Uso de dímeros genéticos del factor de transcripción XylS para la caracterización de la activación del promotor Pm en Pseudomonas putida

xyls is a gene located in the TOL plasmid pWW0 of Pseudomonas putida. Its product XylS is a transcription factor involved in the metabolism of aromatic compounds, and it is induced by this same kind of molecules, especially 3methylbenzoate (3mBz). This transcriptional regulator is widely used in the field of synthetic biology as a key regulatory element in the design of bacterial genetic circuits. Its original host, Pseudomonas putida is also a popular bacterial chassis in synthetic biology projects. The mechanism of induction by XylS has been proposed where two XylS monomers dimerize upon induction and bind to its cognate promoter. However, due to its poor solubility, there are no available experimental structures for this protein, which has hindered the study of this transcription factors and explains why the sequence of events leading to the induction of expression by XylS remains unclear. The aim of this project is to address these two concerns by the characterization of genetic dimers of XylS: synthetic proteins that contain two copies of XylS joined by a linker region. An isodose construction (two independent copies of the protein in the same plasmid) was developed as a control to study the behaviour of three genetic dimer variants, under different concentrations of 3mBz. Induction levels have been assayed by flow cytometry and fluorescence measurement in a plate reader. Each assayed variant differs in the length of the linker region with 40, 24 and 12 amino acids respectively. All assays performed used Pseudomonas putida as a host. Results show that the presence of the linker modifies the behaviour of this transcription factor in terms of peaks of induction, shape of induction curves (induction vs time), expression without 3mBz and dynamic range. Conclusions vary for each variant, but two general trends appear: dynamic range decreased significantly and induction peaks were reached faster when increasing the linker length. The 12 amino acid linker variant showed a marked decrease in peak levels of induction compared to the control, whereas the 40 amino acid linker showed signs of a slight increase. Computational methods hint that these differences between variants might be due to interferences with DNA binding rather than to issues with the dimerization process. The results presented herein have allowed to test the impact of linker length on XylS induction patterns. Furthermore, new hypothesis have been raised upon the results obtained