Multilayered safety framework for living diagnostics in the colon

Introduction: Colorectal cancer is the second most deadly cancer worldwide. Current screening methods have low detection rates and frequently provide false positive results, leading to missed diagnoses or unnecessary colonoscopies. To tackle this issue, the Wageningen UR iGEM team from 2022 developed “Colourectal”, a living diagnostic tool for colorectal cancer. Following a synthetic biology approach, the project used an engineered Escherichia coli Nissle 1917 strain capable of binding to tumour cells that detects two distinct cancer biomarkers, and secretes a coloured protein observable in stool. Due to the utilization of genetically modified bacteria in vivo, precautionary biosafety measures were included within a three level safe-by-design strategy.Results: The first genetic safeguard ensured confinement of the living diagnostic to the colon environment by implementing auxotrophy to mucin that is abundant in the colon lining. For this, a synthetic chimeric receptor was generated to ensure expression of essential genes in the presence of mucin. The second strategy limited the viability of the engineered bacteria to the human body, preventing proliferation in open environments. The use of a temperature sensitive kill switch induced bacterial cell death at temperatures below 37°C. The third biocontainment strategy was installed as an emergency kill switch to stop the Colourectal test at any point. By inducing a highly genotoxic response through CRISPR-Cas-mediated DNA degradation, cell death of E. coli Nissle is triggered.Discussion: While the use of engineered microorganisms in human applications is not yet a reality, the safety considerations of our multi-layered strategy provide a framework for the development of future living diagnostic tools

Estudio sobre VAMP2.1, potencial regulador del canal de potasio KAT1 en Arabidopsis thaliana

[ES] En plantas, muchos eventos fisiológicos dependen del intercambio iónico a través de proteínas específicas de la membrana plasmática. En concreto, los canales de potasio son esenciales en numerosos procesos celulares como, por ejemplo, la regulación de la apertura de los estomas, que son estructuras epidérmicas ubicadas en las hojas y consistentes en dos células oclusivas que rodean un poro. La importancia de estas estructuras radica en que son las responsables de la toma de CO2 para la fotosíntesis, la pérdida agua por transpiración y la entrada de ciertos patógenos en la planta. Por todo ello, la identificación de reguladores fisiológicamente relevantes de los canales de potasio puede ser de ayuda en el diseño de estrategias para mejorar la tolerancia de plantas a la sequía y a la susceptibilidad frente a patógenos. A pesar de que existen diversos canales de potasio, KAT1 se ha caracterizado como uno de los canales altamente selectivos de rectificación interna más importantes expresados en las células oclusivas del estoma. Estos canales controlados por voltaje son responsables del influjo de potasio a largo plazo y juegan un papel clave en la apertura del estoma. Aunque se trata de una proteína bien caracterizada, aún se desconocen muchos detalles acerca de la regulación de KAT1. Las interacciones proteína-proteína tienen un rol fundamental en la regulación de los procesos celulares. Por este motivo, en este estudio se busca caracterizar VAMP2.1, una proteína de membrana asociada a vesículas que interacciona con KAT1 de la planta modelo Arabidopsis thaliana, según ha demostrado un screening Split-ubiquitina realizado previamente en levadura. Por un lado, se estudiará la localización subcelular e interacción proteína-proteína de VAMP2.1 en planta. El conocimiento de la localización de las proteínas y su interacción genera información útil acerca de sus características y regulación. Posteriormente, se estudiará el efecto de la alteración de la expresión de VAMP2.1 sobre la planta. Para ello, se utilizarán mutantes de Arabidopsis thaliana por inserción de T-DNA obtenidos de las colecciones SALK y SAIL. El resultado de este estudio permitirá valorar la manipulación de VAMP2.1 como posible estrategia para mejorar la respuesta de plantas a condiciones de estrés como la sequía.[EN] In plants, many physiological events are dependent on the ion exchange through plasma membrane specific proteins. Specifically, potassium channels are essential in numerous cell processes, such as the stomatal opening regulation. Stomata are epidermal structures located in the leaves and consisting of two guard cells that surround a pore. The importance of these structures lies in that they are responsible for CO2 uptake for photosynthesis, water loss through transpiration and the entry of certain pathogens into the plant. Therefore, the identification of physiologically relevant potassium channel regulators can be helpful in the design of strategies to improve plant drought tolerance and susceptibility to pathogens . Even though there are different kinds of potassium channels, KAT1 has been characterized as one of the most important highly selective inward rectifying channels expressed in guard cells. Those voltage-dependant channels are responsible for the long-term potassium influx into guard cells and play a key role in the stomatal opening. Although KAT1 is a well characterized protein, many details of its regulation are still unknown. Protein-protein interactions have a fundamental role in the regulation of cellular processes, hence this study seeks to characterize VAMP2.1, a vesicle-associated membrane protein that interacts with KAT1 in the model plant Arabidopsis thaliana, according to the results shown in a Split-Ubiquitin screening performed on yeast. On the one hand, the subcellular localization and protein-protein interaction of VAMP2.1 in plant will be studied. Knowing the location of proteins and their interaction generates useful information about their features and regulation. On the other hand, the effect of the alteration of VAMP2.1 expression on the plant will be studied. To do this, Arabidopsis thaliana mutants by insertion of T-DNA obtained from the SALK and SAIL collections will be used. The results of this study will allow to assess the manipulation of VAMP2.1 as a possible strategy to improve the response of plants to stress conditions such as drought.Torregrosa Barragán, L. (2020). Estudio sobre VAMP2.1, potencial regulador del canal de potasio KAT1 en Arabidopsis thaliana. http://hdl.handle.net/10251/150947TFG

