Tuning the Transcriptional Activity of the CaMV 35S Promoter in Plants by Single-Nucleotide Changes in the TATA Box

Synthetic biology uses genetically encoded devices and circuits to implement novel complex functions in living cells and organisms. A hallmark of these genetic circuits is the interaction among their individual parts, according to predefined rules, to process cellular information and produce a circuit output or response. As the number of individual components in a genetic circuit increases, so does the number of interactions needed to achieve the correct behavior, and hence, a greater need to fine-tune the levels of expression of each component. Transcriptional promoters play a key regulatory role in genetic circuits, as they influence the levels of RNA and proteins produced. In multicellular organisms, such as plants, they can also determine developmental, spatial, and tissue-specific patterns of gene expression. The 35S promoter from the Cauliflower Mosaic Virus (CaMV 35S) is widely used in plant biotechnology to direct high levels of gene expression in a variety of plant species. We produced a library of 21 variants of the CaMV 35S promoter by introducing all single nucleotide substitutions to the promoter's TATA box sequence. We then characterized the activity of all variants in homozygous transgenic plants and showed that some of these variants have lower activity than the wild type in plants. These promoter variants could be used to fine-tune the behavior of synthetic genetic circuits in plants.