RESUMEN
BACKGROUND: Synthetic systems that use positive feedback have been developed to control human disease vectors and crop pests. The tTAV system, which has been deployed in several insect species, relies on a positive feedback circuit that can be inhibited via dietary tetracycline. Although insects carrying tTAV fail to survive until adulthood in the absence of tetracycline, the exact reason for its lethality, as well as the transcriptomic effects of an active positive feedback circuit, remain unknown. RESULTS: We engineered the tTAV system in Drosophila melanogaster and investigated the effects of tTAV genome integration locus on the whole fly transcriptome during larval and adult life stages in four transgenic fly strains using gene expression microarrays. We found that while there were widespread effects on the transcriptome, the gene expression differences after removal of tetracycline were not consistent between integration sites. No specific region of the genome was affected, no common set of genes or pathways, nor did the integration site affect the transcripts in cis. CONCLUSION: Although the positive feedback tTAV system is effective at killing insect larvae regardless of where it is inserted in the genome, it does not exhibit a specific, consistent transcriptional signature. Instead, each insertion site is associated with broad, but different, transcriptional effects. Our results suggest that lethality may not be caused by a direct effect on transcription of a set of key genes or pathways. Instead, we propose that rather than a specific action of a tTAV protein, it is the stochastic transcriptional effects specific to each insertion site that contribute to the tTAV-induced mortality.
Asunto(s)
Drosophila melanogaster/genética , Regulación de la Expresión Génica , Genes Sintéticos , Transcripción Genética , Animales , Animales Modificados Genéticamente , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Retroalimentación Fisiológica , Perfilación de la Expresión Génica , Análisis de Secuencia por Matrices de Oligonucleótidos , Especificidad de la Especie , TranscriptomaRESUMEN
Pacific Island countries have experienced periodic dengue, chikungunya and Zika outbreaks for decades. The prevention and control of these mosquito-borne diseases rely heavily on control of Aedes aegypti mosquitoes, which in most settings are the primary vector. Introgression of the intracellular bacterium Wolbachia pipientis (wMel strain) into Ae. aegypti populations reduces their vector competence and consequently lowers dengue incidence in the human population. Here we describe successful area-wide deployments of wMel-infected Ae. aegypti in Suva, Lautoka, Nadi (Fiji), Port Vila (Vanuatu) and South Tarawa (Kiribati). With community support, weekly releases of wMel-infected Ae. aegypti mosquitoes for between 2 to 5 months resulted in wMel introgression in nearly all locations. Long term monitoring confirmed a high, self-sustaining prevalence of wMel infecting mosquitoes in almost all deployment areas. Measurement of public health outcomes were disrupted by the Covid19 pandemic but are expected to emerge in the coming years.
Asunto(s)
Aedes , Virus del Dengue , Dengue , Wolbachia , Infección por el Virus Zika , Virus Zika , Animales , Humanos , Aedes/genética , Aedes/microbiología , Mosquitos Vectores/genética , Mosquitos Vectores/microbiología , Wolbachia/genética , Fiji/epidemiología , VanuatuRESUMEN
From protocellular to societal, networks of living systems are complex and multiscale. Discerning the factors that facilitate assembly of these intricate interdependencies using pairwise interactions can be nearly impossible. To facilitate a greater understanding, we developed a mathematical and computational model based on a synthetic four-strain Saccharomyces cerevisiae interdependent system. Specifically, we aimed to provide a greater understanding of how ecological factors influence community dynamics. By leveraging transiently structured ecologies, we were able to drive community cohesion. We show how ecological interventions could reverse or slow the extinction rate of a cohesive community. An interconnected system first needs to persist long enough to be a subject of natural selection. Our emulation of Darwin's "warm little ponds" with an ecology governed by transient compartmentalization provided the necessary persistence. Our results reveal utility across scales of organization, stressing the importance of cyclic processes in major evolutionary transitions, engineering of synthetic microbial consortia, and conservation biology. IMPORTANCE We are facing unprecedented disruption and collapse of ecosystems across the globe. To have any hope of mitigating this phenomenon, a much greater understanding of ecosystem dynamics is required. However, ecosystems are typically composed of highly dynamic networks of individual species. These interactions are further modulated by abiotic and biotic factors that vary temporally and spatially. Thus, ecological dynamics are obfuscated by this complexity. Here, we developed a theoretical model, informed by a synthetic experimental system, of Darwin's "warm little ponds." This cycling four-species system seeks to elucidate the ecological factors that drive or inhibit interaction. We show that these factors could provide an essential tool for avoiding the accelerating ecological collapse. Our study also provides a starting point to develop a more encompassing model to inform conservation efforts.
