Parts
Here you can see and read more about the part we finished and our process validating its function
Team:Linkoping Sweden/Parts
<groupparts>iGEM2017 Linkoping_Sweden</groupparts>
| Name | Type | Description | Length |
|---|---|---|---|
| BBa_K2474000 | Composite | For expression of a fusion protein with amyloid-beta and mNeonGreen | 2198 |
BBa_K2474000
Press here to read the part’s registry page This is a composite part for expressing the fusion of amyloid-beta and mNeonGreen after inducing with arabinose. The parts contains the pBad promoter from the BBa_K1321333 and the fusion protein is design by us, LiU iGEM 2017, and synthesized by IDT. The purpose of the part was to give the opportunity to study aggregation of amyloid-beta in real time by measuring fluorescence.
The part was sequenced with a 99% match compared to the theoretical version of the part. The validation of the parts function then proceeded with confirming amyloid-beta using a dot-blot and confirming mNeonGreen with spectrophotometric measures from induction in a 96-well-plate.
Dot-blot
Read more about the dot-blot here The results from the dot-blot can be seen in figure 1 compared to figure 2. Table 1 is a scheme explaining figure 1. From these figures, we could confirm that our bacteria was expressing proteins which have epitopes that the antibodies, specific to amyloid-beta, could bind to. This is confirmed by comparing the 4 marks in the second column with the 4 negative marks in the first. By the black marks in figure 1, where we added cell lysate from induced bl21 containing our biobrick, we confirm the expression of amyloid-beta. From the weaker marks we confirm there is some leakage from the promoter. Table 1. scheme over cell lysate applied to the filter.
| Empty Bl21 | uninduced Bl21 with BBa_K2474000 |
| Empty Bl21 | uninduced Bl21 with BBa_K2474000 |
| Empty Bl21 | induced Bl21 with BBa_K2474000 |
| Empty Bl21 | induced Bl21 with BBa_K2474000 |
To the left: Figure 1. Results from dot-blot. To the right: Figure 2. Filter appearance viewed with the naked eye.
Spectrophotometric analysis
E.coli BL21 bacteria cultures with BBa_K2474000 was prepared overnight and the OD was later set to 0.4. The bacteria was distributed to a 96-well plate so that each well contained 200 µl. These were then induced by arabinose to express the fluorescent fusion protein Amyloid-Beta mNeonGreen. The fluorescence was then analysed on a plate reader. The original intent of this experiment was to analyse protein expression over time. However due to technical difficulties this was not possible. Instead figure 3 belowrepresents emission spectra of the fluorescence from Amyloid beta mNeongreen 24 hours after induction.
Figure 3. They were distributed in a 96 well plate in 8 replicate at each of the 4 induction levels (5 mM, 1 mM, 0.2 mM and 0 mM). Empty bacteria (BL21(DE3)) were also included on the plate to serve as a control. All measurements were done with an infinite M1000 pro plate reader. Due to technical difficulties and lack of an mNeonGreen fluorescent standard curve it was impossible to present this data in the preferred unit (µM/OD). The data points represent the mean value of the replicates and the error bars represent 95 % confidence intervals.
From this graph we can clearly see that the promoter and fluorescent parts of our biobrick works as expected as the fluorescence is highly proportional to the arabinose concentration. We can also see that the promoter have a small leakage in its regulation as uninduced bacteria with BBa_K2474000 (purple in figure 3) show more fluorescence than empty bacteria. This graph in combination with the dot-blot above (figure 1) confirms that our biobrick BBa_K2474000 is fully functional.
