Team:METU HS Ankara/Proof

Proof of Concept

The goal of our project was to create a bacterium that is able to bind and terminate cancer cells. Inspired by Team Harvard 2015’s design, we built our construct. The first step was binding. In order to create a bacterium that only binds to cancerous cells, we inserted a colon cancer specific binding peptide (RPMrel) into FimH protein (Kelly & Jones, 2003).

To prove our design and its effect on cancer cells, we first observed specific binding to Caco-2 colorectal cancer cells that was done by Team Harvard BioDesign 2015. Team Harvard had used a dot blot protocol in order to give an accurate image of cancer-specific binding of their bacteria.

The figure above shows the results of Team Harvard 2015’s dot blot protocol.

The second part of the project was termination and for that, we needed to find a substance that is able to eliminate cancer cells. Then, we came across a substance called butyrate. Butyric acid, also found in probiotic substances is a small particulate acid that can be secreted in our intestines that has recently started being used in cancer treatments by scientists (Hassig, Tong & Schreiber, 1997). These effects include promoting differentiation, arresting cell-cycle and inducing apoptosis of transformed colonocytes (Hassig, Tong & Schreiber, 1997). When butyrate enters a colon cancer cell, it inhibits the enzyme, HDAC (Histone deacetylase) which in turn induces p21 transcription, leading the effects mentioned above (Hassig, Tong & Schreiber, 1997). ButCoAT was our best candidate with it’s ability to directly convert Acetyl CoA into butyrate. (Flint et al., 2002).

The figures above show the processes that take place after butyrate (figure a) and vitamin D (figure b) enters the cell. This eventually leads to p21 transcription and then apoptosis. (Hassig, A. C.,Tong, K. J., Schreiber, L. S, 1997)

Our Construct

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According to theses two scientific findings, we designed our construct illustrated below.

Unlike previous studies FimH that we designed is produced continuously, after obtained binding induction of arabinose promoter would give rise to Butyryl CoA: Acetate CoA transferase that forms Butyrate subsequently.


Control group cells only (6 hours)


E.coli K12 (6 hours)


E.coli BL21 (6 hours)

The images above represent our co-culture results that we have treated E. coli K12 and E. coli BL21 with CaCo2 cancer cell lines. The pictures show that the cells are sustainable for 6 hours, which is enough for our project. Our observation showed that optimal time for co-culture in our conditions is 6 hours. Then we optimized 1 hour is enough for binding and our treatment trials going to be performed in 5 hours. We expected to see increase in the cell death in a time dependent manner. According to our observation our BL21 forms clusters. However, Harvard BioDesign used E. coli K12 and it was challenging to see them without staining. therefore, BL21 was the best candidate in our case.

References:

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1. Kelly, A. K., Jones, A.D.. “Isolation of a Colon Tumor Specific Binding Peptide Using Phage Display Selection”. Neoplasia, 5 (2003):437.

2. Hassig, A. C.,Tong, K. J., Schreiber, L. S. “Fiber-derived butyrate and the prevention of colon cancer”. Crosstalk. 4 (1997): 783-789.

3. Flint, J. H., Pryde, E.S, Stewart, S. C., Barcellina, A., Sylvia, H.D. “Acetate Utilization and Butyryl Coenzyme A (CoA):Acetate-CoA Transferase in Butyrate-Producing Bacteria from the Human Large Intestine”. Applied and Environmental Microbiology, 68(10)(2002): 5186–5190.

4. Harvard BioDesign 2015 iGEM Team: Retrieved from: http://2015.igem.org/Team:Harvard_BioDesign/Cancer