Reynolds DHFR Saturation Mutagenesis Libraries
(Pooled Library #1000000194, #1000000195, #1000000196)

• Purpose:

  Saturation mutagenesis library of E. coli dihydrofolate reductase (DHFR) in the background of three different thymidylate synthase (TYMS) variants. The TYMS variants are wild-type (WT) E. coli, TYMS Q33S (a moderate loss-of-function mutation), and TYMS R166Q (a catalytically inactive variant).

  Each of the saturation mutagenesis libraries were constructed as four sub-libraries (SL, to allow for coverage of each sub-library region with short read sequencing). SL1 covers all possible single mutations in positions 2–40 (the start codon is unmutagenized), SL2 covers all possible single mutations to positions 41–80, SL3 covers all possible single mutations to positions 81–120, and SL4 covers all possible single mutations to positions 121–159.

• Vector Backbone:

  pTET-Duet - a modified version of the pACYC Duet vector (Novagen), which we edited to introduce a tet repressor upstream of multiple cloning site 2 (in place of the starting T7 promoter)

• Depositing Labs: Kimberly Reynolds
• Publication: Nguyen et al Nat Commun. 2024 Apr 18;15(1):3351. doi: 10.1038/s41467-024-47671-0.

Library Details

• Species: E.coli
• DHFR variants per library: ~3,002
• Insert size: Each plasmid contains DHFR (477 bp), cloned into multiple cloning site 1 and TYMS (792 bp), cloned into multiple cloning site 2.
• Vector type: Bacterial expression

Library Shipping

Each library will be delivered as four individual sub-libraries as suspended DNA in microcentrifuge tubes on blue ice. The tube contents will not necessarily be frozen. For best results, minimize freeze-thaws.

• Volume: ∼20 µL
• Concentration: 50 ng/µL

Resource Information

• Protocols:
  • Library amplification and PCR protocol (DOCX, 23 KB)
• Depositor Data:
  • Mutational scanning library completeness (PDF, 465 KB)

Terms and Licenses

Academic/Nonprofit Terms:

• UBMTA

Depositor Comments

• Figure 1. DHFR mutagenesis libraries schematic overview. Each sub-library contains all possible single amino acid mutations to DHFR positions 2–40 (SL1), 41–80 (SL2), 81–120 (SL3), and 121–159 (SL4) in one of three TYMS backgrounds (WT, Q33S, or R166Q).

Identification and counting of specific mutants throughout the experiment were performed with the script countingAlleles.py in GitHub . For a more complete description of the data analysis procedure, please see the methods section of our publication. All major analysis scripts are contained in the accompanying GitHub code repository.