# Plant Synthetic Promoters
## 3 Objectives and General Methodology of Synthetic Promoter Creating
### Methods of Synthetic Promoter Generation  
The methods of choice for producing relatively small synthetic promoter sets are PCR-based techniques [204; 205; 206; 207; 208; 209]. Recently, plant synthetic promoters were prepared by PCR-based deletion mutagenesis and subsequent GUS activity assays. In this way, two core promoters were obtained from the _Stellaria media Antimicrobial Peptide1_ (_AMP1_) and _Antimicrobial Peptide2_ (_AMP2_) genes. Although the shorter versions of the _pro-SmAMP1_ and _pro-SmAMP2_ promoters, represented by the \(-58\) and \(-60\) bp fragments, were weak, the longer fragments of \(102\) and \(104\) bp were used as core promoters, as these largely retain the properties of the original promoters [210]. Primer-driven PCR mutagenesis was also used to obtain variants of the synthetic promoter pCL that could regulate the cold-induced sweetening of potatoes. Several point mutations were introduced into the region encompassing the 5\({}^{\prime}\) and 3\({}^{\prime}\) sides of the C-repeat/dehydration-responsive element (CRT/DRT), localized within \(-490\) to \(-500\) bp from TSS. Moreover, the additional cassette of 27 bp containing the second CRT/DRT element was placed 31 bb from the existing CRT/DRE site [211].  
[PERSON] et al. suggest that even discrete point mutations of the repeated G-box (CACGTG) and its 2-4 bp long flanking regions could significantly increase reporter _GFP_ gene expression, opening up the potential for useful and predictable genome editing approaches in the promoter regions [212].  
Besides the presented approaches dealing with the mutagenesis of a single promoter, which provide only a relatively small number of variants, the promoter sequence can be subjected to stepwise-directed evolution using error-prone PCR to obtain larger libraries. It was found that 83 cycles of mutation, construction, screening, and property characterization of the 74-bp-long Ptrc promoter, with 10-20 of the lowest and highest-activity variants sampled after each step, resulted in a library of 3665 clones. Among them, a 454-fold difference was found between the strongest and weakest expression rates [213]. A smaller library containing hundreds of variants has been obtained by shuffling the figwort mosaic virus full-length transcript promoter (F) and the sub-genomic transcript promoter (FS) sequences. Shuffled promoters were weaker than those prepared by hybridization. However, the shuffled libraries contained promoters with a broad spectrum of activities, enabling the application of both weaker and stronger variants in metabolic engineering approaches [209]. To significantly increase the complexity of synthetic promoter libraries, a combination of site-directed mutation with degenerate primers, overlap-extension PCR, and Gibson assembly was proposed. The prepared libraries showed a diversity in the order of \(10^{4}\)-\(10^{7}\) variants and were screened rapidly by performing fluorescence-activated cell sorting (FACS) of the transformed yeast cells [214].  
Construction of larger synthetic promoter libraries composed of over \(10^{8}\) elements is possible by avoiding the use of already-known DNA sequences. Random, 80-bp-long DNA oligos are synthesized and used to build such complex libraries in yeast, theoretically enabling the study of all pairwise interactions between TF (trans-factor) binding sites in the context of orientation and specific spacing constraints. However, the analysis of such large synthetic promoter libraries has problems; for example, the complexity of the synthetic promoter library (\(>\)10\({}^{8}\)) exceeds the number of sorted yeast cells (\(<\)10\({}^{8}\)). Also, the analysis of such large libraries using simplified TF binding models, assuming their position, orientation, and TF-TF interaction independence, explained nearly 93% of the variation in gene expression in the tested libraries; it proved to be insufficient in relation to native yeast genes, where only \(\sim\)16% of variability was demonstrated [46].