Journal: Foods

Article Title: Validation of a Real-Time PCR Assay for Identification of Fresh and Processed Carica papaya Botanical Material: Using Synthetic DNA to Supplement Specificity Evaluation

doi: 10.3390/foods12030530

Figure Lengend Snippet: Target and non-target samples used for validation of Carica papaya assay.

Article Snippet: Curcuma longa , Zingiberaceae , 00031107–328 , Chromadex , Turmeric , Non-Target , Dried root.

Techniques: Biomarker Discovery

Journal: Foods

Article Title: Validation of a Real-Time PCR Assay for Identification of Fresh and Processed Carica papaya Botanical Material: Using Synthetic DNA to Supplement Specificity Evaluation

doi: 10.3390/foods12030530

Figure Lengend Snippet: Target and non-target samples used for validation of Carica papaya assay.

Article Snippet: Spinacia oleracea , Amaranthaceae , 4647 , Chromadex , Spinach , Non-Target , Dried leaf.

Techniques: Biomarker Discovery

Journal: Nucleic Acids Research

Article Title: A genetically encoded fluorescent tRNA is active in live-cell protein synthesis

doi: 10.1093/nar/gkw1229

Figure Lengend Snippet: Expression of a V-Spinach tRNA in E. coli . ( A ) The Spinach aptamer (in green) is fused to the V-loop of E. coli (Ec) tRNA Tyr/CUA (in orange), harboring the amber-reading anticodon 5΄-CUA-3΄. The drawing of the Spinach aptamer is based on the crystal structure of the Fab BL3-6-bound aptamer . The sequence of the Spinach aptamer is as described and it is covalently joined to the V-loop between C 47:2 and A 47:3 as shown by two black up arrow icons. The U71 and U72 substitutions in Ec tRNA Tyr are shown by downward arrows, which replace G1-C72 and G2-C71 base pairs with G1-U72 and G2-U71, respectively. ( B ) Fluorescence microscope images of E. coli cells expressing the amber suppressor form of V-Spinach tRNA Tyr versus cells expressing the Spinach stand-alone. DIC images and scale bars (each representing 2 μm) are shown. On average, when examined in liquid cultured media, 96.4 ± 0.1% of cells expressing the V-Spinach tRNA showed the GFP-like fluorescence ( n = 100).

Article Snippet: The gene for V-Baby-Spinach tRNA Ala/UGC , V-Baby-Spinach tRNA Phe/GAA , and V-Baby-Spinach tRNA Pro/UGG was each synthesized by GenScript, where the sequence of the Baby Spinach aptamer ( ) was inserted into the V-loop of the respective tRNA ( ).

Techniques: Expressing, Sequencing, Fluorescence, Microscopy, Cell Culture

Journal: Nucleic Acids Research

Article Title: A genetically encoded fluorescent tRNA is active in live-cell protein synthesis

doi: 10.1093/nar/gkw1229

Figure Lengend Snippet: Live-cell imaging of the amber-suppressor form of V-Spinach tRNA Tyr /CUA in E. coli . ( A ) Fluorescence microscope images of E. coli cells expressing the amber suppressor form of V-Spinach tRNA Tyr , WT (top) or U71-U72 variant (bottom). The Spinach tRNA fusion was constitutively expressed in CA244 cells. Upon entering log-phase, an aliquot of cells was harvested, washed, suspended in M9 minimal medium and immobilized on a poly-(D)-lysine-coated glass slide. Cells were incubated with DFHBI for 5 min, and washed off the excess ligand. The diffusion of bound DFHBI from cells was limited in the immobilized condition and in minimal media. Green fluorescence was excited by 488 nm Argon ion laser and monitored under a microscope over a time course at a constant temperature of 22.5°C. DIC images and scale bars (each representing 2 μm) are shown. Few cell-to-cell variations are seen in the green channel. ( B ) Quantification of the time courses of fluorescence intensity. Average intensity of the pixels corresponding to the area of a single E. coli cell was quantified by image analysis software (see methods for details). Error bars are expressed as standard errors of mean (SEM), n = 100 for each of the time points. ( C ) The average fluorescence intensity of the native and the U71-U72 variant of V-Spinach tRNA Tyr at 60 min compared to negative controls, where only the Spinach stand-alone was expressed (‘Spinach’) or where V-Spinach tRNA Tyr/CUA was expressed in the absence of DFHBI (‘No DFHBI’). Error bars are expressed as SEM, n = 100 for each of the time points.

Article Snippet: The gene for V-Baby-Spinach tRNA Ala/UGC , V-Baby-Spinach tRNA Phe/GAA , and V-Baby-Spinach tRNA Pro/UGG was each synthesized by GenScript, where the sequence of the Baby Spinach aptamer ( ) was inserted into the V-loop of the respective tRNA ( ).

