Team:NUS Singapore

iGEM 2016         NUS SINGAPORE

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  I. RIOT Sensor II. RIOT Responder III. RIOT Invader
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CD44 is a cell membrane protein involved in normal cell function (cell-cell and cell-matrix adhesion). However, one notable isoform, CD44v6, seems to play a major role in cancer progression, facilitating cell migration and invasion and is commonly upregulated on the surface of cancer cells. The RIOT system uses CD44v6 as a spatial marker, and recognition of this protein via a CD44v6 specific antibody allows anchoring of the engineered bacteria on the surface, triggering the expression of invasin and LLO for subsequent invasion.

Many complex biological processes lead to all forms of cancer, and their significant hallmarks include an ability to resist cell death, prolonged signalling, the origination of angiogenesis and metastasis. Another observable trait of cancer is described by the Warburg effect where a higher rate of glycolysis increases lactic acid production.

In proliferative cell types such as cancer, the bulk of the pyruvate from glycolysis is moved from the mitochondria to create lactate via lactate dehydrogenase - a process usually initiated only when oxygen supply is decreased. This production of lactate in the presence of oxygen is a direct consequence of the Warburg effect, resulting in a microenvironment with an increased lactate concentration.

Invasin is derived from the bacteria Yersinia pestis which allows selective invasion of cells that express β1-integrins. Listeriolysin O (LLO) on the other hand, is expressed in Listeria monocytogenes and allows the bacteria to break out of the endosome after entry into mammalian cells. Combining the traits of these two genes, our engineered bacteria is capable of entering the cytosol of cancer cells to potentially release a cytotoxic drug.

Initially, small amounts of N-Acyl homoserine lactones (AHLs) are produced by our engineered bacteria and they freely diffuse in and out of the cell. In the tumor region where the cell density is expected to be high, the concentration of AHL also increases, and past a threshold level, LuxR binds to AHL. When this happens, the LuxR-AHL complex goes on to further activate expression of LuxI, which then produces more AHL. This results in a positive feedback loop that increases both the concentration of LuxI as well as our desired proteins, invasin and LLO.

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Conventional drug delivery systems are plagued by problems such as non-specific targeting and low bioavailability. Bacterial-based drug delivery systems have gained much interest due to their ability to overcome the issue of non-specific delivery of drugs. This is achieved by engineering bacteria to sense and respond to specific stimuli present in the microenvironment of these pathogenic cells.

In this project, we propose the development of a dual-sensor bacteria which can only survive, and release therapeutics, within the targeted part of the human body (i.e., spatially specific). As our proof of concept, we will be engineering the Escherichia coli bacterium to target cancer tumors with high environmental lactate — see Warburg effect.

To ensure that the bacterium will only affect cancer cells, the bacterium is engineered to detect, and adhere to a cancer specific surface marker. Upon detection, a quorum sensing system and the production of invasin and listeriolysin O is triggered. The invasin and listeriolysin O then allows the bacteria to deliver its payload directly into the cytoplasm of the cancer cell. Since the production of invasin and listeriolysin O cannot occur in the absence of the said marker, this delivery system will only target cancer cells, concentrating drug payload at the intended site.

Our team has been working on cloning our desired biobrick parts and modelling our gene circuit. We hope to be able to characterise and optimise our parts, and demonstrate a functional spatially-specific drug delivery system, as well as complement our wet lab efforts with our model.

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