Team:Kent/Results
Project Results
Atomic force microscopy (AFM)
AFM is a form of scanning probe microscopy, which can be used to image biological specimen to high resolution and magnification. AFM was used to image cells expressing amyloid nanowires. Colonies taken from agar plates were suspended and then placed onto a sample stage from which the scanning probe could operate, as described in the protocols section.
The first samples imaged were of the E. coli VS45 cells expressing CsgAss-SUP35NM protein, which had been induced to express and export the Sup35NM protein capable of forming amyloid fibrils. The cells exhibited long, distinct fibrils which had a tendency to overlap and form a larger mass once they had detached from the cell. The nanowires that assembled were around 20Å wide and varied in length. This shows that the protein could be exported and form part of a longer nanowire.
The second samples we imaged were of the same E. coli VS45 strain but were expressing a fusion protein containing CsgAss-SUP35NM fused with cytochrome b562 in the C-terminus (CsgAss-SUP35NM-b562). When these cells were induced to export the SUP35NM-b562 fusion protein, the fibrils appeared to break off and form smaller curve-linear fibrillar aggregates much more frequently. In addition, it seems the protein assembles frequently as a larger cluster of amyloid rather than distinct fibrils seen in the sample with no cytochrome. Thus, we confirmed that the fusion protein was successfully exported and assembled, but our results show the fusion protein assembled less efficiently compared with their counterparts without the Cytochrome b562.
| Type of sample | Day | Average electric resistance \( \log(k \Omega\)) | Standard deviation of the repeats \( \log( \sigma ) \) |
|---|---|---|---|
| Heme agar plate inoculated with cells containing plasmid with fusion protein CsgAss - Sup35 NM with cytb562 | 1 | 1.1 | - |
| 2 | 2.4 | 2.6 | |
| 3 | 2.3 | 2.6 | |
| 4 | 2.9 | 2.6 | |
| 5 | 2.9 | 2.8 | |
| Heme agar plate inoculated with VS45 cells (negative control plates) | 1 | - | - |
| 2 | 1.1 | - | |
| 3 | 3.0 | 2.2 | |
| 4 | 3.0 | 2.2 | |
| 5 | 3.0 | 2.2 | |
| Heme agar plate inoculated with cells containing plasmid with fusion protein CsgAss - Sup35 NM without cytb562 (BioBrick: BBa_K1739002) | 1 | - | - |
| 2 | 1.1 | - | |
| 3 | 1.1 | -0.3 | |
| 4 | 2.7 | 2.8 | |
| 5 | 2.6 | 2.8 | |
| Heme agar plate inoculated with VS45 cells (negative control plates) | 1 | - | - |
| 2 | 1.1 | - | |
| 3 | 3.0 | 2.2 | |
| 4 | 2.5 | 2.7 | |
| 5 | 2.9 | 2.8 | |
| Heme agar plate inoculated with cells containing plasmid with fusion protein CsgAss - Sup35 NM without cytb562without cytb562 (BioBrick: BBa_K1739002) | 1 | - | - |
| 2 | 1.1 | - | |
| 3 | 1.1 | -0.3 | |
| 4 | 3.0 | 1.1 | |
| 5 | 2.9 | 2.8 |
Table 1: The average resistance calculated for our main heme innoculated cell samples containing plasmid with fusion protein CsgAss - Sup35 NM with cytb562 and 4 other control samples over the period of 5 days. For each day, several repeat resistance measurements were taken (maximum 5). Average electrical resistances were calculated for each measurement. The values above are in 1 decimal places. Note: For some of the days, not enough measurements were taken to account into the average calculations.
Diagram 1: This circuit diagram is a representation of our conductive validation experiment.