Mutagenesis:Article Title: Design of a Histidine Kinase FRET Sensor to Detect Complex Signal Integration within Living Bacteria.
Article Snippet: Table S2: Site directed mutagenesis primers for introduction of CckA point mutants Template Mutation Forward Primer Reverse Primer Annealing Temperature (°C) pMT383 Y514D nSWD98 nSWD99 68 pMT383 D293A nSWD104 nSWD105 65 Table S3: Plasmids utilized in this study Name Description Reference/Addgene # pSWD95 2nd Gen CckA-FRET Sensor This work pSWD155 DivL overexpression plasmid This work pSWD156 DivL A601L overexpression plasmid This work pSWD95Y514D CckA FRET Y514D Mutant sensor This work pSWD95D293A CckA FRET D293A Mutant sensor This work pSWD94 PopZ overexpression plasmid This work pSWD109 2nd Gen CckA 183/184 FRET Sensor This work pSWD110 2nd Gen CckA 192/193 FRET Sensor This work pSWD111 2nd Gen CckA 193/194 FRET Sensor This work pWSC10011 1st Gen CckA CckA 69/70 FRET Sensor This work pWSC10012 1st Gen CckA 91/92 FRET Sensor This work pWSC10013 1st Gen CckA N-terminal FRET Sensor This work pSWD55 Short Fibronectin FRET Standard This work pSWD56 Long Fibronectin FRET Standard This work pSWD275 mRuby3 Intermolecular Sensor This work pSWD276 mClover3 Intermolecular Sensor This work pSWD14 mClover3/mRuby3 Bajar, et al. 8, Addgene# 74252 pSWD63 cckA::cckA-mClover3 This work pMT383 CckA template Shapiro Lab pXCFPC-6 Xylose inducible Vector Thanbichler, et al. 7 pVMCS-1 Vanillate inducible Vector Thanbichler, et al. 7 pBVMCS-2 Vanillate inducible high copy plasmid Thanbichler, et al. 7 pXYFPC-1 Xylose inducible Vector Thanbichler, et al. 7 pXCFPC-2 Xylose inducible Vector Thanbichler, et al. 7 FN_7-8_FN_10 Fibronectin Gene Block Ohashi, et al. 9 Table S4: DNA primers utilized in this study.
Over Expression:Article Title: Design of a Histidine Kinase FRET Sensor to Detect Complex Signal Integration within Living Bacteria.
Article Snippet: Table S2: Site directed mutagenesis primers for introduction of CckA point mutants Template Mutation Forward Primer Reverse Primer Annealing Temperature (°C) pMT383 Y514D nSWD98 nSWD99 68 pMT383 D293A nSWD104 nSWD105 65 Table S3: Plasmids utilized in this study Name Description Reference/Addgene # pSWD95 2nd Gen CckA-FRET Sensor This work pSWD155 DivL overexpression plasmid This work pSWD156 DivL A601L overexpression plasmid This work pSWD95Y514D CckA FRET Y514D Mutant sensor This work pSWD95D293A CckA FRET D293A Mutant sensor This work pSWD94 PopZ overexpression plasmid This work pSWD109 2nd Gen CckA 183/184 FRET Sensor This work pSWD110 2nd Gen CckA 192/193 FRET Sensor This work pSWD111 2nd Gen CckA 193/194 FRET Sensor This work pWSC10011 1st Gen CckA CckA 69/70 FRET Sensor This work pWSC10012 1st Gen CckA 91/92 FRET Sensor This work pWSC10013 1st Gen CckA N-terminal FRET Sensor This work pSWD55 Short Fibronectin FRET Standard This work pSWD56 Long Fibronectin FRET Standard This work pSWD275 mRuby3 Intermolecular Sensor This work pSWD276 mClover3 Intermolecular Sensor This work pSWD14 mClover3/mRuby3 Bajar, et al. 8, Addgene# 74252 pSWD63 cckA::cckA-mClover3 This work pMT383 CckA template Shapiro Lab pXCFPC-6 Xylose inducible Vector Thanbichler, et al. 7 pVMCS-1 Vanillate inducible Vector Thanbichler, et al. 7 pBVMCS-2 Vanillate inducible high copy plasmid Thanbichler, et al. 7 pXYFPC-1 Xylose inducible Vector Thanbichler, et al. 7 pXCFPC-2 Xylose inducible Vector Thanbichler, et al. 7 FN_7-8_FN_10 Fibronectin Gene Block Ohashi, et al. 9 Table S4: DNA primers utilized in this study.
