vitro cell free protein synthesis system  (New England Biolabs)

Journal: Nucleic Acids Research

Article Title: Toehold-VISTA: a machine learning approach to decipher programmable RNA sensor-target interactions

doi: 10.1093/nar/gkag097

Figure Lengend Snippet: Generalization of toehold-VISTA across reporters and expression contexts. ( A ) Schematic of VISTA-designed toehold switches using a LacZ reporter for colorimetric output. ( B ) Endpoint photographs of cell-free transcription-translation reactions showing colorimetric responses for truncated, full-length, and non-cognate SARS-CoV-2 N gene targets. ( C ) Time-course absorbance measurements (570 nm) demonstrating activation kinetics for truncated and full-length targets relative to non-cognate controls. Reaction rate was determined at 60- and 70-min timepoints. Quantification of OFF-state ( D ) and ON-state absorbance for truncated ( E ) and full-length ( F ) RNA targets comparing VISTA-ranked and tsgen2-ranked toehold switches. Fold-change in LacZ reaction rates for truncated ( G ) and full-length ( H ) targets, showing improved fold changes for VISTA-ranked switches. Comparisons made with two-tailed t -test. Bars represent mean and whiskers represent SD.

Article Snippet: In vitro cell-free protein synthesis system (New England Biolabs E6800L) solutions A (0.4) and B (0.3) were combined, with the remaining volume composed of 0.005 U RNase inhibitor (New England Biolabs M0314L), 0.6 mg ml −1 enzymatic substrate chlorophenol red-β-D-galactopyranoside (CPRG, Roche 10884308001 distributed by Sigma), linear DNA templates comprising target or sensor-LacZ, and ultrapure DNase/RNase-free DI water to a total volume of 5 μl.

Techniques: Expressing, Activation Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Dyrk1a inhibition with the Novel Compound DYR533: A Cross-Disease Therapeutic Strategy Targeting Amyloidosis, Tau Pathogenesis, and Neuroinflammation

doi: 10.64898/2026.01.20.700091

Figure Lengend Snippet: (A) Chemical structure of DYR533. Kinome profiling of DYR533 (1 μM) was conducted across 403 wildtype (WT) human kinases using the Eurofins DiscoverX KinomeScan platform (Supp. Table 2). (B) A TREEspot kinase map illustrates binding interactions, with larger circles indicating stronger binding. (C) Selectivity metrics are shown for kinases with percent control <35% ((number of non-mutant kinases with %Ctrl <35) / (number of non-mutant kinases tested)). S-scores represent the fraction of kinases bound at thresholds of <1, <10, and <35, excluding mutant variants. Kinases inhibited >85% are highlighted in bold. Cell-free and in vitro assays confirmed that DYR533 blocks Dyrk1a intramolecular tyrosine autophosphorylation—similar to the established Dyrk1a inhibitor CaNDY—and promotes Dyrk1a degradation. (D) Experimental design for phosphotyrosine (pTyr) assays and (E) Dyrk1a degradation assays. (F) Western blot results from the pTyr assay and (G) corresponding quantification showing reduced pTyr levels following treatment with DYR533 or CaNDY at 3 an 10 μM. (H) Dyrk1a degradation assay results demonstrating decreased Dyrk1a levels after treatment with 1 or 3 μM DYR533 or CaNDY. Data are presented as mean ± SEM. *p < .05, **p<0.01, p<0.0001.

Article Snippet: The NEBExpress® Cell-free E. coli Protein Synthesis System (New England Biolabs, Cat #E5360S) was used to express a Dyrk1a construct comprising the kinase domain (residues 126–490) with an N-terminal His tag.

Techniques: Binding Assay, Control, Mutagenesis, In Vitro, Western Blot, Degradation Assay

Journal: iScience

Article Title: Accessible biocatalyst development by rapid in vitro semi-rational engineering (RISE) of enzymes

doi: 10.1016/j.isci.2025.114257

Figure Lengend Snippet: The concept of RISE (A) General workflow of RISE. (B) Workflow of the cell-free Q5 SDM protocol.

Article Snippet: NEBExpress® Cell-free E. coli Protein Synthesis System , NEB , E5360L.

Techniques:

Journal: iScience

Article Title: Accessible biocatalyst development by rapid in vitro semi-rational engineering (RISE) of enzymes

doi: 10.1016/j.isci.2025.114257

Figure Lengend Snippet: Advantages of RISE (A) Comparison of timelines for DNA manipulation by the standard and cell-free Q5 SDM (for supporting data see ). (B) Comparison of the cost of a 60-member single-point mutant DNA library obtained from commercial sourcing and in-house cell-free Q5 SDM (for supporting data see ). (C) Proposed role of focused enzyme development by RISE in chemical development in the pharmaceutical industry.

Article Snippet: NEBExpress® Cell-free E. coli Protein Synthesis System , NEB , E5360L.

Techniques: Comparison, Mutagenesis