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The Microguards: Overview
Actualmente, existen numerosos procesos que utilizan microorganismos, como Escherichia coli e Saccharomyces cerevisiae, para producir compuestos de interés, como insulina, etanol y diversas enzimas. El éxito de estos procesos depende de un estricto control de contaminación por otros microorganismos en el medio de cultura. La presencia de contaminantes en un proceso fermentativo reduce el rendimiento de producción debido a la competición entre el contaminante y el organismo fermentativo, causando pérdidas de 5% a 10% de la producción total. Para tratar resolver este problema, el objetivo de nuestro proyecto es modificar cepas de E. coli y de S. cerevisiae para que sean capaces de reconocer y destruir contaminantes durante el proceso industrial.
The Coliguard: Nuestro modelo
Nosotros queremos modificar E. coli para que sea capaz de reconocer y destruir contaminantes en el medio de cultura. Para que nuestra bacteria tenga eficiencia máxima durante un proceso industrial, decidimos crear dos diferentes linajes celulares de E. coli: Las “células trabajadoras”, que serán las responsables por ejecutar el proceso industrial, y “las células asesinas” responsables por la detección y eliminación de eventuales contaminantes. Ambos tipos celulares existirán simultáneamente en el medio de cultura pero su proporción variará dependiendo de la presencia o ausencia de contaminantes. En ausencia de microorganismos contaminantes, el número de “células trabajadoras” será mucho mayor que el numero “células asesinas” asegurando de esta manera que el proceso se desarrolle con máxima eficiencia. En contraparte, en presencia de microorganismos contaminantes, “las células asesinas” inducirán a las “células trabajadoras” adyacentes a diferenciarse en mas “células asesinas”, potenciando así su labor. Después de la eliminación de los contaminantes por las células asesinas, la proporción de células trabajadoras y asesinas regresará a su monto inicial.
Basados en nuestro modelo, dividimos el proyecto en tres sub-partes.
- Mecanismo de reconocimiento
- Mecanismo de diferenciación
- Mecanismo "killer"
1) Recognition mechanism
Our idea is based on the premise that the engineered E. coli most be able to recognize contaminants in the culture medium as non-self. As most bacterial species produces AI2 (auto inducer 2) as a secondary metabolite, we decided to use this compound as a recognition factor. Our E. coli will be an AI2- strain and won´t produce native AI2. The presence of AI2 in the culture medium indicates the presence of contaminants, which will be recognized by an AI2 sensitive promoter present in our E. coli.
In the absence of contaminants, the amount of worker cells will be much higher than the number of killer ones, so the industrial process will occur at maximum efficiency.
Contaminants in the culture medium are recognized by the presence of AI2.
2) Differentiation mechanism
The initial differentiation mechanism is based on a random slippage mechanism that will determine the expression of a CRE recombinase in a small percentage of cells. This device is an adaptation from the device presented by the Caltech 2008 iGEM team. When expressed, the CRE recombinase will remove a device from the genome – containing a gene involved in the cell cycle and a gene that represses conjugation – and thus lead to the differentiation into killer cells. Killer cells are unable to reproduce, but able to conjugate. This device is an adaptation of the device presented by the Paris 2007 iGEM team.
Moreover, the presence of AI2 in the culture medium will also trigger the expression of CRE recombinase and thus induce the differentiation of more worker cells into the killer cells, so the proportion of killer cells will be elevated during the decontamination process. After a certain number of generations, the proportion of killer and worker cells will return to its original state, due to the killer cells being unable to reproduce.
3) Killing mechanism
We decided to use conjugation as a sensor that will trigger the killing mechanisms. Our E. coli is going to be an F+ strain, carrying a modified version of the conjugative plasmid pPed100 containing a killing gene. Only the killer lineage will be able to conjugate because in the worker lineage the pPed100 plasmid will be repressed. Since most bacterial strains in nature are F- our F+ E. coli will be able to conjugate with most contaminants. There will be two killing mechanisms, one carried by the modified pPed100 plasmid into the contaminant and another triggered by the conjugation signal. This secondary mechanism is necessary to stall contaminant growth because conjugation may take some time to occur and may be impaired due to culture conditions. This secondary killing mechanism will be triggered by the presence of a diffusible conjugation signal and will be able to induce neighboring killer cells, even when not conjugating. Such diffusible signal and corresponding promoter have never been described, but we’ve found promising candidates and their characterization is the main challenge for this project.
The Yeastguard: Expandind
Industrial ethanol production occurs in open vats with over 500,000 L. This process is mostly hindered by lactobacilli contamination, which produces lactic acid as their main metabolic product. Once contaminated, the whole vat must be discarded and cleaned, thus generating losses ranking in the millions of dollars.
We want our engineered yeast to be able to detect and control the proliferation of lactobacilli by introduction of a simple genetic device. The device must be able to recognize the presence of lactate in the medium during fermentation.
To allow entry of lactate in the cell, we will construct a gene coding for a lactate transporter under the control of a constitutive promoter. Once in the cell, this metabolite will induce the transcription of the gene coding for killer enzyme lysozyme, which is the most widely used antibiotic for lactobacilli decontamination.
The greatest challenge for this project is in characterizing a lactate-inducible promoter that is not subject to glucose catabolic repression, since the device must be activated during the process of sugar fermentation of the diauxic shift of S. cerevisiae.
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