Rongrong ZhangYajia HuangMei LiLei Wang...
450-461页查看更多>>摘要：Synthetic biology relies on the screening and quantification of genetic components to assemble sophisticated gene circuits with specific functions.Microscopy is a powerful tool for characterizing complex cellular phenotypes with increasing spatial and temporal resolution to library screening of genetic elements.Microscopy-based assays are powerful tools for characterizing cellular phenotypes with spatial and temporal resolution and can be applied to large-scale samples for library screening of genetic elements.However,strategies for high-throughput microscopy experiments remain limited.Here,we present a high-throughput,microscopy-based platform that can simultaneously complete the preparation of an 8 × 12-well agarose pad plate,allowing for the screening of 96 independent strains or experimental conditions in a single experiment.Using this platform,we screened a library of natural intrinsic promoters from Pseudomonas aeruginosa and identified a small subset of robust promoters that drives stable levels of gene expression under varying growth conditions.Additionally,the platform allowed for single-cell measurement of genetic elements over time,enabling the identification of complex and dynamic phenotypes to map genotype in high throughput.We expected that the platform could be employed to accelerate the identification and characterization of genetic elements in various biological systems,as well as to understand the relationship between cellular phenotypes and internal states,including genotypes and gene expression programs.
Weilong ShangZhen HuMengyang LiYuting Wang...
462-478页查看更多>>摘要：Focal and systemic infections are serious threats to human health.Preclinical models enable the development of new drugs and therapeutic regimens.In vivo,animal bioluminescence(BL)imaging has been used with bacterial reporter strains to evaluate antimicrobial treatment effects.However,high-sensitivity bioluminescent systems are required because of the limited tissue penetration and low brightness of the BL signals of existing approaches.Here,we report that NanoLuc(Nluc)showed better performance than LuxCDABE in bacteria.However,the retention rate of plasmid constructs in bacteria was low.To construct stable Staphylococcus aureus reporter strains,a partner protein enolase(Eno)was identified by screening of S.aureus strain USA300 for fusion expression of Nluc-based luciferases,including Nluc,Teluc,and Antares2.Different substrates,such as hydrofurimazine(HFZ),furimazine(FUR),and diphenylterazine(DTZ),were used to optimize a stable reporter strain/substrate pair for BL imaging.S.aureus USA300/Eno-Antares2/HFZ produced the highest number of photons of orange-red light in vitro and enabled sensitive BL tracking of S.aureus in vivo,with sensitivities of approximately 10 CFU from mouse skin and 750 CFU from mouse kidneys.USA300/Eno-Antares2/HFZ was a powerful combination based on the longitudinal evaluation of the therapeutic efficacy of antibiotics.The optimized S.aureus Eno-Antares2/HFZ pair provides a technological advancement for the in vivo evaluation of antimicrobial treatment.
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