Reynolds DHFR Saturation Mutagenesis Libraries
(Pooled Library #1000000194, #1000000195, #1000000196)
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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.
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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)
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Depositing Labs
Ordering
Item | Catalog # | Description | Quantity | Price (USD) | ||
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Pooled Library | 1000000194 | WT TYMS library (four tubes)
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1 | $380 | Add to Cart | |
Pooled Library | 1000000195 | R166Q TYMS library (four tubes)
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1 | $380 | Add to Cart | |
Pooled Library | 1000000196 | Q33S TYMS library (four tubes)
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1 | $380 | Add to Cart |
Library Details
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SpeciesE.coli
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DHFR variants per library~3,002
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Insert sizeEach plasmid contains DHFR (477 bp), cloned into multiple cloning site 1 and TYMS (792 bp), cloned into multiple cloning site 2.
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Vector typeBacterial 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.
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Volume∼20 µL
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Concentration50 ng/µL
Resource Information
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Protocols
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Depositor Data
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Terms and Licenses
Academic/Nonprofit TermsIndustry Terms- Not Available to Industry
Trademarks- Zeocin® is an InvivoGen trademark.
Depositor Comments
Identification and counting of specific mutants throughout the experiment were performed with the script countingAlleles.py in GitHub (Link opens in a new window). 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.
These pooled libraries were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the plasmids were described, and include Addgene in the Materials and Methods of your future publications.
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For your Materials & Methods section:
Reynolds DHFR Saturation Mutagenesis WT TYMS Library was a gift from Kimberly Reynolds (Addgene #1000000194)
Reynolds DHFR Saturation Mutagenesis R166Q TYMS Library was a gift from Kimberly Reynolds (Addgene #1000000195)
Reynolds DHFR Saturation Mutagenesis Q33S TYMS Library was a gift from Kimberly Reynolds (Addgene #1000000196) -
For your References section:
The genetic landscape of a metabolic interaction. Nguyen TN, Ingle C, Thompson S, Reynolds KA. Nat Commun. 2024 Apr 18;15(1):3351. doi: 10.1038/s41467-024-47671-0. PubMed 38637543