Techniques: Live Cell Imaging, Fluorescence, Microscopy, Expressing, Variant Assay, Incubation, Diffusion-based Assay, Software

Journal: Nucleic Acids Research

Article Title: A genetically encoded fluorescent tRNA is active in live-cell protein synthesis

doi: 10.1093/nar/gkw1229

Figure Lengend Snippet: Activity of V-Spinach tRNA in protein synthesis. ( A ) Suppression of the lacZ125am locus in E. coli CA244 cells (Yale CGSC stock center) by V-Spinach tRNA Tyr with the 5΄-CUA anticodon (CUA), but not by the Spinach stand-alone, V-Spinach tRNA Tyr with the 5΄-GUA anticodon (GUA), or T- or D-Spinach tRNA Tyr with the CUA anticodon (CUA). E. coli cells expressing each Spinach construct were streaked out on an LB plate with ampicillin. Three to five single colonies with each construct were picked and re-streaked to 1 cm length on a minimal M9 plate with ampicillin and X-gal. This single-colony isolation ensured the homogeneity of each clone of cells. Representative streaks on M9 plates are shown. ( B ) Activity of the wild-type (WT) Ec tRNA Tyr and V-Spinach Ec tRNA Tyr in major steps of protein synthesis, including aminoacylation, stability of the ternary complex, accommodation to the ribosome A-site, and formation of the first peptide bond on an mRNA template with the sequence starting with 5΄-AUG-UAU-3΄. Both V-Spinach tRNA Tyr and Ec tRNA Tyr , each containing the natural Tyr anticodon 5΄-GUA, were isolated from E. coli cells and purified to homogeneity. ( C ) Suppression of the amber codon 5΄-UAG at the lacZ U118Am locus by the amber suppressor form of V-Spinach tRNA Tyr harboring the anticodon 5΄-CUA, leading to synthesis of β-gal. Measurement of β-gal activity encoded in XAC-1 cells expressing a plasmid-borne amber suppressor form of tRNA Tyr (Tyr), amber suppressor form of V-Spinach tRNA Tyr (Spinach), or none. Each activity is reported as a fraction relative to the activity encoded by the lacZ WT gene (no amber mutation) in XAC-1 cells. Error bars denote SD (standard deviation), n = 3. ( D ) Suppression of trmD Am (Y19) by the amber suppressor form of V-Spinach tRNA Tyr leads to synthesis of the growth-dependent TrmD enzyme, which supports E. coli cell viability. The trmD Am (Y19) locus contains an amber codon at the Y19 position, which prevents translation of the trmD gene. This study was conducted in E. coli trmD -knockout ( trmD-KO ) cells where the chromosome-encoded trmD gene was disrupted with an antibiotic marker and cells were maintained viable in the presence of arabinose by a plasmid-borne and arabinose-controlled expression of the human counterpart trm5 gene . ( E ) Examination of viability of E. coli trmD-KO cells expressing a plasmid-borne trmD WT (row 1), a plasmid-borne trmD Am (Y19) (row 2), a plasmid-borne trmD Am (Y19) and the amber suppressor gene of Ec tRNA Tyr (row 3), and a plasmid-borne trmD Am (Y19) and the amber suppressor gene of V-Spinach tRNA Tyr (row 4) in the presence or absence of arabinose (Ara).

Article Snippet: The gene for V-Baby-Spinach tRNA Ala/UGC , V-Baby-Spinach tRNA Phe/GAA , and V-Baby-Spinach tRNA Pro/UGG was each synthesized by GenScript, where the sequence of the Baby Spinach aptamer ( ) was inserted into the V-loop of the respective tRNA ( ).

Techniques: Activity Assay, Expressing, Construct, Isolation, Sequencing, Purification, Plasmid Preparation, Mutagenesis, Standard Deviation, Knock-Out, Marker

Journal: Nucleic Acids Research

Article Title: A genetically encoded fluorescent tRNA is active in live-cell protein synthesis

doi: 10.1093/nar/gkw1229

Figure Lengend Snippet: Localization of V-Spinach tRNA Tyr in living E. coli cells relative to L9-mCherry ribosomes. ( A ) E. coli JM109 cells, expressing L9-mCherry from the chromosome and V-Spinach tRNA Tyr/GUA (top) or the stand-alone Spinach (bottom) from pKK223-3 (induced with IPTG), were observed under microscope as described in Materials and Methods. ( B and C ) Lengthwise intensity scans of mCherry (dotted line) and Spinach fluorescence (solid line) of a representative cell from the two strains. ( D ) E. coli JM109 cells, harboring no plasmid, were imaged for L9-mCherry (left) and with DFHBI (right) and stained with DAPI (middle). ( E ) Lengthwise intensity scans of mCherry (red line), Spinach fluorescence (green line), and DAPI stain (blue line) of a representative cell.

Article Snippet: The gene for V-Baby-Spinach tRNA Ala/UGC , V-Baby-Spinach tRNA Phe/GAA , and V-Baby-Spinach tRNA Pro/UGG was each synthesized by GenScript, where the sequence of the Baby Spinach aptamer ( ) was inserted into the V-loop of the respective tRNA ( ).

Techniques: Expressing, Microscopy, Fluorescence, Plasmid Preparation, Staining