Constitutive expression cassette for TetR +LVA (J23100.B0034.C0040.B0015)

Part Structure

The Biobrick encodes LVA-tagged TetR (BBa_C0040) under control of the constitutive promoter BBa_J23105 followed by the ribosome binding site BBa_B0034. The transcription of tetR is terminated by the double terminator BBa_B0015.

Mode of Action

TetR binds to the pTet regulator (BBa_R0040) and represses transcription. If the inducer [http://openwetware.org/wiki/ATc anhydrotetracycline (ATc)] is added, TetR action is inhibited and the promoter gets derepressed.

Purpose

This Biobrick was designed for the [http://2007.igem.org/ETHZ ETHZ iGEM 2007 project] and belongs to the constitutive part of the system. In the project description, this part is also termed Part 1. The constitutively synthesized TetR interacts with the double promoters BBa_I739102 and BBa_I739106 which are parts of composites BBa_I739015 and BBa_I739010 respectively. A similar (but not used) construct in this context is BBa_Q04400.

Testing

Checked for uniqueness of restriction enzyme cleavage sites:
Eco: ok
Xba: ok
Spe: ok
Pst: ok

Additional characterization of BBa I739001 by [http://2016.igem.org/Team:LMU-TUM_Munich Munich 2016]

BioBrick I739001 BBa I739001 was ligated upstream of BioBrick BBa_K2170221 (TetO-OmpA) which was then ligated upstream of BioBrick BBa_K2170142 (NanoLuc-Strep Tag-double terminator) to assemble BioBricks BBa_K2170141 (TetR-TetO-OmpA) and BBa_K2170053 (TetR-TetO-OmpA-NanoLuc-Strep Tag-double terminator. The functionality of both BioBricks could be confirmed by Nano-Glo assay which is why the functionality of I739001 BBa I739001 could be validated as well.

Results: Testing TetR-TetO system and the bacterial autotransporter system

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.

Discussion: The autotransporter is working and can present small protein cargos on the bacterial surface

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.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 7
Illegal NheI site found at 30
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

↑ Jose, J., Krämer, J., Klauser, T., Pohlner, J., & Meyer, T. F. (1996). Absence of periplasmic DsbA oxidoreductase facilitates export of cysteine-containing passenger proteins to the Escherichia coli cell surface via the Iga β autotransporter pathway. Gene, 178(1), 107-110.

[1] Jose, J., Krämer, J., Klauser, T., Pohlner, J., & Meyer, T. F. (1996). Absence of periplasmic DsbA oxidoreductase facilitates export of cysteine-containing passenger proteins to the Escherichia coli cell surface via the Iga β autotransporter pathway. Gene, 178(1), 107-110.

[1]