Belén Calles

In general, I am very interested in gene expression regulation. During my Ph.D. research I was studying different mechanisms of prokaryotic regulation of transcription using as a model system Bacillus subtilis phage phi29.

In my current work I have also contributed to the study of the regulation of gene expression by the cAMP-CRP system in the soil bacteria Pseudomonas putida.

My research is also strongly focused in developing genetic tools to manipulate bacteria and re-program them to have novel complex behaviors. The main goal is constructing cells endowed with new functioning genetic circuits for the production of added value compounds.

A key problem in such Synthetic Biology approaches is isolating specific metabolic pathways away from their side reactions. To address this question, we have developed a Tn5 based-transposon tool designed for the functionalization of protein of interest with new traits by entering in-frame peptides of different sizes within permissive regions with high efficiency. The system allows saturating the target gene with insertions, which can be screened by a selectable marker and edited by eliminating selected parts of the transposon to produce the desired knock-in into the protein of interest, without loss of the original functionality. One particular transposon variant is intended to produce GFP sandwich fusions and/or to introduce very specific cleavage sites for the plant viral protease PPV-NIa, producing conditional knock-outs. Induction of NIa protease switches-off tagged enzyme(s), causing a proteome rearrangement which in turn re-directs metabolic flux in a rational way. In addition, we have explored the possibility of producing transcriptional factors that not only respond to its natural inducer but also to the NIa protease, which changes its logic operation. This tool can also be used for the functionalization of proteins with any other new trait of interest.

Another important issue when engineering new genetic circuitry is the need of optimized and well characterized regulatory nodes that control gene expression tightly. Regulation of gene expression at appropriate times is, for example, crucial to avoid metabolic burden and toxicity when using cell factories as production platforms. We have developed a combined transcriptional and translational regulatory tool that allows a fine-tuning control of gene expression in a variety of Gram-negative bacteria. Results demonstrated that genes expressed from the engineered regulatory device did not have leaky expression. This feature was instrumental to clone highly toxic colE3 colicin gene in the absence of the cognate immunity gene. In addition, the system showed a clear reduction of cell-to-cell variability. A further benefit of this gene expression device is that it could be integrated under the control of virtually any positive or negative transcriptional regulation system as a plug and play circuit.

Single cell analysis (by flow cytometry assays) of the behavior of the all-or-none synthetic regulatory device compared to the XylS/Pm positive regulation system, in the absence and presence of the inducer 3-methil-benzoate (3mB) after three hours (A) and kinetics of the expression at different time points (B). Top agar plates with Escherichia coli cells transformed with the regulatory device harboring the highly toxic colE3 colicin, without the cognate immunity gene. Note that induction of the expression of the colE3 gene with 3mB kills the bacterium even at low concentrations (50 μM) (C).