Team:UFPR-Curitiba-Brazil/Description
Project Inspiration and Description
Global warming is the main environmental problem to be adressed nowadays. Besides carbon dioxide(CO2), other gases like methane (CH4) and nitrous oxide (N2O) causes harm to the atmosphere andhave greater greenhouse effect than CO2. 58% of all N2O emissions from anthropogenic activities arederived from nitrogen fertilizers (nitrate and ammonium) used in agriculture. Biofertilizers composedof bacteria that associates with plants and promotes their growth can have a positive impact to reducethe use of nitrogen fertilizers, but they don’t completely abolish their addition. Thus, sustainabletechnologies that fertilizes plants and control N2O emissions on field are highly desirable.
In this Project we propose the development of a biosensor for nitric oxide and nitrate detection,precursors of N2O, in soil sample. We will develop a genetic circuit that respond to both compounds by using an “AND” logic gate. Regulators NorR and NarL will be constitutively expressed. NorR is a transcriptional activator sensitive to nitric oxide presence and regulates expression of norVW,while NarL activates transcription of yeaR-yoaG operon in presence of nitrate. Thus, the promoter regions of norV(PnorV) and yeaR(PyeaR) genes will be used to control expression of hrpR and hrpS,respectively. Only in the presence of both proteins occurs the formation of the HrpR-HrpS complex,that binds to the promoter region of hrpL gene, dependent of σ54 factor, with activation and expression of a reporter gene (mht) that codes a protein involved on catalysis of sodium bromide to bromomethane gas. Bromomethane will be quantified by gas chromatography.
References
1. Cheng, Hsiao Ying et al. 2018. “Ratiometric Gas Reporting: A Nondisruptive Approach toMonitor Gene Expression in Soils.” ACS Synthetic Biology 7(3): 903–11.
2. D’Autr ́eaux, Benoˆıt, Nicholas P Tucker, Ray Dixon, and Stephen Spiro. 2005. “A Non-Haem Iron Centre in the Transcription Factor NorR Senses Nitric Oxide.” Nature 437: 769.https://doi.org/10.1038/nature03953.
3. Lin, Hsia Yin, Peggy J. Bledsoe, and Valley Stewart. 2007. “Activation of YeaR-YoaG OperonTranscription by the Nitrate-Responsive Regulator NarL Is Independent of Oxygen-ResponsiveRegulator Fnr inEscherichia ColiK-12.” Journal of Bacteriology 189(21): 7539–48.
4. Wang, Baojun, and Martin Buck. 2014. “Rapid Engineering of Versatile Molecular LogicGates Using Heterologous Genetic Transcriptional Modules.” Chemical Communications 50(79):11642–44.