BioBloom Pooled Libraries
(Pooled Library #1000000273)

• Purpose:

  BioBloom is a method for barcoded saturation mutagenesis of bacterial genomes. The BioBloom Pooled Libraries contain nearly every possible single-base transition and transversion mutation of the Escherichia coli genome, with an identifying “pro-donor” barcode sequence to facilitate pooled selection experiments.

  BioBloom can be used to quickly identify beneficial mutations in a given selection condition that improve E. coli growth, and produce genome-wide datasets describing many such mutations.

  This kit contains 3 tubes, each an independently-prepared replicate of the BioBloom-Ec-2.0 library. Best results are achieved by performing experiments in triplicate, using these three replicate libraries to seed each replicate.

• Vector Backbone:

  pMS_375 (Plasmid #182132)

• Depositing Labs: Pioneer Labs
• Publication: Schubert et al. bioRxiv. 2026 Feb 04. doi: 10.64898/2026.02.04.703572.

Library Details

• Species: E. coli
• Number of genes: ~20 million
• Insert info: The unique sequences used to identify each mutant are 82 bp in length. Each mutant has a unique retron “pro-donor” sequence that can be used to identify the mutant as a barcode, analogous to a CRISPR guide sequence.

Library Shipping

The BioBloom Libraries will be shipped frozen on dry ice.

• Aliquots: 1 mL of confluent glycerol stock (~1 billion cells)*
  *See depositor comments for additional details

Resource Information

• Protocols:
  • Library Amplification and Sequencing Protocol (DOCX, 18 KB)
• Depositor Data:
  • BioBloom Source Data
  • BioBloom Backbone (PNG, 36 KB)
  • BioBloom Library Coverage (PNG, 23 KB)
  • BioBloom SNPs Analysis (PNG, 19 KB)
• Scripts:
  • BioBloom GitHub Page

Terms and Licenses

Academic/Nonprofit Terms:

• UBMTA

Industry Terms:

• Not Available to Industry

Trademarks:

• Zeocin® is an InvivoGen trademark.

Depositor Comments

*The 1 mL glycerol stocks should cover the library well. We recommend recovery/outgrowth of these stocks and immediate use for experiments, but you may wish to cryopreserve material from this outgrowth, to produce “T2” stocks which may not sample a full saturation library as completely, but still be useful for some experiments.

Figure 1: Overview of the BioBloom library method using Retron Library Recombineering (RLR). (a) Each plasmid contains a pro-donor sequence of 82 bp to identify the mutation, a retron reverse transcriptase (RT, orange), and a single-stranded annealing protein (SSAP, purple). The pro-donor sequence is transcribed and targeted by the RT, producing a donor single stranded DNA (ssDNA) including single nucleotide polymorphisms (SNPs) and surrounding homology to the genome (red). The ssDNA donor is recruited to the replication fork where it creates precise edits, catalyzed by the SSAP. The pro-donor is retained on the plasmid after the edit, acting as a barcode to identify the mutant using amplicon-sequencing and specific primers (green arrows). (b) The 82 bp donor sequences were designed for each genomic position containing a degenerate “N” at the central base. These sequences were synthesized with flanking golden gate adaptors (green), assembled into a plasmid library, then transformed and induced in recipient cells to create the barcoded BioBloom libraries.

Figure 2: Schematic representing the saturating mutagenesis coverage at each DNA base in the E. coli genome.

Please visit https://doi.org/10.64898/2026.02.04.703572 for bioRxiv report.