The Sequence of Spacers between the Consensus Sequences Modulates the Strength of Prokaryotic Promoters

We constructed a library of synthetic promoters for Lactococcus lactis in which the known consensus sequences were kept constant while the sequences of the separating spacers were randomized. The library consists of 38 promoters which differ in strength from 0.3 up to more than 2,000 relative units, the latter among the strongest promoters known for this organism. The ranking of the promoter activities was somewhat different when assayed in Escherichia coli, but the promoters are efficient for modulating gene expression in this bacterium as well. DNA sequencing revealed that the weaker promoters (which had activities below 5 relative units) all had changes either in the consensus sequences or in the length of the spacer between the −35 and −10 sequences. The promoters in which those features were conserved had activities from 5 to 2,050 U, which shows that by randomizing the spacers, at least a 400-fold change in activity can be obtained. Interestingly, the entire range of promoter activities is covered in small steps of activity increase, which makes these promoters very suitable for quantitative physiological studies and for fine-tuning of gene expression in industrial bioreactors and cell factories.     

Strategies for development of functionally equivalent promoters with minimum sequence homology for transgene expression in plants: cis-elements in a novel DNA context versus domain swapping

The cauliflower mosaic virus 35S (35S) promoter has been extensively used for the constitutive expression of transgenes in dicotyledonous plants. The repetitive use of the same promoter is known to induce transgene inactivation due to promoter homology. As a way to circumvent this problem, we tested two different strategies for the development of synthetic promoters that are functionally equivalent but have a minimum sequence homology. Such promoters can be generated by (a) introducing known cis-elements in a novel or synthetic stretch of DNA or (b) "domain swapping," wherein domains of one promoter can be replaced with functionally equivalent domains from other heterologous promoters. We evaluated the two strategies for promoter modifications using domain A (consisting of minimal promoter and subdomain A1) of the 35S promoter as a model. A set of modified 35S promoters were developed whose strength was compared with the 35S promoter per se using beta-glucuronidase as the reporter gene. Analysis of the expression of the reporter gene in transient assay system showed that domain swapping led to a significant fall in promoter activity. In contrast, promoters developed by placing cis-elements in a novel DNA context showed levels of expression comparable with that of the 35S. Two promoter constructs Mod2A1T and Mod3A1T were then designed by placing the core sequences of minimal promoter and subdomain A1 in divergent DNA sequences. Transgenics developed in tobacco (Nicotiana tabacum) with the two constructs and with 35S as control were used to assess the promoter activity in different tissues of primary transformants. Mod2A1T and Mod3A1T were found to be active in all of the tissues tested, at levels comparable with that of 35S. Further, the expression of the Mod2A1T promoter in the seedlings of the T1 generation was also similar to that of the 35S promoter. The present strategy opens up the possibility of creating a set of synthetic promoters with minimum sequence homology and with expression levels comparable with the wild-type prototype by modifying sequences present between cis-elements for transgene expression in plants.