Context‐dependency of synthetic minimal promoters in driving gene expression: a case study

: Synthetic promoters are considered ideal candidates in driving robust gene expression. Most of the available synthetic promoters are minimal promoters, for which the upstream sequence of the 5' end of the core region is usually excluded. Although the upstream sequence has been shown to mediate transcription of natural promoters, its impact on synthetic promoters has not been widely studied. Here, a library of chromosomal DNA fragments is randomly fused with the 5' end of the J23119 synthetic promoter, and the transcriptional performance of the promoter is evaluated through β-galactosidase assay, fluorescence intensity and chemical biosynthesis. Results show that changes in the upstream sequence can induce significant variation in the promoter strength of up to 5.8-fold. The effect is independent of the length of the insertions and the number of potential transcription factor binding sites. Several DNA fragments that are able to enhance the transcription of both the natural and the synthetic promoters are identified. This study indicates that the synthetic minimal promoters are susceptible to the surrounding sequence context. Therefore, the upstream sequence should be treated as an indispensable component in the design and application of synthetic promoters, or as an independent genetic part for the fine-tuning of gene expression.