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Operator site library standardization

Even tough transcriptional regulation still plays a pivotal role in synthetic biology, a modular assembly of regulated promoters and characterization of their properties has not been formalized, yet. Even in the Registry, each promoter, though complex, is treated as a “standalone” monolithic element. At present state, the choice of a regulated promoter implies a prefixed sigmoid regulation curve. The only option is to choose a discretional scaling factor in the transfer of the transcription function to the protein through an appropriate RBS. Moreover, the choice of one specific transcription factor limits the choice to one or few possible promoter. The assembly of regulated promoters as the combination of such modular parts, as transcription factor binding sites and operators, could permit the rapid design of devices with desired regulation curves. In fact, in this way, promoter transcriptional strength and repressor binding affinity could be independently fixed.

A first step in the direction of promoting element rationalization has been done the past year with the inclusion in the Registry of a family of constitutive promoters. Each element differs from the other members in the family just for few base pairs in -35 and -10 regions, keeping constant the rest of the sequence and giving rise to a different level of transcription spanning. We decided to use this valuable work as a platform, a starting point for a deeper and more general design strategy.

To pursue this aim, we designed an operator sequence library for four commonly used repressor proteins: LacI, TetR, cI and LexA (link figura). For each of these repressors, we got three sequences from the literature (link) with different repressor binding affinities (link Registry), to get a fine modulation of promoter sensitivity to repressor(see Table 1). Then, we isolated each single operator from the library (link al metodo) to assemble it into standard plasmids from the Registry of standard parts.

Once we get single operators or a combination of them, we can decide to assemble them in different position relatevely to promoter -35 and -10 sequences. It is known from the literature (Cox et al) that the position of an operator site, respectevely to the promoter, plays a crucial role in determining repression or activation. Moreover, even different repression levels can depend on the operator position. The three possible "locations" for operator sequences are:

- the distal region - upstream the -35 sequence - the core region - between the -35 and the -10 - the proximal region- downstream the -10 sequence

Since genomic position affects the operator effect on promoter activation, we decided to take the Berkley's costitutive promoter library as a good "collection" from which we could choose the ideal promoter, depending on the desired transcriptional strengh. Chosen the promoter, we planned to change the operator position to study the effect of position in the design of specific promoter with a derired behaviour.

At the first, we designed a synthetic circuit, composed of the Lac operator sites downstream of the BBa_J23118 promoter controlling the LacI and GFP protein synthesis. These constructs, were meant to allow the characterization of the operator- repressor binding affinity effect on promoter activation.

Single operator isolation