superfolder gfp sfgfp  (Addgene inc)

Journal: Nature Communications

Article Title: Temporal gene regulation enables controlled expression of gas vesicles and preserves bacterial viability

doi: 10.1038/s41467-025-67667-8

Figure Lengend Snippet: Schematic representation of the genetic constructs of the dual-inducer system ( a ) and two single‑reporter constructs ( b ) for fluorescent reporter proteins expression. c , d Fluorescence fold change of dual‑inducer vs. single‑reporter across inducer concentrations. c mCherry-only vs. mCherry-dual: NS at 0 µM IPTG; P = 0.0003, <0.0001 ( P = 0.00000058), and <0.0001 ( P = 0.000003) at 20, 200, and 400 µM. d sfGFP-only vs. sfGFP-dual: NS for all. e , f Fluorescence fold change of the dual-inducer vs. single-reporter without the corresponding inducer. e mCherry-dual vs. mCherry-only. P = 0.00461, 0.0165, <0.0001 ( P = 0.00002) at 0, 50, 500 ng/mL aTc. NS at 1000 ng/mL. f sfGFP-dual vs. sfGFP-only. NS at 0, 20, 200 µM IPTG; P = 0.00461 at 400 μM. Schematic representation of the dual-inducer system induced with varying concentrations of IPTG at fixed aTc concentration ( g ) or with varying concentrations of aTc at fixed IPTG concentration ( j ). Fold change in mCherry ( h ) and sfGFP ( k ). mCherry: 0 vs. 500 ng/mL aTc across IPTG concentrations: NS at 0, 20 µM; P = 0.0238, 0.0044 at 200, 400 µM. sfGFP: 0 vs. 200 µM IPTG across aTc concentrations: NS at 0, 50 ng/mL; P = 0.0089, 0.0012 at 500, 1000 ng/mL. Fold change in sfGFP ( i ) and mCherry ( l ). i sfGFP-dual cultures at 500 ng/mL aTc without IPTG vs. 0–400 µM IPTG: NS at 0 µM; P = 0.0009, 0.0007, 0.0010 at 20, 200, 400 µM. l mCherry-dual cultures at 200 µM IPTG without aTc vs. 0–1000 ng/mL aTc: NS at 0 ng/mL; P = 0.0478, 0.0013, 0.0145 at 50, 500, 1000 ng/mL. Fold change in c – f , h , i , k , l is plotted in arbitrary units (AU, y -axis), data representing mean ± s.d. ( n = 6 biologically independent samples). Statistics: c , d , h , k by two-tailed unpaired Welch t-test; e , f , i , l vs. grey controls by Brown–Forsythe and Welch one-way ANOVA with Dunnett T3 correction.

Article Snippet: The monomeric Cherry red fluorescent protein (mCherry) gene was obtained from Addgene (plasmid #29747), and the superfolder green fluorescent protein (sfGFP) gene was acquired from Addgene (plasmid #85492).

Techniques: Construct, Expressing, Fluorescence, Concentration Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Experimental Evaluation of AI-Driven Protein Design Risks Using Safe Biological Proxies

doi: 10.1101/2025.05.15.654077

Figure Lengend Snippet: (A) PDZ3-CRIPT binding caused reconstitution of a split dihydrofolate reductase (DHFR) enzyme in E. coli , conferring resistance to trimethoprim (TMP). Cell growth in media with TMP was measured as an indicator of PDZ3-CRIPT binding. The assay components were expressed using individual inducer compounds (salicylate/Sal, vanillic acid/Van), allowing for tuning of the system. (B) The URA3 enzyme catalyzes formation of the essential metabolite uridine monophosphate. In the absence of uracil, the chosen yeast host strain grew only with an active URA3 enzyme. The enzyme was expressed from a plasmid with a galactose-inducible promoter, and cell growth in uracilfree media was measured as a reporter of URA3 activity. (C) T7 RNA polymerase (T7 RNAP) is commonly used in cell-free expression (CFE) systems; in this system, T7 RNAP served as both an expression target and a functional component. Linear DNA encoding T7 RNAP was transcribed and translated into protein, then T7 RNAP transcribed superfolder GFP (sfGFP) mRNA from a target plasmid. The sfGFP was then translated by the CFE system, resulting in green fluorescence as a measurable reporter of T7 RNAP activity. All assay measurements were normalized to the response of the associated wildtype proteins and negative controls.