Asunto(s)
Ecosistema , Modelos Teóricos , Evolución Biológica , Consorcios Microbianos , Saccharomyces cerevisiaeRESUMEN
Synthetic biology enables the creative combination of engineering and molecular biology for exploration of fundamental aspects of biological phenomena. However, there are limited resources available for such applications in the educational context, where straightforward setup, easily measurable phenotypes and extensibility are of particular importance. We developed unigems, a set of ten plasmids that enable classroom-based investigation of gene-expression control and biological logic gates to facilitate teaching synthetic biology and genetic engineering. It is built on a high-copy plasmid backbone and is easily extensible thanks to a common primer set that facilitates Gibson assembly of PCR-generated or synthesized DNA parts into the target vector. It includes two reporter genes with either two constitutive (high- or low-level) or two inducible (lactose- or arabinose-) promoters, as well as a single-plasmid implementation of an AND logic gate. The set can readily be employed in undergraduate teaching settings, during outreach events and for training of iGEM teams. All plasmids have been deposited in Addgene.
RESUMEN
BACKGROUND: The introduction of Wolbachia (wMel strain) into Aedes aegypti mosquitoes reduces their capacity to transmit dengue and other arboviruses. Randomised and non-randomised studies in multiple countries have shown significant reductions in dengue incidence following field releases of wMel-infected Ae. aegypti. We report the public health outcomes from phased, large-scale releases of wMel-Ae. aegypti mosquitoes throughout three contiguous cities in the Aburrá Valley, Colombia. METHODOLOGY/PRINCIPAL FINDINGS: Following pilot releases in 2015-2016, staged city-wide wMel-Ae. aegypti deployments were undertaken in the cities of Bello, Medellín and Itagüí (3.3 million people) between October 2016 and April 2022. The impact of the Wolbachia intervention on dengue incidence was evaluated in two parallel studies. A quasi-experimental study using interrupted time series analysis showed notified dengue case incidence was reduced by 95% in Bello and Medellín and 97% in Itagüí, following establishment of wMel at ≥60% prevalence, compared to the pre-intervention period and after adjusting for seasonal trends. A concurrent clinic-based case-control study with a test-negative design was unable to attain the target sample size of 63 enrolled virologically-confirmed dengue (VCD) cases between May 2019 and December 2021, consistent with low dengue incidence throughout the Aburrá Valley following wMel deployments. Nevertheless, VCD incidence was 45% lower (OR 0.55 [95% CI 0.25, 1.17]) and combined VCD/presumptive dengue incidence was 47% lower (OR 0.53 [95% CI 0.30, 0.93]) among participants resident in wMel-treated versus untreated neighbourhoods. CONCLUSIONS/SIGNIFICANCE: Stable introduction of wMel into local Ae. aegypti populations was associated with a significant and sustained reduction in dengue incidence across three Colombian cities. These results from the largest contiguous Wolbachia releases to-date demonstrate the real-world effectiveness of the method across large urban populations and, alongside previously published results, support the reproducibility of this effectiveness across different ecological settings. TRIAL REGISTRATION: NCT03631719.
Asunto(s)
Aedes , Virus del Dengue , Dengue , Wolbachia , Animales , Humanos , Colombia/epidemiología , Ciudades/epidemiología , Incidencia , Análisis de Series de Tiempo Interrumpido , Estudios de Casos y Controles , Reproducibilidad de los Resultados , Control Biológico de Vectores/métodos , Dengue/epidemiología , Dengue/prevención & control , Mosquitos VectoresRESUMEN
BACKGROUND: The wMel strain of Wolbachia has been successfully introduced into Aedes aegypti mosquitoes and has been shown to reduce the transmission of dengue and other Aedes-borne viruses. Here we report the entomological results from phased, large-scale releases of Wolbachia infected Ae. aegypti mosquitoes throughout three contiguous cities located in the Aburrá Valley, Colombia. METHODOLOGY/PRINCIPAL FINDINGS: Local wMel Wolbachia-infected Ae. aegypti mosquitoes were generated and then released in an initial release pilot area in 2015-2016, which resulted in the establishment of Wolbachia in the local mosquito populations. Subsequent large-scale releases, mainly involving vehicle-based releases of adult mosquitoes along publicly accessible roads and streets, were undertaken across 29 comunas throughout Bello, Medellín and Itagüí Colombia between 2017-2022. In 9 comunas these were supplemented by egg releases that were undertaken by staff or community members. By the most recent monitoring, Wolbachia was found to be stable and established at consistent levels in local mosquito populations (>60% prevalence) in the majority (67%) of areas. CONCLUSION: These results, from the largest contiguous releases of wMel Wolbachia mosquitoes to date, highlight the operational feasibility of implementing the method in large urban settings. Based on results from previous studies, we expect that Wolbachia establishment will be sustained long term. Ongoing monitoring will confirm Wolbachia persistence in local mosquito populations and track its establishment in the remaining areas.
