Construction, model-based analysis and characterization of a promoter library for fine-tuned gene expression in Bacillus subtilis

Promoters are among the most-important and most-basic tools for the control of metabolic pathways. However, previous research mainly focused on the screening and characterization of some native promoters in Bacillus subtilis. To develop a broadly applicable promoter system for this important platform organism, we created a de novo synthetic promoter library (SPL) based on consensus sequences by analyzing the microarray transcriptome data of B. subtilis 168. A total of 214 potential promoters spanning a gradient of strengths was isolated and characterized by a green fluorescence assay. Among these, a detailed intensity analysis was conducted on nine promoters with different strengths by reverse-transcription polymerase chain reaction (RT-PCR) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Furthermore, reconstructed promoters and promoter cassettes (tandem promoter cluster) were designed via statistical model-based analysis and tandem dual promoters, which showed strength that was...