Plasmid Preparation:Article Title: Design of a Histidine Kinase FRET Sensor to Detect Complex Signal Integration within Living Bacteria.
Article Snippet: Table S2: Site directed mutagenesis primers for introduction of CckA point mutants Template Mutation Forward Primer Reverse Primer Annealing Temperature (°C) pMT383 Y514D nSWD98 nSWD99 68 pMT383 D293A nSWD104 nSWD105 65 Table S3: Plasmids utilized in this study Name Description Reference/Addgene # pSWD95 2nd Gen CckA-FRET Sensor This work pSWD155 DivL overexpression plasmid This work pSWD156 DivL A601L overexpression plasmid This work pSWD95Y514D CckA FRET Y514D Mutant sensor This work pSWD95D293A CckA FRET D293A Mutant sensor This work pSWD94 PopZ overexpression plasmid This work pSWD109 2nd Gen CckA 183/184 FRET Sensor This work pSWD110 2nd Gen CckA 192/193 FRET Sensor This work pSWD111 2nd Gen CckA 193/194 FRET Sensor This work pWSC10011 1st Gen CckA CckA 69/70 FRET Sensor This work pWSC10012 1st Gen CckA 91/92 FRET Sensor This work pWSC10013 1st Gen CckA N-terminal FRET Sensor This work pSWD55 Short Fibronectin FRET Standard This work pSWD56 Long Fibronectin FRET Standard This work pSWD275 mRuby3 Intermolecular Sensor This work pSWD276 mClover3 Intermolecular Sensor This work pSWD14 mClover3/mRuby3 Bajar, et al. 8, Addgene# 74252 pSWD63 cckA::cckA-mClover3 This work pMT383 CckA template Shapiro Lab pXCFPC-6 Xylose inducible Vector Thanbichler, et al. 7 pVMCS-1 Vanillate inducible Vector Thanbichler, et al. 7 pBVMCS-2 Vanillate inducible high copy plasmid Thanbichler, et al. 7 pXYFPC-1 Xylose inducible Vector Thanbichler, et al. 7 pXCFPC-2 Xylose inducible Vector Thanbichler, et al. 7 FN_7-8_FN_10 Fibronectin Gene Block Ohashi, et al. 9 Table S4: DNA primers utilized in this study.
Blocking Assay:Article Title: Design of a Histidine Kinase FRET Sensor to Detect Complex Signal Integration within Living Bacteria.
Article Snippet: Table S2: Site directed mutagenesis primers for introduction of CckA point mutants Template Mutation Forward Primer Reverse Primer Annealing Temperature (°C) pMT383 Y514D nSWD98 nSWD99 68 pMT383 D293A nSWD104 nSWD105 65 Table S3: Plasmids utilized in this study Name Description Reference/Addgene # pSWD95 2nd Gen CckA-FRET Sensor This work pSWD155 DivL overexpression plasmid This work pSWD156 DivL A601L overexpression plasmid This work pSWD95Y514D CckA FRET Y514D Mutant sensor This work pSWD95D293A CckA FRET D293A Mutant sensor This work pSWD94 PopZ overexpression plasmid This work pSWD109 2nd Gen CckA 183/184 FRET Sensor This work pSWD110 2nd Gen CckA 192/193 FRET Sensor This work pSWD111 2nd Gen CckA 193/194 FRET Sensor This work pWSC10011 1st Gen CckA CckA 69/70 FRET Sensor This work pWSC10012 1st Gen CckA 91/92 FRET Sensor This work pWSC10013 1st Gen CckA N-terminal FRET Sensor This work pSWD55 Short Fibronectin FRET Standard This work pSWD56 Long Fibronectin FRET Standard This work pSWD275 mRuby3 Intermolecular Sensor This work pSWD276 mClover3 Intermolecular Sensor This work pSWD14 mClover3/mRuby3 Bajar, et al. 8, Addgene# 74252 pSWD63 cckA::cckA-mClover3 This work pMT383 CckA template Shapiro Lab pXCFPC-6 Xylose inducible Vector Thanbichler, et al. 7 pVMCS-1 Vanillate inducible Vector Thanbichler, et al. 7 pBVMCS-2 Vanillate inducible high copy plasmid Thanbichler, et al. 7 pXYFPC-1 Xylose inducible Vector Thanbichler, et al. 7 pXCFPC-2 Xylose inducible Vector Thanbichler, et al. 7 FN_7-8_FN_10 Fibronectin Gene Block Ohashi, et al. 9 Table S4: DNA primers utilized in this study.
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