Our project consists of two stages; binding and treatment. For binding stage we used a protein called FimH. FimH is the protein that is present at the end of the type 1 pili, the hair like appendages found outside of E.coli. FimH enables the bacteria to bind to mannose sugar found outside of eukaryotic cells. (Krogfelt, Bergmans & Klemm, 1990) Since that was not a specific type of binding, we inserted RPMrel, a tumor specific binding peptide (Kelly & Jones, 2003) into FimH so that our genetically engineered E.coli will bind specifically to the tumor cells (part: K2052014). For treatment stage we used an enzyme called ButCoAT, the enzyme that directly converts Acetyl CoA into our agent, Butyrate. (Parts: K2052015 and K2052018) ButCoAT can be found in Roseburia intestinalis L1-82.(Duncan, S. H., et al., 2002) To combine those two stages we designed parts K2052016 which consists of FimH and ButCoAT together with terminator (B0015), arabinose promoter (K206000) and RBS (B0034).
The figure shows our plasmid design.
We made a part collection which enables E.Coli to bind to cancerous cells in the colon flora and secrete Butyrate when all parts work together as a group. We wanted to ensure that our E.Coli doesn’t secrete Butyrate without binding to cancer cells, so we made our part collection considering the consistency of basic parts.
On the other hand, we used the part FimH + RPMrel ( K1850010 ) in order to bind our E.Coli to cancerous cells. But we should use some other basic and composite parts to form a collection and make our part work perfectly and in an order. BBa_B0034 is used as RBS which is needed to make the bacteria read the part perfectly and produce the proteins. BBa_B0015 is used as a terminator to stop the reading and producing process. BBa_K206000 is an arabinose promoter which is really important for our part. When FimH + RPMrel production starts and E.Coli binds to cancer cells , Arabinose Induced Promoter ( K206000 ) becomes activated with the addition of arabinose into environment. The fact that we shouldn't produce ButCoAT without binding to cancerous cells, arabinose promoter is a really helpfull part for our collection.
1. Krogfelt, K.A., Bergmans H, Klemm P. (1990). “Direct evidence that the FimH protein is the mannose-specific adhesin of Escherichia coli type 1 fimbriae.”
2. Kelly, K. A., Jones, D. A. (2003). “Isolation of a Colon Tumor Specific Binding Peptide Using Phage Display Selection”
3. Duncan, S. H., Hold G. L., Barcenilla A., Stewart C. S., Flint H. J. (2002). “Roseburia intestinalis sp. nov., a novel saccharolytic, butyrate-producing bacterium from human faces”