We recently developed a high-throughput functional genomics approach, named ‘SorTn-seq’, to identify factors affecting expression of any gene of interest in bacteria. Our approach facilitates high-throughput screening of complex mutant pools, a task previously hindered by a lack of suitable techniques. SorTn-seq combines high-density, Tn5-like transposon mutagenesis with fluorescence-activated cell sorting of a strain harboring a promoter-fluorescent reporter fusion, to isolate mutants with altered gene expression. The transposon mutant pool is sorted into different bins on the basis of fluorescence, and mutants are deep-sequenced to identify transposon insertions. DNA is prepared for sequencing by using commercial kits augmented with custom primers, enhancing ease of use and reproducibility. Putative regulators are identified by comparing the number of insertions per genomic feature in the different sort bins, by using existing bioinformatic pipelines and software packages. SorTn-seq can be completed in 1–2 weeks and requires general microbiology skills and basic flow cytometry experience.
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This work was supported by the Marsden Fund from the Royal Society of New Zealand and the School of Biomedical Sciences Bequest Fund from the University of Otago. L.M.S. was supported by a University of Otago Doctoral Scholarship and Postgraduate Publishing Bursary. We thank M. Wilson for help with cell sorting and A. Jeffs of the Otago Genomics Facility for assistance with sequencing. We thank J. Ussher and R. Hannaway for help with flow cytometry. We thank H. Hampton, D. Mayo-Muñoz, N. Birkholz and members of the Fineran laboratory for helpful discussions.
The authors declare no competing interests.
Peer review information Nature Protocols thanks the anonymous reviewers for their contribution to the peer review of this work.
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Smith, L. et al. Nat. Microbiol. 6, 162–172 (2021): https://doi.org/10.1038/s41564-020-00822-7
Extended Data Fig. 1 Gating on a secondary reporter eliminates noise from the primary reporter fluorescence distribution.
The primary reporter (eYFP) fluorescence distribution is shown for without (a) or with (b) gating on a secondary reporter (mCherry). In a and b, events are first gated on SSC and FSC parameters area (A) and height (H) to isolate individual bacteria (‘singlets’ and ‘cells’). a, A characteristic secondary peak is observed centered around zero. Many of the events comprising this secondary peak exhibit negative fluorescence levels and therefore cannot be sorted. This non-fluorescent population is probably comprised of dead/dormant cells, cellular debris, bubbles or electronic noise generated by the instrument. b, The addition of a gate around mCherry-positive (mCherry+) events results in the removal of the secondary peak from the eYFP fluorescence distribution. This improved distribution allows for more accurate placement of gates during cell sorting.
Type III-A CRISPR-Cas expression was measured from an eYFP fusion to 250 nt upstream of the csm operon. Key features in the promoter are indicated (−35 and −10) as are the transcription start site (+1), native RBS and the start codon (ATG, green). An IPTG-inducible 2nd fluorophore (mCherry) is under the control of the T5-lac promoter (PT5-lac). The pBR322 origin of replication facilitates ~15–20 copies of the reporter per cell.
a, Organization of the pKRCPN2 transposon delivery vector. b, Schematic of the transposon Tn-DS1028uidAKm, which contains a transcriptional uidA reporter, origin of replication (R6Kγ) and kanamycin resistance gene (KmR).
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Smith, L.M., Jackson, S.A., Gardner, P.P. et al. SorTn-seq: a high-throughput functional genomics approach to discovering regulators of bacterial gene expression. Nat Protoc 16, 4382–4418 (2021). https://doi.org/10.1038/s41596-021-00582-6