Synthetic Biology─High Time to Deliver?

Synthetic biology (SynBio) has attracted like no other recent development the attention not only of Life Science researchers and engineers but also of intellectuals, technology think-tanks, and private and public investors. This is largely due to its promise to propel biotechnology beyond its traditional realms in medicine, agriculture, and environment toward new territories historically dominated by the chemical and manufacturing industries─but now claimed to be amenable to complete biologization. For this to happen, it is crucial for the field to remain true to its foundational engineering drive, which relies on mathematics and quantitative tools to construct practical solutions to real-world problems. This article highlights several SynBio themes that, in our view, come with somewhat precarious promises that need to be tackled. First, SynBio must critically examine whether enough basic information is available to enable the design or redesign of life processes and turn biology from a descriptive science into a prescriptive one. Second, unlike circuit boards, cells are built with soft matter and possess inherent abilities to mutate and evolve, even without external cues. Third, the field cannot be presented as the one technical solution to many grave world problems and so must avoid exaggerated claims and hype. Finally, SynBio should pay heed to public sensitivities and involve social science in its development and growth, and thus change the technology narrative from sheer domination of the living world to conversation and win-win partnership.

Refactoring the Conjugation Machinery of Promiscuous Plasmid RP4 into a Device for Conversion of Gram-Negative Isolates to Hfr Strains.

Chromosomal exchange and subsequent recombination of the cognate DNA between bacteria was one of the most useful genetic tools (e.g., Hfr strains) for genetic analyses of E. coli before the genomic era. In this paper, yeast assembly has been used to recruit the conjugation machinery of environmentally promiscuous RP4 plasmid into a minimized, synthetic construct that enables transfer of chromosomal segments between donor/recipient strains of P. putida KT2440 and potentially many other Gram-negative bacteria. The synthetic device features [i] a R6K suicidal plasmid backbone, [ii] a mini-Tn5 transposon vector, and [iii] the minimal set of genes necessary for active conjugation (RP4 Tra1 and Tra2 clusters) loaded as cargo in the mini-Tn5 mobile element. Upon insertion of the transposon in different genomic locations, the ability of P. putida-TRANS (transference of RP4-activated nucleotide segments) donor strains to mobilize genomic stretches of DNA into neighboring bacteria was tested. To this end, a P. putida double mutant ΔpyrF (uracil auxotroph) Δedd (unable to grow on glucose) was used as recipient in mating experiments, and the restoration of the pyrF+/edd+ phenotypes allowed for estimation of chromosomal transfer efficiency. Cells with the inserted transposon behaved in a manner similar to Hfr-like strains and were able to transfer up to 23% of their genome at frequencies close to 10-6 exconjugants per recipient cell. The hereby described TRANS device not only expands the molecular toolbox for P. putida, but it also enables a suite of genomic manipulations which were thus far only possible with domesticated laboratory strains and species.

A Standardized Inverter Package Borne by Broad Host Range Plasmids for Genetic Circuit Design in Gram-Negative Bacteria.

Genetically encoded logic gates, especially inverters-NOT gates-are the building blocks for designing circuits, engineering biosensors, or decision-making devices in synthetic biology. However, the repertoire of inverters readily available for different species is rather limited. In this work, a large whole of NOT gates that was shown to function previously in a specific strain of Escherichia coli, was recreated as broad host range (BHR) collection of constructs assembled in low, medium, and high copy number plasmid backbones of the SEVA (Standard European Vector Architecture) collection. The input/output function of each of the gates was characterized and parametrized in the environmental bacterium and metabolic engineering chassis Pseudomonas putida. Comparisons of the resulting fluorescence cytometry data with those published for the same gates in Escherichia coli provided useful hints on the portability of the corresponding gates. The hereby described inverter package (20 different versions of 12 distinct gates) borne by BHR plasmids thus becomes a toolbox of choice for designing genetic circuitries in a variety of Gram-negative species other than E. coli.

Recombination-Independent Genome Editing through CRISPR/Cas9-Enhanced TargeTron Delivery.

