Ma-PylRS-lib1 Pooled Library
(Pooled Library #224585)

• Purpose:

  This library contains active site mutants of a Methanomethylophilus alvus pyrrolysine tRNA-synthetase, which can be used in life/death/fluorescence-based selections in order to identify variants with substrate specificity for non-canonical amino acids. This allows researchers to generate new genetic code expansion (GCE) systems that site-specifically encode new non-canonical amino acids into proteins with altered side chain structures and novel chemistries.

• Vector Backbone:

  pBK- Ma PylRS WT (Plasmid #197573)

• Depositing Labs: Ryan Mehl
• Publication: Avila-Crump et al. ACS Chem Biol. 2022 Dec 16;17(12):3458-3469. doi: 10.1021/acschembio.2c00639.

Library Details

• Species: Methanomethylophilus alvus
• PylRS variants per library: 3.2 million
• Insert size: 834 bp
• Vector type: Bacterial expression

Library Shipping

Each library is delivered in a microcentrifuge tube on blue ice. The tube's contents will not necessarily be frozen. For best results, minimize freeze/thaws.

• Volume: ∼25 µL
• Concentration: 100 ng/µL

Resource Information

• Protocols:
  • Library Transformation Protocol (DOCX, 150 KB)
• Depositor Data:
  • Nucleotide frequency within mutagenized sites (XLSX, 10 KB)
  • Ma-PylRS-lib1 Primers (DOCX, 15 KB)

Terms and Licenses

Academic/Nonprofit Terms:

• UBMTA

Industry Terms:

• Not Available to Industry

Trademarks:

• Zeocin® is an InvivoGen trademark.

Depositor Comments

This library is used in E. coli and expresses variants of the Methanomethylophilus alvus pyrrolysine tRNA synthetase protein under a low-level constitutive GlnS promoter. Five sites in the amino acid substrate binding pocket of the synthetase have been mutated to all 20 amino acids. There are theoretically 11 million unique genetic variants. However, due to redundancy of the genetic code and the strategy used for library generation, these genetic variants express 3.2 million unique protein variants.

The vector backbone (pBK- Ma PylRS WT, Plasmid #197573) and plasmids required for life (Ma-pREP, Plasmid #197571), death (Ma-Barnase, Plasmid #197572), and fluorescence-based screening (pALS2-sfGFP 150TAG, Plasmid #197574) are also available.

Figure 1: The wild-type M. alvus pyrrolysine (Pyl) synthetase with a Pyl analogue docked in yellow (PDB: 6JP2; 2Q7G). Five sites, shown in green, in the amino acid substrate binding pocket of the synthetase were mutated to all 20 amino acids. Degenerate codons NNK were introduced at residues N166 and V168, while mutations at residues L125, A223, and W239 were generated using the 22-c codon trick (22c trick). The theoretical diversity, or product of the possible codons at each site, is 32² × 22³ = 10.9 million variants.