iGEM Unesp Brazil

Project Description

Background

Designer probiotics have the power to revolutionize the way we diagnose, treat, cure and prevent diseases. Acting as biofabrics in the human's gut, genetically engineered probiotics produce and delivery therapeutics to treat metabolic disorders and infections directly in the patient's microbiome. They provide a promising alternative to produce pharmaceuticals, eliminating costly steps of downstream processes such as final product purification. Not to mention the major benefit to patients by offering a treatment that can be used via oral instead of intravenous administration, commonly used for biopharmaceuticals.

In 2017, we came up with the goal of revolutionizing the treatment for type I diabetes, to end the daily needs for insulin injection and all the invasive treatment that comes with it. For that, we proposed an orally administered probiotic, capable of producing insulin directly inside the person’s gut that has diabetes, according to the glucose levels coming from the diet. When released from the human body, a mechanism of kill switch would be activated by the light present in the environment, inducing the death of the microorganism and avoiding its spread into the environment.

The Insubiota project, as it was called, was prone to an improvement. A broader approach to reach other diseases that, like diabetes, make the lives of thousands of people difficult and require continuous treatment to suppress a metabolic disorder. Due to the lack of well-established platforms and protocols to engineer probiotics, our team designed a robust framework that enables access to the iGEM community to the full potential of this novel approach called Hope.

As in the Insubiota project, our goal with Hope is to engineer a bacterium, Bacillus subtilis, to sense the body needs for medication, and produce and deliver it only when necessary. Insubiota is a proof of concept for our new approach, going from design, fine-tuned expression and biocontainment to real-world application.

Our engineered probiotic is meant to be administered as a fermented milk or a lyophilized supplement to be mixed with other beverages. That would be a much less invasive procedure to the patient needing daily injections. Moreover, it would lessen the frequency of medicine administration compared to the conventional treatment. After ingestion, the bacteria would set up in the intestine lumen where it will sense and produce the corresponding molecule.

Sense and control

To make it possible to sense and respond to the patient’s needs, we built a fine-tuned regulatory circuit engineered to display an output signal after computing specific inputs, allowing it to be interchangeable and adapted to different signals. The regulatory circuit turns gene expression ON in the presence of the desired signal molecule, regulating at both transcriptional and translational levels. As proof of concept, we built a regulatory circuit based on the TetR and the T7 RNA Polymerase, which makes our device fully orthogonal.

Production

On this level, we built a standardized device designed to improve and facilitate the secretion and uptake of the molecule of interest in the human gut. The device employs the yncM signal peptide to drive exportation by B. subtilis and the cell-penetrating peptide penetratin to increase its absorption by the intestine epithelium. Absorbed proteins then reach the bloodstream to perform its function in the body.

Biocontainment

To avoid spreading GMOs in the environment, a biocontainment system responsive to light and based on the CRISPR/Cas machinery was designed to target specific DNA sequences in the genome. The Cas9 nuclease was split into n-Cas9 and c-Cas9 and fused to nMag and pMag, respectively. Thus, bacteria released from the person’s body will be exposed to light, which induces dimerization of nMag and pMag, and consequently rebuilds a fully functional Cas9 nuclease.

Gut simulation

To evaluate how our engineered probiotics survive and function in a real-world application, we designed and built a DIY bioreactor using a 3D printer and low-cost materials. The design reproduced the intestinal proportion and internal flow. We set a mixed culture under anaerobic condition in two DIY bioreactors operated as chemostats using a continuous flow for intake and outtake. Samples were periodically withdrawn for growth and fluorescence analysis. We successfully operated our bioreactors for 20h continuously. The mixed culture was kept stable during cultivation, and our GFP-producing B. subtilis could be tracked within the mixture throughout the culture period.

Navigate through our design and hardware pages to learn more!