t7 terminator  (Integrated DNA Technologies)

Journal: bioRxiv

Article Title: Efficient cell-free evolution of RNA polymerases by droplet microfluidics

doi: 10.64898/2026.02.23.707346

Figure Lengend Snippet: (a) Optimization of elongation factor P (EF-P) and chaperone conditions. (b) Optimization of the linker length between Sso7d and SP6 RNA polymerase. (c) Crystal structure of T7 RNA polymerase initiation state (PDB ID: 2PI4). (d) Crystal structure of T7 RNA polymerase intermediate state (PDB ID: 3E2E). (e) Crystal structure of T7 RNA polymerase elongation state (PDB ID: 1H38). (f) Predicted structure of SP6 RNA polymerase by ColabFold. N-terminal domains of RNA polymerases (Residues 1-240 in SP6 RNAP, residues 1-266 in T7 RNAP) are colored in purple, other regions are colored in gray. RNAs are colored in yellow and template DNAs are colored light purple, and non-templated DNAs are in pink.

Article Snippet: Resulting gene fragment was introduced by NEBuilder HiFi DNA Assembly into the vector prepared by inverse PCR with Oligo32, Oligo33 and pET22b-FKBP-C-SP6 RNAP Lib5-3-#01 (198+) (v4)_N-SP6 RNAP-v3 (−197). pET22b-Rapa-spT7 RNAP: The gene fragment encoding N-29-1 split N-terminal T7 RNAP-FRB was amplified by PCR using Oligo34 and Oligo35 with p5-79 N-29-1 split N-terminal T7 RNAP variant-GGSGSGSS-FRB expression plasmid (#118087, Addgene) as templates.

Techniques:

Journal: bioRxiv

Article Title: Efficient cell-free evolution of RNA polymerases by droplet microfluidics

doi: 10.64898/2026.02.23.707346

Figure Lengend Snippet: (a) Crystal structure of T7 RNA polymerase elongation state (PDB ID: 1H38). Split site of previously reported spT7 RNAP is between K179 (red sphere) and K180 (blue sphere). Protein is colored in green, RNAs are colored orange, template DNAs are colored light purple, and non-templated DNAs are in pink. (b) Predicted structure of SP6 RNA polymerase by ColabFold. Split sites tried in this study are shown in red and blue spheres. (c) Rapamycin-induced spSP6 RNAP activities measured by mNG reporter assay in PURE system. The data are presented as the mean ± SD from three independent experiments. (d, e) Histograms of the fluorescence signal distribution of droplets screened for spSP6 RNAP Library 4 ( d ) or Library 5 ( e ) in FADS. Overlaid histograms are the same as or 3c. (f, g) Sequence alignment of evolved variants identified from Library 4.3 ( f ) and Library 5.3 ( g ). Residue numbers correspond to SP6 RNAP-wt (UniProt: P06221). The same residues relative to SP6 RNAP-v3 are shown in dots. ( h, i ) Rapamycin-induced spSP6 RNAPs’ activity measured by mNG reporter assay in PURE system. The data are presented as the mean ± SD from three independent experiments.

Article Snippet: Resulting gene fragment was introduced by NEBuilder HiFi DNA Assembly into the vector prepared by inverse PCR with Oligo32, Oligo33 and pET22b-FKBP-C-SP6 RNAP Lib5-3-#01 (198+) (v4)_N-SP6 RNAP-v3 (−197). pET22b-Rapa-spT7 RNAP: The gene fragment encoding N-29-1 split N-terminal T7 RNAP-FRB was amplified by PCR using Oligo34 and Oligo35 with p5-79 N-29-1 split N-terminal T7 RNAP variant-GGSGSGSS-FRB expression plasmid (#118087, Addgene) as templates.

Techniques: Reporter Assay, Fluorescence, Sequencing, Residue, Activity Assay

Journal: bioRxiv

Article Title: Efficient cell-free evolution of RNA polymerases by droplet microfluidics

doi: 10.64898/2026.02.23.707346

Figure Lengend Snippet: (a) Optimization of elongation factor P (EF-P) and chaperone conditions. (b) Optimization of the linker length between Sso7d and SP6 RNA polymerase. (c) Crystal structure of T7 RNA polymerase initiation state (PDB ID: 2PI4). (d) Crystal structure of T7 RNA polymerase intermediate state (PDB ID: 3E2E). (e) Crystal structure of T7 RNA polymerase elongation state (PDB ID: 1H38). (f) Predicted structure of SP6 RNA polymerase by ColabFold. N-terminal domains of RNA polymerases (Residues 1-240 in SP6 RNAP, residues 1-266 in T7 RNAP) are colored in purple, other regions are colored in gray. RNAs are colored in yellow and template DNAs are colored light purple, and non-templated DNAs are in pink.

Article Snippet: The gene fragment encoding FKBP-split C-terminal T7 RNAP was amplified by PCR using Oligo36, Oligo37, Oligo38, Oligo39 with p5-39 FKBP-TSGGSG-Split C-terminal T7 RNAP expression plasmid (#118088, Addgene) as templates.

Techniques:

Journal: bioRxiv

Article Title: Efficient cell-free evolution of RNA polymerases by droplet microfluidics

doi: 10.64898/2026.02.23.707346

Figure Lengend Snippet: (a) Crystal structure of T7 RNA polymerase elongation state (PDB ID: 1H38). Split site of previously reported spT7 RNAP is between K179 (red sphere) and K180 (blue sphere). Protein is colored in green, RNAs are colored orange, template DNAs are colored light purple, and non-templated DNAs are in pink. (b) Predicted structure of SP6 RNA polymerase by ColabFold. Split sites tried in this study are shown in red and blue spheres. (c) Rapamycin-induced spSP6 RNAP activities measured by mNG reporter assay in PURE system. The data are presented as the mean ± SD from three independent experiments. (d, e) Histograms of the fluorescence signal distribution of droplets screened for spSP6 RNAP Library 4 ( d ) or Library 5 ( e ) in FADS. Overlaid histograms are the same as or 3c. (f, g) Sequence alignment of evolved variants identified from Library 4.3 ( f ) and Library 5.3 ( g ). Residue numbers correspond to SP6 RNAP-wt (UniProt: P06221). The same residues relative to SP6 RNAP-v3 are shown in dots. ( h, i ) Rapamycin-induced spSP6 RNAPs’ activity measured by mNG reporter assay in PURE system. The data are presented as the mean ± SD from three independent experiments.

Article Snippet: The gene fragment encoding FKBP-split C-terminal T7 RNAP was amplified by PCR using Oligo36, Oligo37, Oligo38, Oligo39 with p5-39 FKBP-TSGGSG-Split C-terminal T7 RNAP expression plasmid (#118088, Addgene) as templates.

Techniques: Reporter Assay, Fluorescence, Sequencing, Residue, Activity Assay