People

Inés Merino

In simple terms, my role is to keep things running smoothly and make sure the lab’s day-to-day work flows as easily as possible. I really enjoy it because it’s so varied and gives me the chance to work closely with everyone on the team, helping solve problems and keep things moving forward.

I take care of the whole hiring process from start to finish, and I make a point of supporting new joiners so they can settle in quickly and feel comfortable from day one.

I also organize group events like seminars, meetings, and retreats, which is something I particularly enjoy since it brings together planning, coordination, and working with people.

From an administrative and financial perspective, I keep track of projects, monitor budgets, and make sure everything stays on track. I try to anticipate potential issues whenever I can, so things don’t get stuck later on.

I handle purchasing as well, both equipment and consumables, and look after maintenance contracts, making sure everything is available and working when it’s needed.

I also coordinate visits, paying attention to the small details to make sure people have a great experience. I support the administrative side of publications as well, and more generally handle any non-technical issues that come up in the lab. That come up.

I work closely with researchers, technicians, and administrative staff, often acting as a link between them. I like to think I help bring a bit of structure, clarity, and calm to the team’s day-to-day work.

That unpredictable side of the role is actually one of the things I enjoy most—every day is different, and there are always new challenges to tackle.

Overall, I find the role really rewarding because it lets me contribute directly to how well the lab runs, while helping others focus on what they do best

My research interests are mostly related to regulation of biological processes. In particular, my Ph.D. research was about different mechanisms of prokaryotic regulation of transcription, using Bacillus subtilis phage phi29 as model system. More recently, I have contributed to the study of the regulation of gene expression by the cAMP-CRP system and the role of the major RNA chaperone Hfq in the soil bacteria Pseudomonas putida.

Understanding biological processes is also important for Synthetic Biology applications to manipulate bacteria to perform novel complex behaviors. New organisms that satisfy human needs can be constructed by re-progamming extant cellular pathways to produce added value compounds.  A key problem in such approaches is isolating specific metabolic pathways away from their side reactions.

To address these questions, we have developed a Tn5 based-transposon designed for the functionalization of target proteins with new traits. The tool enters in-frame peptides of different sizes within permissive regions with high efficiency. The system allows saturating the gene of interest with insertions that can be then screened to select for the desired knock-in into the protein of interest, without loss of the original functionality. One particular transposon variant was designed to produce GFP sandwich fusions and/or to insert very specific NIa protease cleavage sites, producing conditional knock-outs. Induction of the cognate protease cleaves and switches-off NIa 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 protease, which changes its logic operation by cleaving different parts of the regulatory protein. This transposon-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. Regulation of gene expression at appropriate times is, for example, crucial to avoid metabolic burden and toxicity when using cell factories as production platforms. By taking advantage of our standardized SEVA platform we have made a systematic study and comparison of the performance of the expression systems available to date, that will be very useful when selecting the most appropriate regulator/promoter pair.

To further improve fine-tuning of gene expression we have developed a translational regulatory tool that can be combined with different transcriptional regulation systems allowing tight control in Gram-negative bacteria, e.g. E. coli and Pseudomonas putida. The engineered module includes a sRNA that inhibits translation of leaky mRNAs. Expression of this regulatory sRNA is controlled by a cross-inhibition bistable circuit. Genes controlled under this regulatory circuit showed negligible leaky expression. This feature was instrumental to clone highly toxic colE3 colicin gene in the absence of the cognate immunity protein. 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.

Schematic representation of the regulatory device tailored to have an all-or-none expression behavior of the gene of interest. The engineered module, which is SEVA-platform compatible, includes a sRNA that inhibits translation of leaky mRNAs. Expression of this regulatory sRNA is controlled by a cross-inhibition bistable circuit (A) Top agar plates with Escherichia coli cells transformed with the regulatory device harboring the highly toxic colE3 colicin, without the cognate immunity gene under the control of the XylS/Pm transcriptional regulation system.  Note that induction of the expression of the colE3 gene with 3-methilbenzoate (3mB) kills the bacterium even at low concentrations (50 mM) (B). Basal expression of GFP cloned in AlkS/pAlkB regular expression system (239M) or the same transcriptional control in combination with the translational regulatory device (239M-SR) in Pseudomonas putida EM42 strain (the so called cell factory) in solid agar plates and liquid cultures. Note that 237M cells, which harbor the plasmid backbone with GFP but no expression system, were used as control of autofluorescence.

I got a PhD in Biology under the supervisión of Dr. Perera (Faculty of Biology-UCM, 2000), where I studied the biology of a Thiobacillus plasmid. After that my profesional track included experience in a biotech company (PharmaMar S.A.-Madrid, 2001-2004), technical work in the Unit of Genomics of Scientific Park of Madrid (PCM, 2004-2009), a post-doc period in the CBMSO (Madrid, 2010) under the supervisión of Dr. García-Ballesta and also teaching activities as Associate Professor in UCM and UFV universites. Although involved in different research fields along my career, I have been mainly focused in Microbiology and Bacterial Genetic Engenieering.

In Victor´s Lab (CNB-Madrid, 2013-), I have a technical position aimed to give support for general laboratory activities, focusing on P. putida KT2440 strain engineering and pSEVA platform development. In this regard I colaborate in a number of specific projects such as recombieering and CRISPR/Cas9 assisted GE, Yeast Assembly, contruction of a variety of shuttle vectors (Gram+, yeast, streptomyces) and new expression systems design.

