People

Inés Merino

I am the executive assistant of the Laboratory. Among my duties are the follow up of the Lab projects, organization of workshops and scientific meetings.

I am the lab technician. I have a degree in Biological Sciences from the Autonoma University of Madrid and I have been in the laboratory for over 25 years tuning up multiple techniques of molecular biology. Among other tasks, I keep the stock of laboratory strains plus the new pSEVA collection. “I am the person who knows where all things in the lab”.

In the last time, we are looking for different strategies to create artificial communities of bacteria modifying the adhesive properties of P. putida. This could have an enormous biotechnological potencial.

I created in the lab the nanoPad platform, an important factory in order to produce and select highly specific adhesins. These adhesins are monomeric recombinant antibodies or nanobodies. The nanobodies against different proteins are using as tools to detect and manipulate different biological activities as well as the adhesive properties of P.putida.

P.putida, our bacteria model due to its plasticity; its capacity to use different organic compounds as carbon sources, including important contaminants such as lindane, toluen, etc.

These monomeric recombinant antibodies can be packaged into phage M13 to achieve new rounds of panning, secreted into the extracellular medium, expressed intracellularlly or bacteria surface expression, allowing us the binding with other bacteria or solid surfaces.

In the laboratory, we also have external membrane anchoring systems that can express surface proteins such as intimin and autotransporters.

The last technique that I used to generate specific adhesion is QCM, quartz crystal microbalance. This allows us to see if there is physical binding between bacteria and substrate with a serie of advantages: I deposit the substrate in the crystal with the gold in a vertical position and create a constant flow of bacteria to prevent the non-specific binding of these.

QCM measures mass-frequency relationship. Frequency change is translated in an increase in the thickness.

In the future, we are planning to select VHHs recognizing different proteins from strains of interest, in order to generate Bacterial Consortia.

Schematic representation of the use of nanobodies as adhesins in P. putida. The OM anchoring system used is intimin. A. Bacteria binding to a substrate or solid surface. B. Adhesión bacteria with other bacteria.

1. Detection of nanobodies again fibrinogen. Only those bacteria that express this nanobody will stick, thus increasing the mass deposited on the crystal and lowering the frequency.

See more in: Fraile S et al. Proteomics. 2013 Oct;13(18-19):2766-75.

I received my PhD in Microbiology from the Autonomous University of Madrid. My PhD project was focused on following a Protein Engineering approach to expand the fluorescent protein “color palette” suitable as cellular localization tools in thermophiles. First, rational design was used to obtain cyan, cyan-green, green and yellow variants from the scaffold of superfolder GFP. Second, a method of directed molecular evolution by random mutagenesis allowed to obtain libraries to screen for derivatives of mCherry. This work yielded several proteins that were characterized and validated as localization tools in Thermus thermophilus. In the current episode of my scientific adventure at Víctor de Lorenzo´s laboratory in the National Center of Biotechnology, I am developing a platform based on Synthetic Biology to obtain variants of Antibodies.

Since my incorporation to Victor de Lorenzo´s laboratory I am nourished with a broad repertoire of resources that I can use to develop new molecular technologies hosted in microbes. In this context, my main project at Victor de Lorenzo´s laboratory aims at creating a device able to work in vivo to generate diversification of specific target sequences of interest while the accumulation of undesired mutations in the host chromosome is prevented.

Antibodies are proteins produced by the immune system and are very efficient at binding molecules specifically. Although this property makes them the tools of choice in a wide number of applications, both antibodies and antigen-binding fragments are difficult and expensive to produce with current procedures. This is the reason why we set out to design and implement an alternative technology to produce antibodies in a fashion reminiscent of the immune system but assembled with Synthetic Biology approaches. To this end, we embarked in setting up a platform made of parts assembled in bacteria that behaves as an immune-system-like machine. With this setup, single-chain camel antibodies (nanobodies) are generated and displayed on the bacterial surface—thus enabling a large number of uses in medical and industrial Biotechnology. First, autonomous functional modules are assembled in the genomic chassis of an Escherichia coli strain to allow the display of nanobody scaffolds on the surface of the cell. Second, the conditional diversification with mutagenesis of target-binding sequences of the nanobody lead to affinity maturation. Finally, binders with improved affinity and specificity towards desired molecular targets are selected. The hereby described artificial immune system (ARISYS) will dramatically simplify the production of recombinant antibodies.

