Part:BBa_K2306000:Design
TorA-GFP
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 770
Design Notes
We, iGEM 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 (2). We looked at several methods and pathways and found that Trondheim 2013 had also used the Tat translocation pathway that we wanted to use. We noticed that they had some problems in translocating two fluorescence protein (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 by fusing the same transport tag with only a GFP protein giving us this basic part (BBa_K2306000). The composite part used (BBa_K2306001) for characterization is combined with a LacI promoter (BBa_R0010), a ribosome binding site (BBa_B0030) and a double terminator (BBa_B0015) to control its expression.
Source
The TorA sequence originated from BBa_K1082001, a composite part made up of a tat-GFP-RFP sequence. From this part we took the tat sequence for our part. We then combined this sequence with the GFP sequence from BBa_E0040 (basic part).