I am a biologist who has always been enthusiastic about applied research. I obtained my PhD (2011) within the “Biotechnology of the rhizosphere” group lead by F. Javier Gutiérrez Mañero (Universidad CEU San Pablo, Madrid). In that period, I participated in several R&D projects related to different ways to improve plant-microbe interactions through various biotechnological applications, such as the exploitation of metagenomics to discover new genes and enzymes, to increase the production of interesting bioactive compounds, biofertilizers, biocontrol agents. During that time I spent four months in the Austrian Centre of Industrial Biotechnology (ACIB GmbH, Graz, Austria).

After my PhD I completed a Master in Biotechnology, (2016, Aliter-Escuela Internacional de Negocios de Madrid) oriented to gain expertise in the management part of science.

Then, I joined Victor’s group to contribute to the molecular tool repertoire and to exploit and engineer P. putida KT2440 to produce new chassis with increased performance for synthetic biology.

More specifically, we have developed new SEVA plasmids that will expand the already established collection. Moreover, we followed the SEVA standard to construct a new broad-host-range cosmid which can be used to carry out functional screenings of metagenomics libraries in different hosts.

Figure 1. Schematic representation of the cargo region of the broad-host-range SEVA cosmid vector with the RK2 replicon. The COS site allows phage packaging of the DNA, while the multicloning site (MCS) is placed into the “Killer” gene ccdB allowing us to minimize the selection of empty vectors. Functional screenings could be done by inducing the expression of the P_T7/ P_T3 promoters and its transcriptional termination is enforced by the presence of the T_t7 and the T₀ of the SEVA backbone, respectively. The system has the SEVA format with the advantages of its modularity.

On the other hand, I also have two basic science related projects associated to P. putida KT2440. However, we plan to use that knowledge to minimize horizontal gene transfer (HGT) and to increasing resistance to desiccation of this bacterium.

For the first purpose, we constructed a double ΔungΔdut mutant strain, which has its DNA enriched in uracil. We expect that when a strain with a high uracil content transfers plasmid DNA (U enriched) to a wild type host, that DNA would be degraded by the host base excision repair mechanism. Ideally, that double mutant strain could be used as a “biosafe strain” to minimize horizontal transfer events.

The second topic is related to desiccation stress in KT2440. First, by studying the desiccation stress response of this bacterium, and particularly by the characterization of a promoter (P₄₇₀₇) which specifically responds to desiccation conditions. Finally, we would like to endow P. putida with a higher resistance to desiccation by heterologous expression of genes coming from different organisms (tardigrades, Deinococcus, Arthrobacter…) that naturally have a great potential to endure those conditions.

Figure 2. Representative image of the P₄₇₀₇ characterization in P. putida KT2440. The image shows a colony section (xy plane) of KT2440 taken by a confocal microscopy. KT2440 constitutively expresses cherry and harbors a plasmid with a transcriptional fusion of the P₄₇₀₇ promoter to GFP (pSEVA237 plasmid), growing on agarose plates with low humidity (less than 30% RH) for 48h. From left to right, GFP channel, mCherry channel and merged image, where the xz plane is also shown at the bottom. The specific response of that promoter (GFP) to dessication conditions is shown in the left picture of the image.

The TOL plasmid encodes a regulatory network that links substrate sensing to the degradation of aromatic compounds. Upon detection of m-xylene, XylR activates the upper pathway, leading to the formation of intermediates such as 3-methylbenzoate (3MB). This compound induces XylS and the meta-cleavage pathway, but can also enter the chromosomally encoded ortho route, creating a potential metabolic conflict. The regulatory architecture of the system anticipates this scenario, coordinating gene expression to minimize the accumulation of toxic intermediates and ensure efficient flux through the appropriate degradation pathway, illustrating how regulatory design contributes to metabolic robustness by preventing the accumulation of harmful intermediates.

My name is Guillermo Gómez García. I studied Biochemistry at the Universidad Autónoma de Madrid and later completed a Master’s in Biotechnology there as well. I’ve always been fascinated by microbiology and the many ways microbes can be used in biotechnology, so I decided to dive deeper and start a PhD at the Centro Nacional de Biotecnología (CNB), in Fernando Rojo’s lab. My PhD focused on understanding how a transposon is regulated in Pseudomonas putida KT2440.

After finishing my doctorate, I had the chance to join Víctor de Lorenzo’s group, where I continue exploring bacteria from different angles. I’m also collaborating with Alfonso Jaramillo’s lab at I2SysBio in Valencia, working on improving the electrical conductivity of Escherichia coli.

In the lab, I’m currently working on three main projects. One looks at how the distance between a promoter and its regulator in the genome affects gene expression, using the XylS/Pm system and transposon insertions. Another focuses on how the mutation rate of the pyrF gene changes depending on where it sits in the chromosome. And finally, a project that started from a casual observation: studying airborne communication between different P. putida cultures and how this might influence the development of antibiotic resistance.

Figure. Experimental setup to assess the influence of KT2440 LB cultures on the growth of antibiotic-treated cultures on solid media. (A) The right compartment of the split Petri dish (inducer side) contains LB agar overlaid with a soft agar layer inoculated with Pseudomonas putida KT2440. The left compartment (inducible side) contains LB agar supplemented with Nx50, also overlaid with soft agar inoculated with KT2440. For visual distinction of possible cell transfer between compartments, the inducer strain express mCherry, while inducible strain express GFP. A separate negative control plate, physically isolated from other cultures, replicates the conditions of the inducible side without exposure to VOCs (left plate). (B) Same experimental design as panel A, but with inverted fluorescent markers in a three-compartment split.