Asunto(s)
Aedes , Wolbachia , Animales , Humanos , Ciudades , Colombia , Ambiente , Mosquitos VectoresRESUMEN
BACKGROUND: The filamentous fungus Trichoderma reesei (Hypocrea jecorina) is an important source of cellulases for use in the textile and alternative fuel industries. To fully understand the regulation of cellulase production in T. reesei, the role of a gene known to be involved in carbon regulation in Aspergillus nidulans, but unstudied in T. reesei, was investigated. RESULTS: The T. reesei orthologue of the A. nidulans creB gene, designated cre2, was identified and shown to be functional through heterologous complementation of a creB mutation in A. nidulans. A T. reesei strain was constructed using gene disruption techniques that contained a disrupted cre2 gene. This strain, JKTR2-6, exhibited phenotypes similar to the A. nidulans creB mutant strain both in carbon catabolite repressing, and in carbon catabolite derepressing conditions. Importantly, the disruption also led to elevated cellulase levels. CONCLUSIONS: These results demonstrate that cre2 is involved in cellulase expression. Since the disruption of cre2 increases the amount of cellulase activity, without severe morphological affects, targeting creB orthologues for disruption in other industrially useful filamentous fungi, such as Aspergillus oryzae, Trichoderma harzianum or Aspergillus niger may also lead to elevated hydrolytic enzyme activity in these species.
Asunto(s)
Celulasa/metabolismo , Celulosa/metabolismo , Regulación Fúngica de la Expresión Génica , Microbiología Industrial/métodos , Trichoderma , Ubiquitina Tiolesterasa/genética , Secuencia de Aminoácidos , Aspergillus niger/genética , Aspergillus oryzae/genética , Prueba de Complementación Genética , Datos de Secuencia Molecular , Mutagénesis Insercional , Plásmidos , Homología de Secuencia de Aminoácido , Transducción Genética , Trichoderma/enzimología , Trichoderma/genética , Ubiquitina Tiolesterasa/deficiencia , Regulación hacia ArribaRESUMEN
Apis mellifera (honeybees) are a well-established model for the study of learning and cognition. A robust conditioning protocol, the olfactory conditioning of the proboscis extension response (PER), provides a powerful but straightforward method to examine the impact of varying stimuli on learning performance. Herein, we provide a protocol that leverages PER for classroom-based community or student engagement. Specifically, we detail how a class of high school students, as part of the Ryukyu Girls Outreach Program, examined the effects of caffeine and dopamine on learning performance in honeybees. Using a modified version of the PER conditioning protocol, they demonstrated that caffeine, but not dopamine, significantly reduced the number of trials required for a successful conditioning response. In addition to providing an engaging and educational scientific activity, it could be employed, with careful oversight, to garner considerable reliable data examining the effects of varying stimuli on honeybee learning.
RESUMEN
Culex quinquefasciatus mosquitoes are a globally widespread vector of several human and animal pathogens. Their biology and behavior allow them to thrive in proximity to urban areas, rendering them a constant public health threat. Their mixed bird/mammal feeding behavior further offers a vehicle for zoonotic pathogens transmission to people and, separately, poses a threat to the conservation of insular birds. The advent of CRISPR has led to the development of novel technologies for the genetic engineering of wild mosquito populations. Yet, research into Cx. quinquefasciatus has been lagging compared to other disease vectors. Here, we use this tool to disrupt a set of five pigmentation genes in Cx. quinquefasciatus that, when altered, lead to visible, homozygous-viable phenotypes. We further validate this approach in separate laboratories and in two distinct strains of Cx. quinquefasciatus that are relevant to potential future public health and bird conservation applications. We generate a double-mutant line, demonstrating the possibility of sequentially combining multiple such mutations in a single individual. Lastly, we target two loci, doublesex in the sex-determination pathway and proboscipedia, a hox gene, demonstrating the flexibility of these methods applied to novel targets. Our work provides a platform of seven validated loci that could be used for targeted mutagenesis in Cx. quinquefasciatus and the future development of genetic suppression strategies for this species. Furthermore, the mutant lines generated here could have widespread utility to the research community using this model organism, as they could be used as targets for transgene delivery, where a copy of the disrupted gene could be included as an easily scored transgenesis marker.
