Insertion of the butyrate gene pathway
Gene deletion of carbon-draining enzymes
Addition of colon cancer binding ability
Our primary BioBrick was designed to contain an operon with a combination of 5 enzymes shown to facilitate butyrate production. A similar pathway has been shown by other investigators, but no journal publication describes this pathway being employed in E. coli Nissle 1917. We thus repurposed the pathway for probiotic application. The result of this aim is a part that is a functional improvement of Toulouse iGEM 2015 butyrate pathway biobricks (composite part BBa_K1587004 and BBa_K1587005). We designed our butyrate pathway gblock (gene block) as two segments due to the 5Kb sequence length. A ribosome binding site (RBS) calculator was used to create moderate strength RBS between each gene.
We also wanted E. coli Nissle 1917 to under-express enzymes that drain carbon and reducing equivalents (ex. NADH) away from butyrate production. Acetyl-CoA is the starting point for the pathway. Theoretically, taking away the wild-type enzymes that also use acetyl-CoA, NADH, or any of the intermediate byproducts could increase end-product yields of our desired SCFA. Gene deletion is an underutilized route in iGEM team projects as it can pose significant difficulties, especially for constitutive or ‘housekeeping’ genes. Nevertheless, we included it in our experimental design, as it is needed for metabolic engineering. Butyrate production of all engineered strains can be assessed through high-performance liquid chromatography (HPLC) and anaerobic fermentation. Presence of other metabolites (fumarate, glucose, succinate, lactate, formate, acetate, ethanol) will also be measured to assess efficiency of gene deletions.
Lastly, we wanted to utilize an engineered fimH type-one pillus derived cancer binding protein. FimH, if correctly expressed, will attach at the pili and will allow bacterial cells to naturally bind to the sugar mannose on epithelial cells. However, in order to make our bacteria bind specifically to colon cancer cells, we chose the RPMrel colon cancer specific binding peptide part already designed by Harvard BioDesign iGEM 2015 (BBa_K1850010). It is to be inserted under control of its included promoter, downstream of the butyrate operon terminator. This will then be added to the edited E. coli Nissle 1917. The binding assay performed by the METU HS ANKARA 2016 iGEM team will be altered to show that our construct effectively binds Caco-2 colon cancer cells in vitro. A red fluorescent protein will be inserted next to FimH to visualize cancer cell binding.