Difference between revisions of "Part:BBa K2552005"

Revision as of 07:09, 17 October 2018

Ompa-Oligo peptide T-1

Ompa-Oligo peptide T-1 part uses Ompa system to display oligo peptide T-1 on the surface of E.coli cells.

Usage and Biology

It’s an improvement of OmpA(BBa_K1489002 ). OmpA is widely utilized to anchor otherwise soluble proteins in the outer cell membrane of bacteria like E.coli. Thus, OmpA functions as a carrier that carry different protein or domain to the surface of bacterial cells. Considering more and more teams are focusing on cancer targeting, we added a 15-AA oligo peptide to the C-terminal of OmpA. The oligo peptide has the ability to bind to Thomsen–Friedenreich antigen (T antigen) that exist on many kinds of cancer cells by a mechanism similar to antigen-antibody binding reaction, so it‘s helpful in cancer targeting. On the other hand, the oligo peptide is so short that its influence on the function of OmpA is limited. That is, when OmpA is carrying other peptides or protein domains at the C terminal, there is little chance that the peptide will cause adverse effects such as misfolding.

experiment design

Experiments have been conducted by Team SJTU-BioX-Shanghai. We used cell-climbing cover glasses as the solid basement on which the cell-bacteria adhesion experiment was carried out. Cell line HT29(human colorectal cancer cell) was chosen for the experiment, which grew over the glasses. We certificate the expression of T antigen by FACS previously. After cell HT29 grew and covered the glasses， we mixed it with E.coli BL21(DE3) cultures which expressed OmpA-peptide fusion protein (egfp was expressed as reporter gene ). The mixture glasses were incubated at 4°C for hours(results of 12h are shown) after which cells were cleaned with normal saline to wash away unconjugated bacteria. These cover glasses were carefully observed via the fluorescent microscope.

Protein structure modeling

We use Swiss Model to predict the structure of our OmpA-peptide fusion protein. The result shows us that the oligo peptide don’t influence the correct folding of the β-barrel of OmpA.

Experimental results

Fig1. The results of our experiments Sequence and Features