Temporally resolved assays of bacterial gene expression using printed fluorescence imaging boxes (PFIboxes) are non-destructive, inexpensive and simple to prepare. Herein, we describe a full experimental pipeline wherein PFIbox parts are modified and 3D printed, electronics assembled and used to study transcriptional responses of Escherichia coli to chemical stressors. A chemical probe is added to agar growth medium, and a promoter–fluorophore fusion library is arrayed in high density on the agar slab. With high temporal resolution, the reporter library is imaged in PFIboxes, then quantified using promoter activity as a measure of gene expression. PFIboxes have advantages over conventional transcriptomic approaches such as RNA-seq, as the non-destructive nature permits a high-resolution temporal dimension in the data. This results in rapid measurement of transcriptional responses to chemical or physical stimuli. Each time-course gene expression assay costs about US$2 to run, in triplicate, using this method. Printing time depends on printer and settings, but once printed, PFIboxes can be fully assembled, programmed and loaded with samples in less than 1 h. Experimental durations and sampling frequency are set according to user need, but can be run in the duration of a microbial growth curve.
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All files listed in this protocol, including 3D models and image analysis macros, can be found on the PFIbox GitHub repository at https://github.com/sfrench007/pfibox. The raw data from Figs. 3, 9 and 10 can be found on Mendeley Data at https://doi.org/10.17632/6hjcdr3td3.1. The dataset for the French et al.4 PFIbox manuscript can also be found on Mendeley Data at https://doi.org/10.17632/fmv6vv4bsf.1. All files and data are freely available, and licensed under the BSD-3 clause.
Analysis code can be found on the PFIbox GitHub repository at https://github.com/sfrench007/pfibox. The code in this protocol has been peer reviewed.
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This research was supported by a Foundation grant from the Canadian Institutes for Health Research (FRN 143215), by the Natural Sciences and Engineering Research Council of Canada, by infrastructure funding from Canada Foundation for Innovation, and by a Tier I Canada Research Chair award to E.D.B. A.B.Y.G. was funded by an award from the Canadian Institutes for Health Research.
The authors declare no competing interests.
Peer review information Nature Protocols thanks Geoff Baldwin, Jan-Willem Veening and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Key reference using this protocol
French, S., Coutts, B. E. & Brown, E. D. Cell Systems 7, 339–346.e3 (2018): https://doi.org/10.1016/j.cels.2018.07.004
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French, S., Guo, A.B.Y. & Brown, E.D. A comprehensive guide to dynamic analysis of microbial gene expression using the 3D-printed PFIbox and a fluorescent reporter library. Nat Protoc 15, 575–603 (2020). https://doi.org/10.1038/s41596-019-0257-0