Article Snippet: Plasmid pJL1 expressing superfolder GFP (sfGFP), a gift from Michael Jewett (Addgene plasmid 69496), was used as the reporter plasmid. sfGFP was selected as the fluorescent output in cell-free reactions because it matures rapidly and produces a strong signal.

Techniques: Binding Assay, Plasmid Preparation, Activity Assay, Expressing, Functional Assay, Fluorescence

Journal: ACS applied bio materials

Article Title: Encapsulation of Transketolase into In Vitro -Assembled Protein Nanocompartments Improves Thermal Stability.

doi: 10.1021/acsabm.3c01153

Figure Lengend Snippet: Figure 2. In vitro sfGFP cargo loading specificity and scaffolding effect. (A) Workflow of in vitro sfGFP cargo loading method. Reassembly of encapsulin proteins was initiated by 10 times dilution of denaturant condition with reassembly buffer (0.3 M Tris-Cl pH 7.5, 0.15 M NaCl) to a final monomer concentration of 10 μM. (B, C) sfGFP loading into the Tm_encap (B) and sfGFP loading into the Mx_encap (C). Molar ratio of sfGFP to encapsulin monomer 0.2, 1, 5, and 10 to 1, respectively. M = molecular weight marker. Top black and white image shows fluorescence signal of sfGFP. Bottom image shows Coomassie-stained BN-PAGE gel. Full BN-PAGE is shown in Figure S2. (D) sfGFP cargo loading into Mx_encap (disassembled in 8 M urea) at increasing concentrations of sfGFP showing a decrease of assembly with high sfGFP concentration. Top black and white image shows fluorescence signal of sfGFP, bottom image shows Coomassie-stained BN-PAGE gel. M = molecular weight marker, vivo = in vivo-loaded encapsulins, A = assembled (before denaturation), and numbers in lanes indicate molar ratio of sfGFP to encapsulin monomer.

Article Snippet: The superfolder GFP (sfGFP) sequence was ordered from Addgene in the pJL1 expression vector under a T7 promotor (Addgene:69496).

Techniques: In Vitro, Scaffolding, Concentration Assay, Molecular Weight, Marker, Fluorescence, Staining, In Vivo

Journal: ACS applied bio materials

Article Title: Encapsulation of Transketolase into In Vitro -Assembled Protein Nanocompartments Improves Thermal Stability.

doi: 10.1021/acsabm.3c01153

Figure Lengend Snippet: Figure 3. In vitro sfGFP cargo loading and scaffolding effect on M. xanthus encapsulin. All data shown are from SEC purified samples. (A, B) SDS- PAGE densitometry of in vitro- versus in vivo-loaded encapsulin. Schematic below A and B shows summary of loading capacity of in vitro versus in vivo sfGFP-loaded encapsulins. Data were derived from a single experiment. (C−G) TEM micrographs of the following: in vitro-loaded Tm_encap with sfGFP-TmCLP, molar ratios indicated (C, D); in vivo-loaded Tm_encap with sfGFP-TmCLP (E); in vitro-loaded Mx_encap with sfGFP-MxCLP (F); and in vivo-loaded Mx_encap with sfGFP-MxCLP (G). TEM scale bar = 100 nm. (H) Violin plot of TEM diameter measurement of capsids before and after in vitro loading of sfGFP versus in vivo loading. Measurements of 100 particles per sample. Associated T = 3 and T = 1 size range indicated. (I) sfGFP:capsid monomer concentration affects in vitro capsid assembly efficiency and capsid properties. Increasing sfGFP concentrations lead to misformed Tm_encap and higher proportion of T = 3 over T = 1 Mx_encap species (scaffolding effect), followed by a decrease in Mx_encap assembly when ratio is above 5:1.

Article Snippet: The superfolder GFP (sfGFP) sequence was ordered from Addgene in the pJL1 expression vector under a T7 promotor (Addgene:69496).

Techniques: In Vitro, Scaffolding, Purification, SDS Page, In Vivo, Derivative Assay, Concentration Assay