Group II introns were developed some time ago as tools for the construction of knockout mutants in a wide range of organisms, ranging from Gram-positive and Gram-negative bacteria to human cells. Utilizing these introns is advantageous because they are independent of the host's DNA recombination machinery, they can carry heterologous sequences (and thus be used as vehicles for gene delivery), and they can be easily retargeted for subsequent insertions of additional genes at the user's will. Alas, the use of this platform has been limited, as insertion efficiencies greatly change depending on the target sites and cannot be predicted a priori. Moreover, the ability of introns to perform their own splicing and integration is compromised when they carry foreign sequences. To overcome these limitations, we merged the group II intron-based TargeTron system with CRISPR/Cas9 counterselection. To this end, we first engineered a new group-II intron by replacing the retrotransposition-activated selectable marker (RAM) with ura3 and retargeting it to a new site in the lacZ gene of E. coli. Then, we showed that directing CRISPR/Cas9 toward the wild-type sequences dramatically increased the chances of finding clones that integrated the retrointron into the target lacZ sequence. The CRISPR-Cas9 counterselection strategy presented herein thus overcomes a major limitation that has prevented the use of group II introns as devices for gene delivery and genome editing at large in a recombination-independent fashion.

Volatilization of arsenic from polluted soil by Pseudomonas putida engineered for expression of the arsM Arsenic(III) S-adenosine methyltransferase gene.

Even though arsenic is one of the most widespread environmental carcinogens, methods of remediation are still limited. In this report we demonstrate that a strain of Pseudomonas putida KT2440 endowed with chromosomal expression of the arsM gene encoding the As(III) S-adenosylmethionine (SAM) methyltransfase from Rhodopseudomonas palustris to remove arsenic from contaminated soil. We genetically engineered the P. putida KT2440 with stable expression of an arsM-gfp fusion gene (GE P. putida), which was inserted into the bacterial chromosome. GE P. putida showed high arsenic methylation and volatilization activity. When exposed to 25 µM arsenite or arsenate overnight, most inorganic arsenic was methylated to the less toxic methylated arsenicals methylarsenate (MAs(V)), dimethylarsenate (DMAs(V)) and trimethylarsine oxide (TMAs(V)O). Of total added arsenic, the species were about 62 ± 2.2% DMAs(V), 25 ± 1.4% MAs(V) and 10 ± 1.2% TMAs(V)O. Volatilized arsenicals were trapped, and the predominant species were dimethylarsine (Me2AsH) (21 ± 1.0%) and trimethylarsine (TMAs(III)) (10 ± 1.2%). At later times, more DMAs(V) and volatile species were produced. Volatilization of Me2AsH and TMAs(III) from contaminated soil is thus possible with this genetically engineered bacterium and could be instrumental as an agent for reducing the inorganic arsenic content of soil and agricultural products.

Genetically modified organisms for the environment: stories of success and failure and what we have learned from them / Organismos modificados para el medio ambiente: historias de éxito y fracaso, y qué hemos aprendido de ellas

The expectations raised in the mid-1980s on the potential of genetic engineering for in situ remediation of environmental pollution have not been entirely fulfilled. Yet, we have learned a good deal about the expression of catabolic pathways by bacteria in their natural habitats, and how environmental conditions dictate the expression of desired catalytic activities. The many different choices between nutrients and responses to stresses form a network of transcriptional switches which, given the redundance and robustness of the regulatory circuits involved, can be neither unraveled through standard genetic analysis nor artificially programmed in a simple manner. Available data suggest that population dynamics and physiological control of catabolic gene expression prevail over any artificial attempt to engineer an optimal performance of the wanted catalytic activities. In this review, several valuable spin-offs of past research into genetically modified organisms with environmental applications are discussed, along with the impact of Systems Biology and Synthetic Biology in the future of environmental biotechnology (AU)

Las expectativas surgidas a mediados de la década de 1980 sobre las posibilidades de la ingeniería genética para remediar in situ problemas de contaminación ambiental no se han cumplido totalmente. A pesar de ello, se ha aprendido mucho sobre la expresión de las vías catabólicas por parte de las bacterias en sus hábitat naturales y sobre la influencia que ejercen las condiciones ambientales en la expresión de las actividades catalíticas deseadas. Las numerosas opciones entre los nutrientes y las respuestas al estrés forman una red compleja de regulación transcripcional que, debido a la redundancia y la robustez de los circuitos que intervienen, no se puede ni descifrar mediante un análisis genético estándar ni programar artificialmente de manera simple. Los datos disponibles sugieren que la dinámica de poblaciones y el control fisiológico de la expresión catabólica de los genes prevalece sobre cualquier intento de buscar artificialmente el funcionamiento óptimo de las actividades catalíticas deseadas. En esta revisión se comentan varios efectos valiosos de la investigación en organismos modificados genéticamente para su aplicación ambiental, así como el impacto de la Biología de Sistemas y de la Biología Sintética en el futuro de la biotecnología ambiental (AU)