Coding

Part:BBa_K2306000

Designed by: Gabriella Tany   Group: iGEM17_TUDelft   (2017-10-04)


TorA-GFP

This biobrick is the basic part of the composite part (BBa_K2306001). This composite part is combined with a LacI promoter (BBa_R0010), a ribosome binding site (BBa_B0030) and a double terminator (BBa_B0015) to control its expression. Further information of our project can be found on our results page.

Usage and Biology

We, TU Delft 2017 , tried to transport proteins from the cytoplasm into the periplasm and eventually into vesicles. Several transport mechanisms exist that facilitate the export of either folded or unfolded proteins, and typically they require an export tag. There are different export pathways, but the most common ones are the twin arginine translocation (Tat) pathway and the general secretory (Sec) pathway (Baker et al. 2014).

We looked at several methods and pathways and found that Trondheim 2013 had also used the Tat translocation pathway that we wanted to use. This pathway translocate folded proteins from the cytoplasm into the periplasm. We noticed that they had some problems in translocating two fluorescence proteins (GFP and RFP) which they fused together with the transport tag. As a proof of concept of our design, we would like to transport GFP into the periplasm. We thus decided to modify their biobrick (BBa_ K1082001) by fusing the same transport tag with only a GFP protein.


Figure 1 Representation of the Tat pathway. (1) The tag at the N terminus of the protein will dock at site TatC. (2) TatB, which forms a complex with TatC, will act as a mediator and translocate protein P to TatA. TatA forms a ring structure which functions as a pore through the cytoplasmic membrane. (3) Protein P will translocate through TatA pore. (4) When translocated into the periplasm, the tag at the N terminus will be cleaved off, therefore releasing protein P in the periplasm.

The Tat pathway transports folded proteins across the cytoplasmic membrane into the periplasm. This pathway, shown in figure 1, consists of three components: TatA, TatB and TatC. The transport tag, which is located on the N terminus of the protein sequence, will interact with the initial docking site TatC, after which TatB will act as a mediator and translocate the protein to TatA. TatA will form a ring structure to make a pore through which the protein can be translocated. Passage through the cytoplasmic membrane into the periplasm occurs after polymerization of TatA. Once in the periplasm, the tag will be cleaved off. A key characterization of this pathway is that the pore sizes of TatA range from 100 kDa to over 500 kDa (Oates, J., et al, 2004).


Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 770


References

Baker, J.L. et al, 2014. Microbial biosynthesis of designer outer membrane vesicles. Curr Opin Biotechnol. 29, pp 76-84.

Oates, J., et al, 2004. The Escherichia coli twin-arginine translocation apparatus incorporates a distinct form of TatABC complex, spectrum of modular TatA complexes and minor TatAB complex. J Mol Biol. 346(1), pp 295-305.

[edit]
Categories
//cds/reporter/gfp
Parameters
None