研究目的
To expedite the discovery of novel pyrrolysyl-tRNA synthetase (PylRS) variants that can charge non-natural amino acids into proteins site-specifically.
研究成果
The FACS-based screening protocol enables rapid identification of functional PylRS variants within a week, demonstrating its efficiency and applicability for charging various non-natural amino acids into proteins. The method offers advantages in sensitivity, flexibility, and speed over traditional selection methods.
研究不足
The method is not practical for screening libraries larger than 1 × 10^8 due to FACS limitations. It may require optimization for larger libraries through intelligent mutagenesis. The protocol is specific to bacterial systems and may not directly apply to other organisms without modification.
1：Experimental Design and Method Selection:
The protocol involves constructing a PylRS library using Gibson assembly, selecting functional mutants via fluorescence-activated cell sorting (FACS), and validating them using a fluorescent reporter protein in Escherichia coli.
2：Sample Selection and Data Sources:
The PylRS library is constructed by randomizing residues Tyr306, Leu309, Cys348, and Tyr
3：Acella? cells (a derivative of BL21(DE3)) are used as the bacterial strain, pretransformed with a reporter plasmid. List of Experimental Equipment and Materials:
3 Includes plasmids (e.g., pEvol-mmPylRS, pET-sfGFP-204TAG), enzymes (e.g., Q5 hot start high-fidelity 2× master mix, Phusion high-fidelity DNA polymerase), reagents (e.g., BocK, SphK, IPTG, arabinose), and equipment like flow cytometers and plate readers.
4：Experimental Procedures and Operational Workflow:
Steps include library construction via PCR and Gibson assembly, transformation into Acella cells, induction of protein expression with non-natural amino acids, FACS analysis and sorting, and verification of activity through fluorescence measurement and DNA sequencing.
5：Data Analysis Methods:
FACS data analyzed using Flowing 2.5.1 software; fluorescence intensity measured with a microtiter plate reader; DNA sequencing for variant identification.
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Nanodrop 2000c spectroscopy
2000c
Thermo Fisher Scientific
Used for determining plasmid concentration.
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Q5 hot start high-fidelity 2× master mix
New England Biolabs
Used for PCR amplification in the construction of the PylRS library.
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Phusion high-fidelity DNA polymerase
New England Biolabs
Used for PCR assembly of DNA fragments.
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HiFi DNA Assembly master mix
2×
New England Biolabs
Used for Gibson assembly of DNA fragments.
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NEB5α cells
New England Biolabs
Electrocompetent cells used for transformation in library construction.
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Acella? cells
EdgeBio
Bacterial strain used for expression and amplification, derived from BL21(DE3).
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BD LSR Fortessa X-20 flow cytometer
LSR Fortessa X-20
BD Biosciences
Used for FACS analysis of cell fluorescence.
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BD FACSAria II cell sorter
FACSAria II
BD Biosciences
Used for sorting fluorescent cells based on GFP expression.
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Synergy H1 plate reader
H1
BioTek Instruments
Used for measuring GFP fluorescence in cell lysates.
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BugBuster Protein Extraction Reagent
EMD Millipore
Used for lysing cells to extract proteins.
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