Team:METU HS Ankara/Composite Part
Design Notes of K2052014
As our project consists of two stages we digested the part that we’ve divided into two sections. This part made up of protein coding region; FimH, a double terminator (B0015) and an arabinose induced promoter (K206000).
We got our protein coding region from Harvard Bio Design iGEM 2015 Team. without its arabinose dependent promoter region. This part enables E.coli to bind cancerous cells in colon flora. “Fim” gene expression in some substranes of E.coli results hairlike appendages on the bacteria. At the end of the pilus, there is a protein “FimH” which is from the Fim family. It binds naturally to sugar mannose.
RPMrel is a nine-amino-acid, disulfide-constrained peptide. By the help of RPMrel, a colon tumor specific binding peptide, our E.coli has the ability to bind specifically to tumor cells in colon flora
The double terminator (B0015) and arabinose induced promoter that we’ve used is gotten from the iGEM part library.
Source
FimH; Escherichia coli k12 mg1665
3D Structure of FimH together with RPMrel
3D Structure of FimH together with RPMrel can be seen below as Harvard BioDesign 2015 submitted in part registry :
Visual demonstration of interaction between FimH and its ligand
Our simulations were designed by the NAMD program, and CHARMM force field was applied. This CHARMM technique was used to calculate the energy usage in the simulations. The FimH and HETA models were used in the simulation were taken from an cristal model. After 100 ns, the protein could not disassociate from the ligand. We can understand that there is a strong interaction between the protein and the ligand.
Design Notes of K2052015
Our RBS and protein coding region –butCoaT- are digested from our whole construct with the enzymes SpeI, PstI an ligated with pSB1C3. This part enables our E.coli to secrete ButCoaT which is the enzyme that converts Acetyl-CoA to Butyrate.
Butyrate is a four carbon, short chain fatty acid, is formed in the human colon by bacterial fermentation of carbohydrates (including dietary fiber), and putatively suppresses colorectal cancer (CRC). It induces apoptosis and differentiation, inhibits proliferation of tumorous cells in colon flora. At the intestinal level, butyrate plays an administrative part on the transepithelial liquid transport, improves mucosal aggravation and oxidative status, strengthens the epithelial protection obstruction, and regulates instinctive affectability and intestinal motility. As it is claimed to suppress colorectal cancer, Butyrate is our agent to defeat cancerous cells.
D3D Structure of ButCoAT
Butyrate can be formed by numerous ways as in the figure below;
As Acetyl-CoA is readily present in the cells, we used an enzyme that directly converts Acetyl-CoA to our agent; Butyrate. Enzyme name is But-Coat, our protein coding region. Here we have schematic demonstration of reaction;
Source
Design Notes of K2052016
Our construct consists of a protein coding region (FimH), a double terminator (B0015), an arabinose induced promoter (K206000), a ribosome binding site (B0034), and another protein coding region (ButCoaT).
Our construct have 11 restriction enzyme cut sides. The prefix that we have designed includes EcoRI, NotI, XbaI. On the other hand, SpeI, NotI, PstI is included in our suffix. We entailed NotI for confirming the size of our construct while we're making our experiments. Therewithal, we added NcoI and HindIII so that we can clone our construct into Pet28a. To determine that both FimH and ButCoaT sections function well, we included SpeI, SalI, XbaI restriction enzyme cut sides. By this way, we conduct our experiments for FimH and ButCoaT separately. We put these three restriction enzyme cut sides after our arabinose induced promoter to determine if our construct is working with that promoter or not. We decided to work with Arabinose Induced Promoter in the middle of our construct to understand, if FimH works without putting arabinose and if ButCoaT works with arabinose in the medium.
We want our bacteria to stick to cancer cells and release butyric acid in order to suppress tumor formation and prevent colon cancer in early stages. We use RPMrel and FimH in order to provide our bacteria to stick to cancer cells as RPMrel is a colon tumor specific binding peptide. After, our bacteria sticks to the cancerous cells in colon flora, it will secrete butyrate which induces apoptosis and differentiation and also inhibits proliferation.
Design Notes of K2052017
The construct K2052016 was cloned into an cloning vector pUC57.
Plasmid view:
References:
1. Kelly, K. A., Jones, D. A., (2003). Isolation of a Colon Tumor Specific Binding Peptide Using Phage Display Selection
2. Hassig, C.A., Tong, J.K., Schreiber, S.L. (1997). Fiber-derived Butyrate and the Prevention of Colon Cancer
3. Canani, R. B., Costanzo, M. D., Leone, L., Pedata, M., Meli, R., Calignano, A. (2011). Potential beneficial affects of butyrate in intestinal and extraintestinal diseases
4. Kelly, K., Alencar, H., Funovics, M., Mahmood, U., Weissleder, R. (2004).Detection of invasive colon cancer using a novel, targeted, library-derived fluorescent peptide.
5. Sauer MM, Jakob RP, Eras J, Baday S,, Eriş D, Navarra G, Bernèche S, Ernst B, Maier T, Glockshuber R. (2016 March 7). “Catch-bond mechanism of the bacterial adhesin FimH” Retrieved from: http://www.ncbi.nlm.nih.gov/pubmed/26948702
6. Hassig, C.A., Tong, J.K., Schreiber, S.L. (1997). Fiber-derived Butyrate and the Prevention of Colon Cancer
7. Roberto Berni Canani, Margherita Di Costanzo, Ludovica Leone, Monica Pedata, Rosaria Meli, Antonio Calignano. (2011). Potential beneficial affects of butyrate in intestinal and extraintestinal diseases
8. Duncan,S. H., Barcenilla, A., Stewart, C. S., Pryde, S. E., Flint, H. J. (2002). Acetate Utilization and Butyryl Coenzyme A (CoA):Acetate-CoA Transferase in Butyrate-Producing Bacteria from the Human Large Intestine