DataSheet1_Multilayered safety framework for living diagnostics in the colon.XLSX

Introduction: Colorectal cancer is the second most deadly cancer worldwide. Current screening methods have low detection rates and frequently provide false positive results, leading to missed diagnoses or unnecessary colonoscopies. To tackle this issue, the Wageningen UR iGEM team from 2022 developed “Colourectal”, a living diagnostic tool for colorectal cancer. Following a synthetic biology approach, the project used an engineered Escherichia coli Nissle 1917 strain capable of binding to tumour cells that detects two distinct cancer biomarkers, and secretes a coloured protein observable in stool. Due to the utilization of genetically modified bacteria in vivo, precautionary biosafety measures were included within a three level safe-by-design strategy.Results: The first genetic safeguard ensured confinement of the living diagnostic to the colon environment by implementing auxotrophy to mucin that is abundant in the colon lining. For this, a synthetic chimeric receptor was generated to ensure expression of essential genes in the presence of mucin. The second strategy limited the viability of the engineered bacteria to the human body, preventing proliferation in open environments. The use of a temperature sensitive kill switch induced bacterial cell death at temperatures below 37°C. The third biocontainment strategy was installed as an emergency kill switch to stop the Colourectal test at any point. By inducing a highly genotoxic response through CRISPR-Cas-mediated DNA degradation, cell death of E. coli Nissle is triggered.Discussion: While the use of engineered microorganisms in human applications is not yet a reality, the safety considerations of our multi-layered strategy provide a framework for the development of future living diagnostic tools.</p

Image1_Multilayered safety framework for living diagnostics in the colon.PDF

Introduction: Colorectal cancer is the second most deadly cancer worldwide. Current screening methods have low detection rates and frequently provide false positive results, leading to missed diagnoses or unnecessary colonoscopies. To tackle this issue, the Wageningen UR iGEM team from 2022 developed “Colourectal”, a living diagnostic tool for colorectal cancer. Following a synthetic biology approach, the project used an engineered Escherichia coli Nissle 1917 strain capable of binding to tumour cells that detects two distinct cancer biomarkers, and secretes a coloured protein observable in stool. Due to the utilization of genetically modified bacteria in vivo, precautionary biosafety measures were included within a three level safe-by-design strategy.Results: The first genetic safeguard ensured confinement of the living diagnostic to the colon environment by implementing auxotrophy to mucin that is abundant in the colon lining. For this, a synthetic chimeric receptor was generated to ensure expression of essential genes in the presence of mucin. The second strategy limited the viability of the engineered bacteria to the human body, preventing proliferation in open environments. The use of a temperature sensitive kill switch induced bacterial cell death at temperatures below 37°C. The third biocontainment strategy was installed as an emergency kill switch to stop the Colourectal test at any point. By inducing a highly genotoxic response through CRISPR-Cas-mediated DNA degradation, cell death of E. coli Nissle is triggered.Discussion: While the use of engineered microorganisms in human applications is not yet a reality, the safety considerations of our multi-layered strategy provide a framework for the development of future living diagnostic tools.</p