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A comprehensive guide to dynamic analysis of microbial gene expression using the 3D-printed PFIbox and a fluorescent reporter library


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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Fig. 1: Broad overview of the time-course gene expression pipeline using a fluorescent promoter–reporter library.
Fig. 2: Detailed experimental overview of creating PFIboxes and using them to generate time-course gene expression datasets.
Fig. 3: Example dataset illustrating promoters most active when E. coli is grown in MOPS minimal medium.
Fig. 4: PFIbox assembly, filter attachment and Raspberry Pi mounting steps.
Fig. 5: Basic relay wiring for PFIbox transilluminator control.
Fig. 6: Camera focusing modifications for Raspberry Pi camera.
Fig. 7: ImageJ workflow for image quantification.
Fig. 8: Overview of processing steps for data outputs from ImageJ quantifications.
Fig. 9: Time-course data analysis overview summarizing steps involved in clustering promoter activity over time.
Fig. 10: Comparison of overall gene expression fingerprints, comparing LB and MOPS minimal growth media.

Data availability

All files listed in this protocol, including 3D models and image analysis macros, can be found on the PFIbox GitHub repository at The raw data from Figs. 3, 9 and 10 can be found on Mendeley Data at The dataset for the French et al.4 PFIbox manuscript can also be found on Mendeley Data at All files and data are freely available, and licensed under the BSD-3 clause.

Code availability

Analysis code can be found on the PFIbox GitHub repository at 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.

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Authors and Affiliations



S.F. designed and created the PFIbox hardware and software, and developed the protocol. A.B.Y.G. refined the software in the protocol, optimized hardware settings and ran the experimental pipeline to generate data. E.D.B. supervised the work, and provided guidance and direction on all components of the protocol.

Corresponding author

Correspondence to Eric D. Brown.

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The authors declare no competing interests.

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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.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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French, S., Coutts, B. E. & Brown, E. D. Cell Systems 7, 339–346.e3 (2018):

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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).

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