Modified Pili Expression
Over the duration of the project we endeavored to answer a number of questions experimentally. Can we modify FimH with large proteins? Can the modified FimH be exported and can pilus biogenesis occur when large proteins have been fused? Can we modify the FimH protein at the N-terminal, after the signal peptide cleavage point? Can we drive expression of the fim operon with a constitutive promoter?
The T7_FimH_225_sfGFP construct exhibited fluorescence when examined using a plate reader (Tecan) and an Amnis ImageStream ISX. Protein expression was demonstrated using a Western blot and TEM-Immunogold techniques. The results in sum make a compelling case to suggest that a protein as large as sfGFP can be fused with FimH without losing the integrity of the tertiary structure or the robustness of pilus biogenesis.
The P_Rha_FimH_1_His part was shown to be successfully expressed in four different E. coli. The protein produced had an affinity for anti-His antibodies. A molecular weight differential was observed in the separation of bands in the polyacrylamide gel which supports the case that the signal peptide is cleaved upon successful export of FimH. Not only did this evidence serve to imply that we had produced the protein in the pili on the surface of the cell, but it also strengthened the argument that the first amino acid position is a good placement for a binding domain.
While transmission electron microscopy, coupled with negative staining, did not provide definitive evidence, it showed a lack of pili on the expected cells and a positive result on the positive control. There were certainly structures suggestive of pili on the surface of the cells in the P_Ara_FimOperon sample.
The hydrocyclone that we designed, built and tested, separated sediment from water with 97.4% efficiency. That is to say, of the contaminated solution that was pumped through the hydrocyclone, only 2.6% of the sediment was left in the filtered water.
In the future, we would like to explore the possibility of filtering smaller particulates from water with a higher efficiency by adapting the geometry of the hydrocyclone. By doing so, we could easily adapt the design to solve a variety of different problems.
Metal Binding Reactor
Experiments surrounding the metal binding reactor found that currently a maximum of 26.4% of a solute can be removed from a solution, through design of experiment (DOE). In the future, we would like to conduct further iterations of the DOE to improve the accuracy of the optimization. We would then carry out the same work using our modified bacteria to test their extractive power on metal ions.
It was also found that a 0.1% concentration of a chlorhexidine gluconate surfactant can be used to induce biofilm growth on a surface.
BiosecurityThe results we obtained showed that a 10 minute exposure to UV was not able to cause sufficient cell death and therefore is not suitable for use as a biosecurity mechanism in our filtration system. In order to achieve the industry required escape frequency, which is of the order 10-8, the water would have to be irradiated for a much more significant period of time which is not practical for our intended use. Therefore we need to explore possible alternatives, such copper alginate beads as detailed on the Applied Design page.