Engineering escherichia coli for nanoparticle synthesis and targeting of colon cancer

• Main Author: Pasula, Rupali Reddy
• Other Authors: Lim Sierin
• Format: Theses and Dissertations
• Language: English
• Published: 2018
• Subjects: DRNTU::Engineering::Chemical engineering
• Online Access: http://hdl.handle.net/10356/75835
• Institution: Nanyang Technological University

The past decade and a half stands witness to a remarkable growth in the techniques of forward genetic engineering in living organisms, thereby engendering synthetic biology as a major player in disease diagnosis and treatment among other biomedical applications such as vaccine development and microbiome engineering. There is a growing interest to combine both nanotechnology and synthetic biology in cancer treatment due the appealing features of drug delivery and diagnosis offered by miniature programmable robots. Synthetic biology has been rapidly producing tools used in biomedicine by leveraging the various genetic tools at its disposal. This strategy has been extended to nanotechnology leading to the design of novel organisms capable of producing nanoscale materials with high precision. Microbes have been engineered to produce various natural and unnatural nanoparticles by borrowing the various available synthetic biology tools. Here, we report the first attempt to employ a synthetic biology approach to engineer the bacterium Escherichia coli to increase the iron loading in Archaeoglobus fulgidus Ferritin (AfFtn) by co-expressing an iron influx protein (FeoB) and knocking out an iron efflux protein (fieF). We exploit the natural iron storage function of ferritin to sequester iron and store it in the internal cavity to produce iron nanoparticles. Chimeric ferritin (AfFtn-m6A) has also been constructed to impart magnetotactic properties to E. coli where bacterial motility can be observed under the influence of an external magnetic field. These iron nanoparticles with protein corona have also been produced in vitro in ambient conditions and their properties such as size, thermal stability and the effect of iron loading on the structure of the protein has been characterized. These ferritin protein nanocages, which can also be employed as carriers, have been loaded with photosensitizer acridine orange and have been shown to be cytotoxic upon exposure to light for potential use in photodynamic therapy as assistive surgery. We envision a potential usage of the engineered nanoparticle-producing E. coli for magnetic resonance imaging of colorectal cancer. To complement the bacterium with specificity towards colorectal cancer, truncated membrane protein invasin was engineered to bind to colorectal cancer cells. The engineered bacterium was shown to bind to the β1-integrins and internalized into the colorectal cancer cells within 2 hours of co-incubation. Multiple functionalities of tumor imaging and therapy have been engineered in E. coli and its derivative entities for the management of colorectal cancer by employing a synthetic biology based approach.