Abstract
High-throughput screening is a popular tool for collating biological data which would otherwise require the use of excessive resources. In this study, an artificial genetic circuit sensor responding to dimethylallyl diphosphate (DMAPP) was constructed based on a modified L-arabinose operon for high-throughput screening and isoprene synthase (ispS) evolution in Escherichia coli (E. coli). As a first step, the DNA sequence of the L-arabinose ligand-binding domain (LBD) was replaced with an ispS gene to enable the AraC operon responding to DMAPP, which is the substrate of the IspS enzyme. Then, an enhanced GFP (eGFP) was also introduced as a reporter for pBAD promoter. The expression level of the reporter was monitored using either of the two tools: flow cytometer (FCM) and microplate reader. Sequentially, we observed that a high DMAPP concentration led to low eGFP fluorescence, and the overexpression of ispS gene, which consumes DMAPP, resulted in a high eGFP expression. These results demonstrated that the artificial genetic circuit sensor responded directly to the intracellular concentration of DMAPP, and the expression of IspS enzyme could be positively correlated to the expression level of eGFP. Finally, we identified two IspS mutants with different activities from an ispS gene library and further validated the screening method.
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Acknowledgements
This work was supported by the State key laboratory of organic–inorganic composites, the National Basic Research Program of China (973 program) (2013CB733600), and the National Nature Science Foundation of China (21390202, 21476017). The authors also thank David Luo (Lawrence Berkeley National Lab, Joint Bio-Energy Institute) and Jan-Christer Janson (Uppsala University) for revising the language of this manuscript.
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Liu, CL., Cai, JY., Bi, HR. et al. A novel DMAPP-responding genetic circuit sensor for high-throughput screening and evolving isoprene synthase. Appl Microbiol Biotechnol 102, 1381–1391 (2018). https://doi.org/10.1007/s00253-017-8676-8
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DOI: https://doi.org/10.1007/s00253-017-8676-8