the widely-used food preservative, propionate in Pseudomonas aeruginosa. 04.01.18 - 03.31.20. PI: Prof. Rafael Silva-Rocha and Prof. Martin Welch, University of Cambridge.

Past

MCTI/CNPq Universal: Deciphering the cis-regulatory elements related to the expression of cellulase-coding genes in Trichoderma reesei. 12.18.14 - 11.18.17

Cellulase producing microorganisms are of great interest for several biotechnological applications such as the production of bioethanol from plant biomass. In this sense, there is an increasing focus on the development of new technologies targeting the engineering of microorganisms to enhance the production of this enzyme class. This proposition aims the combination of different computational tools together with experimental setups in order to decipher the regulatory mechanisms related to the expression of cellulolytic enzymes in the filamentous fungus Trichoderma reesei. The project will focus on the identification of cis-regulatory elements in groups of co-regulated genes trigged by different growth conditions (such as in the presence of the cellulase-inducers cellulose or sophorose, or in the presence of the repressor glucose) identified using available RNA-seq data. The DNA motifs identified will be used to search databases for well-characterized cis-regulatory elements in order to allow the screening of new transcriptional factors potentially related to the control of cellulase expression in T. reesei. The new cis-regulatory elements will be characterized in vivo using transcriptional fusions of target promoters to the reporter gene GFP, while candidate transcriptional factors will be analyzed through the construction of mutant strains of T. reesei. In this way, the new regulatory information will be used to construct computational models aimed to simulate the response of the regulatory network to new environments, and the resulting predictions will be validated in vivo. The accomplishment of the proposition will potentially result in the generation of new knowledge on the regulatory mechanisms ruling cellulase expression in T. reesei, which will be crucial for future attempts to engineer this organism for application in the bioethanol industry.

Newton International Exchanges: High-throughput analysis of cis-regulatory logic in synthetic bacterial promoters. 03.10.15 - 03.09.17

Living cells can be programmed by incorporating integrated genetic circuits into their DNA. To date, circuits used to engineer conditional responses have been based on the combination of a limited number of genetic regulatory elements. Therefore, the aim of this project is to expand the toolbox of inputs that can be integrated into a synthetic circuit. In order to do that, we plan to design and engineer a library of single promoters that can act as individual logic gates by integrating new combinations of signals. The in silico design will be followed by an experimental validation 100 of these synthetic promoters using a specialized dual-reporter vector and a high-throughput robotic platform intended to measure gene expression at translational level.