Journal: Nucleic Acids Research

Article Title: Functional replacement of the endogenous tyrosyl-tRNA synthetase–tRNA Tyr pair by the archaeal tyrosine pair in Escherichia coli for genetic code expansion

doi: 10.1093/nar/gkq080

Figure Lengend Snippet: The tyrS complementation tests with pMjYS, pScYS, pMjYSYR and pScYSYR ( A ). The mutant cells transformed with these plasmids were inoculated on the LB plates containing 1% d -glucose and 34 µg/ml Cm. The three sectors of each half plate represent dilutions of the cells. An illustration of the genetic modifications and plasmid systems in FT3 and FB3 cells ( B ). The absence of the E. coli TyrRS activity in the strain FT1 ( C ). TOP10 and FT1 cells were each transformed with pACamKsupF and were then inoculated on the LB plate with 10 µg/ml Cm.

Article Snippet: Escherichia coli strains TOP10, BL21(DE3) and DH5α were purchased from Invitrogen, Novagen and Toyobo (Tokyo, Japan), respectively.

Techniques: Mutagenesis, Transformation Assay, Plasmid Preparation, Activity Assay

Journal: Nucleic Acids Research

Article Title: Functional replacement of the endogenous tyrosyl-tRNA synthetase–tRNA Tyr pair by the archaeal tyrosine pair in Escherichia coli for genetic code expansion

doi: 10.1093/nar/gkq080

Figure Lengend Snippet: E. coli DH5α cells, an E. coli K-12 strain, expressing the wild-type E. coli TyrRS from plasmid pEcYS (filled circles) and those expressing iodoTyrRS- ec from plasmid pEcIYS (open circles) were grown in the presence of 3-bromo- l -tyrosine (0.5 mg/ml) ( A ) and 3-azido- l -tyrosine (0.3 mg/ml) ( B ). FT3 cells expressing iodoTyrRS- ec were grown in the absence (filled circles) and presence (open circles) of 3-bromo- l -tyrosine ( C ). The growth was monitored upon the addition of the non-natural amino acid to the growth media.

Article Snippet: Escherichia coli strains TOP10, BL21(DE3) and DH5α were purchased from Invitrogen, Novagen and Toyobo (Tokyo, Japan), respectively.

Techniques: Expressing, Plasmid Preparation

Journal: Cell

Article Title: Structural insights into the process of GPCR-G protein complex formation

doi: 10.1016/j.cell.2019.04.021

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Bacterial and Virus Strains E. coli cells BL21(DE3) CWBIO CW0809S E. coli cells TOP10 CWBIO CW0807S E. coli cells Rossetta 2 (DE3) EMD Millipore 70954 E. coli cells DH10Bac Invitrogen 10361012 Chemicals, Peptides, and Recombinant Proteins Benzamidine Sigma Cat#B6506 Leupeptin Sigma Cat#L2884 n-dodecyl-beta-D-maltopyranoside (DDM) Anatrace Cat#D310 Lauryl Maltose Neopentyl Glycol (MNG) Anatrace Cat#NG310 Cholesterol hemisucinate (CHS) Sigma Cat#C6512 ANTI-FLAG M1 Agarose Affinity Gel Sigma-Aldrich Cat#A4596 1-Oleoyl-rac-glycerol (monoolein) Sigma Cat#M7765 Cholesterol Sigma Cat#C8667 POPG Avanti Cat# 840457 FLAG peptide Sigma-Aldrich Cat# F3290 Alprenolol Sigma-Aldrich Cat# A8676 BI-167107 Custom N/A Isoproterenol Tocris Cat# 1747 GDP Sigma Cat# 7127 ESF921 culture medium Expression Systems Cat# 96–001 Monobromobimane Invitrogen Cat# M1378 FBS VWR Cat#97068–085 PNGase F New England Biolabs Cat# P0708 Lambda Protein Phosphatase (Lambda PP) New England Biolabs Cat# P0753 TCEP Sigma-Aldrich Cat# C4706 Antarctic Phosphatase New England Biolabs Cat# M0289 CIP New England Biolabs Cat# M0290 DpnI New England Biolabs Cat# R0176 Dihydroalprenolol hydrochloride, Levo-[ring, propyl- 3 H(N)] PerkinElmer Cat# NET720001MC Polyethylenimine Sigma Cat# 408727 cOmpleteTM, EDTA-free Protease Inhibitor Cocktail Roche Cat# 30307800 CK28 beads Bertin Corp. Cat# 03961CK28 GF/B unifilters, Whatmann PerkinElmer Cat# 6005177 Deposited Data β2AR-T4L-GsCT-CC structure This paper PDB: 6E67 Gs GDP structure This paper PDB: 6EG8 Experimental Models: Cell Lines Insect cell line Sf9 Expression Systems N/A Insect cell line High Fives (Tni) Expression Systems N/A BHK-21 cell line ATCC Cat# ATCC® CCL-10TM Recombinant DNA pfastbac-wtβ2AR This study N/A pfastbac-mutβ2AR This study N/A pfastbac-Gαs/βγ This study N/A Pfastbac-mutGαs/βγ This study N/A pET28a-ApoA1 Denisov et al., 2004 Addgene Plasmid# 20060 pET28a-Gαs C3S This study N/A pVLdual_Gβγ C68S This study N/A pcDNA3.1(+)-β2AR This study N/A pcDNA3.1(+)-β2AR-cpep This study N/A pcDNA3.1(+)-β2AR-cpep mutants This study N/A Software and Algorithms COOT Emsley et al., 2010 www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot XDS Kabsch, 2010 http://xds.mpimf-heidelberg.mpg.de/ Phaser McCoy et al., 2007 http://www.ccp4.ac.uk Phenix Adams et al., 2010 https://www.phenix-online.org PyMOL Schrodinger LLC https://www.pymol.org/2/ KAMO Yamashita et al., 2018 https://github.com/keitaroyam/yamtbx Prism v.6.0 GraphPad Software, Inc. https://www.graphpad.com Open in a separate window KEY RESOURCES TABLE An active β2AR structure was obtained by fusion with residues 381–394 of Gs (GsCT).

Techniques: Virus, Recombinant, Expressing, Protease Inhibitor, Plasmid Preparation, Software