Composite

Part:BBa_K2306001

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


TorA-GFP (LacI promoter, RBS, double terminator)

TU Delft 2017 designed a basic part encoding the TorA-GFP sequence (BBa_K2306000), and then combined this fusion 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 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 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 (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).

Experiment Design

For characterization, TorA-GFP was transformed into both the E.coli BW25133 strain E. coli BW25133 strain with (KEIO, from Keio collection) and without (WT) the deletion of TolA. The different combinations of strains and plasmid are shown in figure 2. The KEIO strain was proven to hypervesiculate, which would help in our end goal, which was transporting GFP into vesicles. Widefield microscope images were made to visualize whether the addition of the transport tag impaired the GFP structure and therefore its fluorescence. Additionally, an osmo-shock was performed to harvest the periplasmic fraction of the cell. After that, the plate-reader was used to determine the GFP levels in the periplasm and cytoplasm. This will tell us whether the construct is functional thereby translocating the GFP into the periplasm. The goal was to have GFP transported into the vesicles. Therefore, a final experiment was done in which the GFP fluorescence was measured in vesicles.

Figure 2 Overview of the parental strain of Keio (WT) and the Keio strain (KEIO) and GFP-TA. In combination with the GFP-TA plasmid, there will be more GFP fluorescence in the periplasm in WT. In KEIO the GFP fluorescence from the periplasm will go to the vesicles.

Verifying functionality of GFP

To verify whether the addition of TorA tag to GFP did not impair the functionality of GFP, widefield images were taken with the widefield protocol. As shown in figure 3, GFP fluorescence was both observed in the KEIO and WT strain. Consequently, It is shows that more GFP is present in WT. Furthermore, you can see that the shape of the KEIO strain cells is elongated. This is probably caused by the deletion of TolA, which destabilizes the membrane (ref).

Figure 3 Widefield images of GFP-TA in the KEIO (left) and WT (right) strain.

Verifying functionality of TorA-GFP

For GFP to be able to be transported into vesicles, it needs to be present in the periplasm. Therefore, an osmoshock was performed using the osmoshock protocol , to check the periplasmic fraction of the KEIO and WT strain, both with and without TorA-GFP. After the osmoshock the fluorescence of the GFP in the periplasm and cytoplasm was measured in the plate-reader.


Results

In figure 4, the fluorescence intensity in the periplasm in WT is significantly higher than in the cytoplasm. However, this difference in intensity is much less pronounced in the KEIO strain. A possible explanation for this, is the previously demonstrated vesicle production. We measured that the KEIO strain produces a large amount of vesicles, while the WT strain produces none. This result suggests that in the KEIO strain GFP is transported into the vesicles, which reduces the amount of GFP in the periplasm. Another possibility could be that both the growth and the protein production is greatly impaired in the KEIO-strain, due to its mutation. This means that the overall GFP production is lower, leading to a lower concentration in the periplasm as well. All in all, we see that GFP-TA is transported into the periplasm.


Figure 4 Widefield images of GFP-TA in the KEIO (left) and WT (right) strain.

Confirming the presence of GFP in vesicles

The goal was to transport proteins into vesicles. One of the things we have shown is that hypervesiculation occurs in E.coli BW25113 of the Keio strain with a TolA deletion through Dynamic Light Scatter (DLS) experiments. Additionally, our experiments have demonstrated that GFP is transported to the periplasm with the TorA tag by measuring fluorescence intensity in the cytoplasm and periplasm. To determine if GFP was transported into vesicles, we combined the two plasmids and measured the intensity of GFP in vesicles. Modeling determined that after 25 minutes post induction the concentration of GFP in the vesicles did not grow anymore and reached its maximum. On top of this, it we had shown that the amount of vesicles produced was higher after 20 hours. Combining this, the vesicles we harvested after 20 hours post induction. The same purification method as in the DLS experiments (DLS Protocol) was used, after which fluorescence was measured in the plate reader.


Results

Evidently, GFP is present in the purified sample of GFP-TA in WT, even though previous experiments showed that WT produces little to no vesicles, suggesting that those values are a background from the protocol used, due to a cell destruction during the centrifugation step (Figure 5).To test this hypothesis, the protocol could be adjusted to see whether GFP is still present at lower centrifugation speeds. Regardless of these possible changes, the fluorescence intensity of both GFP-TA with and without TolR in KEIO is significantly higher than GFP-TA in WT. Taken together, we can conclude that GFP is transported into the vesicles.


Figure 5 Intensity of GFP in vesicles. The fluorescence intensity of the samples TorA-GFP with TolR in the KEIO strain, TorA-GFP in the KEIO and WT strain and the empty backbone in the WT strain.

Conclusion

In this part of the project, we successfully transformed the GFP-TA plasmid into the KEIO and WT strain. With the wideifield images, we have shown that the functionality of GFP to fluoresce is still working. The osmo-shock experiment shows that the intensity of GFP-TA is significantly higher in the periplasm than in the cytoplasm in the WT strain, while there was no pronounced difference in the KEIO strain. On top of this it is proven that GFP is present in the vesicles, when vesicles are produced. All in all, we can conclude that TorA-GFP is translocated to the periplasm, after which GFP is transported into the vesicles.



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 999


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