ARISYS: Artificial Immune System. Nanobodies from camels are light antibodies consisting on one heavy variable region (V_HH). Libraries of vhh genes are cloned in bacteria. Only the ARISYS clones that specifically bind the antigen of interest are selected. Then, the genetic diversity-generating device mutates this vhh in order to generate a new library ready for a new maturation step. In the end, there are clones with greater specific binding ability against the antigen of interest

My name is Angeles Hueso-Gil and I am doing my PhD at Victor’s lab. I was always interested in life sciences, so studied Biology and a Biotechnology master in Valencia, my hometown, and later I moved to Madrid to make my PhD.

My PhD project consists of the development of several tools and devices that will be placed into the ARYSIS circuit, which has the aim of producing affibodies (the variable part of camel antibodies) inside bacteria as Pseudomonas putida. In this circuit, the affibodies will be produced and diversified in a similar way that happens inside mammal cells, getting as a result a final affibody with a higher affinity for the antigen.

I have three main tasks involved with this project.

1-Developing a reversible light switch to control the conditional diversification of the affibodies. For this purpose I used the CcaS-CcaR green light system from Synecocystis, which was previously adapted to Escherichia coli. After several mutagenesis cycles of the construction carrying all the CcaS-CcaR components, we selected an optimized clone for P. putida and we are using it for its implementation into the ARYSIS circuit and the control of biofilm production.

2-Developing a surface contact switch which triggers the mechanism of the circuit once bacteria find their antigen and attach to the surface. I am characterizing the transcriptomic response of P. putida to solid surfaces and some of its natural contact systems of P. putida, as for example wsp complex, in order to know about possible tools suitable for our needs.

3-Expression of proteoRhodopsin and the production of its cofactor, the retinal, inside P. putida. This transmembrane protein is able to transform light energy into a proton gradient that will be used for ATP production inside the bacteria. The idea is to increase the energy supply inside our bacteria for the new functions of the ARYSIS circuit using the proteoRhodopsin activity.

Behaviour inside Pseudomonas putida of the original construction carrying the CcaS-CcaR system and the selected clone after diversification. GFP production is induced when cultures

My name is Elena Velázquez and I studied Biotechnology at the Technical University of Madrid (UPM). The reasons for this were my desire to understand how living organisms work and be able to tune them for something useful and new. That is why, after hearing the term “Synthetic Biology”, I was really attracted to this field. At this point, I was lucky enough to be accepted in Victor’s lab to perform my master thesis and, finally, I decided to start my PhD with a FPU fellowship.
In the lab, I have two main goals:

1. Development of a biosensor able to detect 2,4-Dinitrotoluene (2,4-DNT): This compound is one of the main degradation products of anti-personnel landmines. In order to do this, I am currently implementing in Pseudomonas putida a technology based on a cytosine deaminase fused to the T7 RNA polymerase. This deaminase mutates DNA by turning cytosine into uracil, which is finally fixed as adenine in the DNA strand.I plan to use this fusion protein as the driving force to alter the specificity of a transcriptional factor in P. putida and find a version which is able to detect 2,4-DNT at high efficiency.

2. Setting of a DNA-barcoding platform: In collaboration with the group of Dr. Natalio Krasnogor in Newcastle, we are developing a system meant to insert a particular, short and traceable sequence into the bacterial genome. This fragment of DNA codifies important information that you could seek for in a data base. With this purpose, we have engineered a group II intron from Lactococcus lactis to bear this “barcodes” and deliver them to precise sites of the chromosome.

Figura 1. Schematic representation of our barcoding platform. From left to right: expression plasmid bearing the group II intron with the “DNA-barcode” inserted at MluI site. After induction, this group II intron integrates into the selected locus, labelling the strain.

Figure 1. Group II intron integration assay. After induction of group II intron expression, this molecule is able to recognize lacZ gene sequence and insert in the designated place. After integration, the kanamycin resistant gene inside of it is activated and insertion mutants can be selected by plating the culture in the presence of this antibiotic. (A) Left plates: viability check. Right plates: selection of integration mutants. White colonies, a priori, have the group II intron inserted in the right position, disrupting lacZ gene. Blue colonies resistant to kanamycin are off-target integration mutants, with the intron inserted in other parts of the genome. (B) Test of correct integration through PCR: The presence of the intron in the correct position of lacZ gene produces a 719 bp amplicon while its absence leads to not amplification.

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.