Asunto(s)
Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Culex/genética , Edición Génica , Técnicas de Inactivación de Genes , Ingeniería Genética , Animales , Culicidae/genética , Edición Génica/métodos , Marcación de Gen , Ingeniería Genética/métodos , Sitios Genéticos , Microinyecciones , Mutación , Fenotipo , ARN Guía de KinetoplastidaRESUMEN
Skilled motor behavior requires bihemispheric coordination, and participation of striatal outputs originating from two neuronal groups identified by distinctive expression of D1 or D2 dopamine receptors. We trained mice to reach for and grasp a single food pellet and determined how the output pathways differently affected forelimb trajectory and task efficiency. We found that inhibition and excitation of D1-expressing spiny projection neurons (D1SPNs) have a similar effect on kinematics results, as if excitation and inhibition disrupt the whole ensemble dynamics and not exclusively one kind of output. In contrast, D2SPNs participate in control of target accuracy. Further, ex vivo electrophysiological comparison of naive mice and mice exposed to the task showed stronger striatal neuronal connectivity for ipsilateral D1 and contralateral D2 neurons in relation to the paw used. In summary, while the output pathways work together to smoothly execute skill movements, practice of the movement itself changes synaptic patterns.
Asunto(s)
Cuerpo Estriado/fisiología , Miembro Anterior/fisiología , Movimiento/fisiología , Animales , RatonesRESUMEN
Hybrid sterility maintains reproductive isolation between species by preventing them from exchanging genetic material1. Anti-recombination can contribute to hybrid sterility when different species' chromosome sequences are too diverged to cross over efficiently during hybrid meiosis, resulting in chromosome mis-segregation and aneuploidy. The genome sequences of the yeasts Saccharomyces cerevisiae and Saccharomyces paradoxus have diverged by about 12% and their hybrids are sexually sterile: nearly all of their gametes are aneuploid and inviable. Previous methods to increase hybrid yeast fertility have targeted the anti-recombination machinery by enhancing meiotic crossing over. However, these methods also have counteracting detrimental effects on gamete viability due to increased mutagenesis2 and ectopic recombination3. Therefore, the role of anti-recombination has not been fully revealed, and it is often dismissed as a minor player in speciation1. By repressing two genes, SGS1 and MSH2, specifically during meiosis whilst maintaining their mitotic expression, we were able to increase hybrid fertility 70-fold, to the level of non-hybrid crosses, confirming that anti-recombination is the principal cause of hybrid sterility. Breaking this species barrier allows us to generate, for the first time, viable euploid gametes containing recombinant hybrid genomes from these two highly diverged parent species.
Asunto(s)
Hibridación Genética , Meiosis/genética , Recombinación Genética , Saccharomyces cerevisiae/genética , Saccharomyces/genética , Aneuploidia , Segregación Cromosómica , Proteína 2 Homóloga a MutS/genética , RecQ Helicasas/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
By virtue of complex ecologies, the behavior of mutualisms is challenging to study and nearly impossible to predict. However, laboratory engineered mutualistic systems facilitate a better understanding of their bare essentials. On the basis of an abstract theoretical model and a modifiable experimental yeast system, we explore the environmental limits of self-organized cooperation based on the production and use of specific metabolites. We develop and test the assumptions and stability of the theoretical model by leveraging the simplicity of an artificial yeast system as a simple model of mutualism. We examine how one-off, recurring, and permanent changes to an ecological niche affect a cooperative interaction and change the population composition of an engineered mutualistic system. Moreover, we explore how the cellular burden of cooperating influences the stability of mutualism and how environmental changes shape this stability. Our results highlight the fragility of mutualisms and suggest interventions, including those that rely on the use of synthetic biology.IMPORTANCE The power of synthetic biology is immense. Will it, however, be able to withstand the environmental pressures once released in the wild. As new technologies aim to do precisely the same, we use a much simpler model to test mathematically the effect of a changing environment on a synthetic biological system. We assume that the system is successful if it maintains proportions close to what we observe in the laboratory. Extreme deviations from the expected equilibrium are possible as the environment changes. Our study provides the conditions and the designer specifications which may need to be incorporated in the synthetic systems if we want such "ecoblocs" to survive in the wild.
