title: An efficient design strategy for a whole-cell biosensor based on engineered ribosome binding sequences.
abstract: In prokaryotes, the ribosome binding sequence (RBS), located in the 5' untranslated region (5' UTR) of an mRNA, plays a critical role in enhancing mRNA translation and stability. To evaluate the effect of the RBS on the sensitivity and signal intensity of an environmental whole-cell biosensor, three Escherichia coli-based biosensors that respond to benzene, toluene, ethylbenzene, and the xylenes (BTEX) were constructed; the three biosensors have the same Pu promoter and xylR regulator from the Pseudomonas putida TOL plasmid but differ in the engineered RBS in their reporter genes. The results from time and dose-dependent induction of luminescence activity by 2-chlorotoluene showed that the BTEX-SE and BTEX-SD biosensors with engineered RBS had signal intensities approximately 10-35 times higher than the primary BTEX-W biosensor. The limits of detection (LOD) of the BTEX-SE and BTEX-SD biosensors were also significantly lower than the LOD of the BTEX-W biosensor (20 ± 5 μmol L(-1) and 25 ± 5 μmol L(-1) vs. 120 ± 10 μmol L(-1)). Moreover, the BTEX-SE and BTEX-SD biosensors responded three times more rapidly to the analytes. These results suggest that rationally designed RBS in the 5' UTR of a reporter gene may be a promising strategy for increasing the sensitivity, signal intensity, and response speed of whole-cell biosensors.
authors.affiliation: Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Yu
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
authors.forename: Anzhou
authors.initials: A
authors.lastname: Ma
authors.forename: Weifeng
authors.initials: W
authors.lastname: Liu
authors.forename: Hailin
authors.initials: H
authors.lastname: Wang
authors.forename: Guoqiang
authors.initials: G
authors.lastname: Zhuang
mesh_terms: D020121:5' Untranslated Regions; D001554:Benzene / Q000032:analysis; D001555:Benzene Derivatives / Q000032:analysis; D015374:Biosensing Techniques / Q000379:methods*; D004787:Environmental Pollution / Q000032:analysis*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000379:methods*; D057230:Limit of Detection; D049449:Luminescence; D010957:Plasmids; D011401:Promoter Regions, Genetic; D016958:Pseudomonas putida / Q000737:chemistry / Q000235:genetics; D012107:Research Design; D012270:Ribosomes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D059010:Single-Cell Analysis / Q000379:methods*; D014050:Toluene / Q000032:analysis; D014992:Xylenes / Q000032:analysis
chemical_list: D020121:5' Untranslated Regions; D001555:Benzene Derivatives; D014992:Xylenes; D014050:Toluene; D001554:Benzene; C004912:ethylbenzene