Optimized Replication of Arrayed Bacterial Mutant Libraries Increases Access to Biological Resources

Abstract

Biological collections, including arrayed libraries of single transposon (Tn) or deletion mutants, greatly accelerate the pace of bacterial genetic research. Despite the importance of these resources, few protocols exist for the replication and distribution of these materials. Here, we describe a protocol for creating multiple replicates of an arrayed bacterial Tn library consisting of approximately 6,800 mutants in 96-well plates (73 plates). Our protocol provides multiple checkpoints to guard against contamination and minimize genetic drift caused by freeze/thaw cycles. This approach can also be scaled for arrayed culture collections of various sizes. Overall, this protocol is a valuable resource for other researchers considering the construction and distribution of arrayed culture collection resources for the benefit of the greater scientific community. IMPORTANCE Arrayed mutant collections drive robust genetic screens, but few protocols exist for replication of these resources and subsequent quality control. Increasing the distribution of arrayed biological collections will increase the accessibility and use of these resources. Developing standardized techniques for replication of these resources is essential for ensuring their quality and usefulness to the scientific community.

Keywords: Enterococcus faecalis; biological collections; functional genomics; genetic tools; scientific rigor; transposon mutagenesis.

FIG 1

Overview of arrayed library replication and quality control checkpoints. SmarT library plates were used to inoculate deep-well plates. Cultures were grown, examined for contamination, and then dispensed into 15 individual prelabeled, preloaded plates for new library sets. This image was created using BioRender.

FIG 2

Evidence that the sealing and mixing process does not introduce intraplate contamination. (A) A test plate with glycerol and bromophenol blue was sealed with foil, mixed by inversion, vortex-mixed, and incubated overnight on a shaking platform to ensure that the inversion process used to mix glycerol and bacterial cultures would not create contamination between wells. (B) A 96-well plate was inoculated with alternating wells of E. faecalis OG1RF (~10⁷ CFU/mL) or medium blanks. To mimic library plate processing, the plate was capped with a foil seal and mixed by inversion. The plate was incubated at 37°C overnight, after which an OD₆₀₀ measurement was taken to evaluate growth. The heatmap values are the averages of three independent biological replicates.

FIG 3

Comparison of the SmarT library pooled from liquid cultures and that from the original pooled library format. (A) Relative abundances of Tn mutants in each library. (B) Distribution of relative abundances. (C) Abundances of low-abundance mutants from the new pooled library, relative to abundances in the original library. (D) Abundances of low-abundance mutants from the original library, relative to abundances in the new pooled library.