A comprehensive guide to dynamic analysis of microbial gene expression using the 3D-printed PFIbox and a fluorescent reporter library.
Shawn FrenchAmelia Bing Ya GuoEric D BrownPublished in: Nature protocols (2020)
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.
Keyphrases
- gene expression
- rna seq
- dna methylation
- single cell
- fluorescence imaging
- high density
- escherichia coli
- high resolution
- microbial community
- high throughput
- transcription factor
- crispr cas
- photodynamic therapy
- physical activity
- mental health
- mass spectrometry
- drug delivery
- machine learning
- single molecule
- staphylococcus aureus
- candida albicans
- heat shock protein
- multidrug resistant
- data analysis