RESUMEN
Ecosystems are complex networks of interacting individuals co-evolving with their environment. As such, changes to an interaction can influence the whole ecosystem. However, to predict the outcome of these changes, considerable understanding of processes driving the system is required. Synthetic biology provides powerful tools to aid this understanding, but these developments also allow us to change specific interactions. Of particular interest is the ecological importance of mutualism, a subset of cooperative interactions. Mutualism occurs when individuals of different species provide a reciprocal fitness benefit. We review available experimental techniques of synthetic biology focused on engineered synthetic mutualistic systems. Components of these systems have defined interactions that can be altered to model naturally occurring relationships. Integrations between experimental systems and theoretical models, each informing the use or development of the other, allow predictions to be made about the nature of complex relationships. The predictions range from stability of microbial communities in extreme environments to the collapse of ecosystems due to dangerous levels of human intervention. With such caveats, we evaluate the promise of synthetic biology from the perspective of ethics and laws regarding biological alterations, whether on Earth or beyond. Just because we are able to change something, should we?
RESUMEN
Sex with another species can be disastrous, especially for organisms that mate only once, like yeast. Courtship signals, including pheromones, often differ between species and can provide a basis for distinguishing between reproductively compatible and incompatible partners. Remarkably, we show that the baker's yeast Saccharomyces cerevisiae does not reject mates engineered to produce pheromones from highly diverged species, including species that have been reproductively isolated for up to 100 million years. To determine whether effective discrimination against mates producing pheromones from other species is possible, we experimentally evolved pheromone receptors under conditions that imposed high fitness costs on mating with cells producing diverged pheromones. Evolved receptors allowed both efficient mating with cells producing the S. cerevisiae pheromone and near-perfect discrimination against cells producing diverged pheromones. Sequencing evolved receptors revealed that each contained multiple mutations that altered the amino acid sequence. By isolating individual mutations, we identified specific amino acid changes that dramatically improved discrimination. However, the improved discrimination conferred by these individual mutations came at the cost of reduced mating efficiency with cells producing the S. cerevisiae pheromone, resulting in low fitness. This tradeoff could be overcome by simultaneous introduction of separate mutations that improved mating efficiency alongside those that improved discrimination. Thus, if mutations occur sequentially, the shape of the fitness landscape may prevent evolution of the optimal phenotype--offering a possible explanation for the poor discrimination of receptors found in nature.
Asunto(s)
Evolución Biológica , Péptidos/genética , Receptores de Feromonas/genética , Saccharomyces cerevisiae/genética , Sustitución de Aminoácidos/genética , Factor de Apareamiento , Datos de Secuencia Molecular , Plásmidos/genética , Reproducción/fisiología , Saccharomyces cerevisiae/fisiologíaRESUMEN
The asymmetrical inheritance of plasmid DNA, as well as other cellular components, has been shown to be involved in replicative aging. In Saccharomyces cerevisiae, there is an ongoing debate regarding the mechanisms underlying this important asymmetry. Currently proposed models suggest it is established via diffusion, but differ on whether a diffusion barrier is necessary or not. However, no study so far incorporated key aspects to segregation, such as dynamic morphology changes throughout anaphase or plasmids size. Here, we determine the distinct effects and contributions of individual cellular variability, plasmid volume and moving boundaries in the asymmetric segregation of plasmids. We do this by measuring cellular nuclear geometries and plasmid diffusion rates with confocal microscopy, subsequently incorporating this data into a growing domain stochastic spatial simulator. Our modelling and simulations confirms that plasmid asymmetrical inheritance does not require an active barrier to diffusion, and provides a full analysis on plasmid size effects.
Asunto(s)
Modelos Biológicos , Plásmidos/genética , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , ADN de Hongos/genética , DifusiónRESUMEN
The idea of introducing genetic modifications into wild populations of insects to stop them from spreading diseases is more than 40 years old. Synthetic disease refractory genes have been successfully generated for mosquito vectors of dengue fever and human malaria. Equally important is the development of population transformation systems to drive and maintain disease refractory genes at high frequency in populations. We demonstrate an underdominant population transformation system in Drosophila melanogaster that has the property of being both spatially self-limiting and reversible to the original genetic state. Both population transformation and its reversal can be largely achieved within as few as 5 generations. The described genetic construct {Ud} is composed of two genes; (1) a UAS-RpL14.dsRNA targeting RNAi to a haploinsufficient gene RpL14 and (2) an RNAi insensitive RpL14 rescue. In this proof-of-principle system the UAS-RpL14.dsRNA knock-down gene is placed under the control of an Actin5c-GAL4 driver located on a different chromosome to the {Ud} insert. This configuration would not be effective in wild populations without incorporating the Actin5c-GAL4 driver as part of the {Ud} construct (or replacing the UAS promoter with an appropriate direct promoter). It is however anticipated that the approach that underlies this underdominant system could potentially be applied to a number of species.