Difference between revisions of "Part:BBa K2170051"

For the testing of our autotransporter constructs we transformed all three autotransporter constructs together with a secreted NanoLuc luciferase, which served as a control, into E. coli JK321^[1]. Transformed cells were grown in LB-medium supplemented with chloramphenicol (pSB1C3-vector) at 30°C and 180 RPM. In the morning a stationary over night culture was used to inoculate 50 mL fresh medium with 1 mL of culture. The cultures were grown to OD₅₅₀ = 0.5 and then the induction of the protein production was started using aTc: "non" = non-induced; "low" = 25 ng/mL aTc; "high" = 200 ng/mL. After induction the cultures were grown for further 5 h at the same concitions and cells were afterwards stored at 4°C until they were used for analysis or bioprinting.
After the cell harvest the optical density (OD₅₅₀) was determined again as it is known that a high expression of EspP makes the outer membrane of E. coli permeable and this drastically affects cellular viability. For the scAvidin-autotransporter and the secreted BAP-NanoLuc autotransporter at high inducer concentration show clearly reduced viability as they did not show any growth after induction at OD₅₅₀ = 0.5 (see Fig. A). In order to analyze the functionality of the whole design and these new BioBricks we subjected each of the twelve cultures to three different treatments and determined subsequently the amount of NanoLuc luciferase using the manufacturers substrate kit (Thanks to our sponsor Promega ;). At first the total NanoLuc luciferase content was determined in samples that were just washed once with PBS and contained the whole bacterial cell (see Fig. B), bacterial cells that were incubated with PPA-buffer (periplasmic extraction buffer; 500 mM sucrose, 100 mM Tris/HCl pH 8.0, 1 mM EDTA) that extracts the proteins present in the periplasm (see Fig. C) and finally the culture supernatant of the cultivated bacterial cells (see Fig. D). The total NanoLuc activity is only present in bacteria that are transformed with a plasmid encoding the luciferase and there is a clear dependency of inducer concentration a detected luciferase activity. In the samples where a periplasmic extract was prepared the luciferase activity is highest in the samples of the secreted luciferase as the EspP-fused BAP-NanoLuc is attached to the cells and only proteins from lysed cells are present in the periplasmic extract. In the case of the samples where the supernatant of the cultures was analyzed the luciferase activity is present in both "high" induced samples.

As the luciferase activity only gave an indication for the subcellular localization of the cargo-autotransporter fusion protein, we also performed flow cytometry measurements. For this purpose the same cells were stained with app. 10 µM A3C5-Fab that was coupled to the fluorescent dye Dy634-NHS and 100 µM of the chemical compound biotin-atto488. Cells were incubated for 2 h at 4°C, washed twice with PBS and analyzed using a FACSAria II flow cytometer (BD bioscience, voltages for fluorescence channels were all at 750 V).

For the channels FITC (biotin-atto488) and Dy634 (A3C5-F_ab-Dy634, detected using a HeNe laser (633 nm) and a 660/20 band pass filter) the fluorescence signal was measured and mean values are given. The cells showed a homogeneious population in the FSC/SSC plot. The A3C5-F_ab staining detected high signals up til a mean of over 4000 for the eMA-autotransporter dependent on the inducer concentration. For the same bacterial cells the binding of biotin-Atto488 could be confirmed as well. The single chain Avidin (scAvidin) cells showed absolutely no A3C5 signal but a low Biotin-Atto488 background signal that could be caused by biotin-importers that transport biotin into the cells or the slow diffusion into the periplasm of E. coli. In contrast the BAP-Receptor also shows a high surface presentation as seen in the A3C5 signal of nearly 4000 for the "high" induced sample. The elevated biotin-Atto488 is unexpected but could be explained by the weakened and partly permeable out membrane that does not stop the small-molecule dye from entering the periplasm.

The performed experiment confirm the functionality of the device including the tetracyclin repressed promoter, the secretion into the bacterial periplasm as well as the presentation of the protein cargon on the bacterial outer membrane. In agreement with the expectation the 62 kDa big scAvidin with its extremely strong fold is too big in size for the autotransporter and can not be presented on the bacterial surface. In contrast the smaller BAP-NanoLuc (19 kDa for the NanoLuc) or the eMA (15 kDa) both can be efficiently presented on the surface. The enzymatic biotinylation of the Biotin Acceptor Peptide (BAP) using recombinant BirA biotin ligase is well established but could not be show before the wiki freeze. In Summary the whole device works great but there seems to be a limitation concerning the size of proteindomains that can be presented with the EspP autotransporter. We are curious to see what future iGEM teams will use the EspP Autotransporter in the future.