pTriEx-mTFP1(cp175)-Luciferase-Venus(cp173) (called here MLucV) was created by using NEB Gibson assembly (NEBuilder HiFi DNA Assembly_E5520S) taking pTriEx-mTFP1(cp175)-Barnase-Venus(cp173) as template plasmid27 (link),28 (link) in which Barnase was replaced by mutant Luciferase6 (link).
To construct plasmid with a low leaky MLucV expression in Escherichia coli, MLucV was cloned into the pSE380 plasmid. MLucV was PCR amplified from the mTFP1-Luciferase-Venus-pTriEx4 plasmid as DNA template, using primers 5′-AGGAAACAGAATGGGCGGCCACCACCGC-3′ and 5′-CGCCAAAACATTAGATGTTGTGGCGGATCTTGAAGTTGG-3′. The pSE380 vector was PCR amplified using primers 5′-CAACATCTAATGTTTTGGCGGATGAGAG-3′ and 5′-GGCCGCCCATTCTGTTTCCTGTGTGAAATTG-3′ and pSE380-MLucV was created using the NEB Gibson assembly kit (NEBuilder HiFi DNA Assembly_ E5520S). All constructs were confirmed by Sanger sequencing.
GrpE and DnaJ were cloned into pET28-Smt3 expression vector. GrpE and DnaJ were first PCR amplified using E. coli genomic DNA then assembled by Gibson assembly (NEBuilder HiFi DNA Assembly_E5520S). The complete maps and sequences of the plasmids described above are available at 10.6084/m9.figshare.20502495.
To construct plasmid with a low leaky MLucV expression in Escherichia coli, MLucV was cloned into the pSE380 plasmid. MLucV was PCR amplified from the mTFP1-Luciferase-Venus-pTriEx4 plasmid as DNA template, using primers 5′-AGGAAACAGAATGGGCGGCCACCACCGC-3′ and 5′-CGCCAAAACATTAGATGTTGTGGCGGATCTTGAAGTTGG-3′. The pSE380 vector was PCR amplified using primers 5′-CAACATCTAATGTTTTGGCGGATGAGAG-3′ and 5′-GGCCGCCCATTCTGTTTCCTGTGTGAAATTG-3′ and pSE380-MLucV was created using the NEB Gibson assembly kit (NEBuilder HiFi DNA Assembly_ E5520S). All constructs were confirmed by Sanger sequencing.
GrpE and DnaJ were cloned into pET28-Smt3 expression vector. GrpE and DnaJ were first PCR amplified using E. coli genomic DNA then assembled by Gibson assembly (NEBuilder HiFi DNA Assembly_E5520S). The complete maps and sequences of the plasmids described above are available at 10.6084/m9.figshare.20502495.
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