Difference between revisions of "Team:Exeter/Lab Conclusion"
Rahannazeer (Talk | contribs) |
Rahannazeer (Talk | contribs) |
||
| Line 49: | Line 49: | ||
<p> The P_Rha_FimH_1_His part was shown to be successfully expressed in four different <i>E. coli</i>. 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. </p> | <p> The P_Rha_FimH_1_His part was shown to be successfully expressed in four different <i>E. coli</i>. 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. </p> | ||
| + | |||
| + | <p> 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. </p> | ||
| + | |||
<h2 id="h2">Applied Design</h2> | <h2 id="h2">Applied Design</h2> | ||
Revision as of 23:57, 1 November 2017
Conclusion
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.
Applied Design
Hydrocyclone
Metal Binding Reactor
Biosecurity
The 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.
