Team:Caltech/Project/Results

Testing Export of Signaling Ligands

To determine whether or not the signaling ligands in these quorum sensing systems are exported, we Western blotted both the supernatant and cell lysate from cell cultures expressing the constructs we named pTG004 and pTG005 (lam system with 3xFLAG at the N-terminus of the lamD prepeptide sequence, and fsr system with 3xFLAG at the N-terminus of the GBAP domain within the fsrB protein sequence, respectively).

After running the Western blots, we observed multiple bands in both the supernatant and cell lysate for the samples originating from cells expressing the fsr quorum sensing system, offering promise that the GBAP signaling ligand is synthesized and exported. The bands observed appeared indicate the presence of proteins roughly 30 kDa, 25 kDa, and 5 kDa in size both in the supernatant and in the cell lysate. The 30 kDa fragment appears to correlate to entire, uncleaved fsrB membrane protein (see Figure 2 in "The Experiments"), while the 25 kDa fragment appears to correlate to the cleaved membrane-bound component of fsrB (the third fragment). Due to the faintness of the Western C ladder in the cell lysate blot and the crookedness of the supernatant blot, it was difficult to ascertain the size of the smallest band present in the fsr samples, but it is hypothesized to be close to 5 kDa, which, would suggest it is the GBAP signaling molecule with the 3xFLAG, and possibly the remainder of the fsrB membrane protein, attached to it (the last two fragments). While the presence of bands in the Westerns demonstrates that the fsr export system’s genes are being expressed in E. coli, the presence of large bands in the supernatant as well is troublesome, since those bands should correspond to fragments still embedded in the membrane and should have been pelleted and not present in the supernatant. It is possible that some cells were not fully spun down, and so the fsrB fragments embedded in their membranes were still present in the supernatant sample.

In contrast, no bands at all appeared on the Western blot for samples originating from cells expressing the lam system. This indicates that, barring some experimental error, none of the fragments detailed in Figure 1 (in "The Experiments") were synthesized by the cells, much less exported, suggesting that the ligand synthesis and export components of the lam QS system is incapable of being implemented in E. coli.