pCB4270BU-amy

Protein Expression Prokaryotic cells - L. citreum amy

Experiment
Protein Expression Prokaryotic cells - L. citreum amy
Product
pCB4270BU-amy from Ki Jun Jeong, Department of Chemical and Biomolecular Engineerin
Manufacturer
Ki Jun Jeong, Department of Chemical and Biomolecular Engineerin

Protocol tips

Protocol tips
The plasmids were purified from E. coli library using GeneAll® Hybrid-QTM Plasmid Rapidprep kit (GeneAll, Seoul, Korea) and transformed into L. citreum CB2567 by electroporation using the following parameters: 25 μF, 1.0 kV, and 400 Ω49. Transformed cells were recovered by incubation on MRS agar plates with 10 mg/L chloramphenicol at 30 °C for 36 h.

Publication protocol

For the construction of SD2 library, pCB4270B-sfGFP was used as the template DNA, and SD2 region was randomized by PCR with three primers (F2-BCD-et, F1-BL, and R-sfG). The PCR product was digested with XhoI and SalI and ligated into pCB4270B-sfGFP. For the construction of promoter library, pCB4270BU-sfGFP was used as the template, and P710 promoter region was randomized by PCR with two primers (F-P710RL and R-sfG). PCR product was digested with HindIII and SalI, and further was ligated into pCB4270BU-sfGFP. Each library was transformed into E. coli XL1-Blue by electroporation. The plasmids were purified from E. coli library using GeneAll® Hybrid-QTM Plasmid Rapidprep kit (GeneAll, Seoul, Korea) and transformed into L. citreum CB2567 by electroporation using the following parameters: 25 μF, 1.0 kV, and 400 Ω49. Transformed cells were recovered by incubation on MRS agar plates with 10 mg/L chloramphenicol at 30 °C for 36 h.

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