Cell-based Results
The only sub system of the cell based method that was tested was the MF(alpha)1 activation of the yeast mating system. The reason no other subsystems, let alone the full cell based system, was tested was due to issues with cloning, both for E.coli and S. cerevisiae. Our systems are composed of many parts, both coding genes and different promotors and terminators. Throughout the summer, a lot of assemblies and transformations failed at all different stages. Besides problems with cloning, we were not able to go into the lab until July because of maintenance reducing the time we had available in the lab. By the time we had planned to finish, at the end of August, we had only barely finished making our assemblies. Most of September we spent attempting to finish transforming our assemblies into yeast, while also trying to remake unsuccessful assemblies in E.coli. There was very little time to finish the systems before the wikifreeze.
The MF(alpha)1 subsystem was tested by measuring GFP fluorescence in the yeast strain.
The yeast strain we used had a GFP gene that is activated when the mating system is activated by the mating factor α-peptide.
The MF(alpha)1 gene produces mating factor α-peptide when expressed.
Therefore, the yeast strain with a promotor constantly expressing MF(alpha)1 should also express GFP,
as the mating system is activated by the mating peptide.
Using a plate reader to measure fluorescence showed that the MF(alpha)1 yeast strain indeed expressed GFP,
see table below.
- Issues with cloning, both in E.coli and S. cerevisiae, not working properly. This was very time consuming.
- Our cloned MF(alpha)1 gene works for activating the yeast mating system in S. cerevisiae , inducing GFP expression.
- Not enough time to clone violacein pathway into yeast, and therefore no experimental data on the violacein system produced.
- NubG and Cub “activation by binding target DNA ….
| Replicate | 1 | 2 | 3 |
|---|---|---|---|
| MF(alpha)1 | 2938 | 2825 | 2807 |
| D4 | 1757 | 1462 | 1468 |
Cell-free Results
- The experiment to test if the transformed S. cerevisiae expressed FRET failed. The plate reader displayed no difference in fluorescence between the blank, control and the test.
The test to quantify FRET of pair fluorescent proteins (mNeongreen-mRuby3) failed. The blank, control and the S. cerevisiae transformed with FRET showed essentially the same fluorescence. This indicates that there was no evidence that FRET was expressed. The colony PCR indicated that the gene had the right length, but the sequencing results were terrible. In the future, it would be good to use common fluorescent proteins, such as GFP, that the available instruments would be able to measure more easily.
- The experiment to test if the designed Inhibited Beta-galactosidase worked gave strange and unsatisfactory results. A conclusion is that something within the lysed cell slurry of S. cerevisiae can degrade X-gal.
The experimental setup tested the mixtures of X-gal and cell-lysates from S. cerevisiae transformed with corresponding protein is shown in table 1 and table 2. Each column corresponds to one transformation of S. cerevisiae with TEV and each row makes up one transformation with the Inhibited Beta-galactosidase.
Experiment
Table 1. Experimental setup of in the figures below.| Date: 2/9 | TEV5.4.2 | TEV5.4.5 | TEV5.4.6 | NO TEV |
|---|---|---|---|---|
| iGal_1.2 | X | X | X | X - TEV |
| iGal_3.1 | X | X | X | X - TEV |
| iGal_3.3 | X | X | X | X - TEV |
| iGal_3.4 | X | X | X | X - TEV |
| iGal_4.1 | X | X | X | X - TEV |
| iGal_4.2 | X | X | X | X - TEV |
| iGal_4.3 | X | X | X | X - TEV |
| No iGal | X - iGal | X - iGal | X - iGal | Only X-gal and MQ |
X :10 μL X-gal stock (20 mg/ml), 30 μL iGal lysed cell slurry , 30 μL TEV lysed cell slurry , 20 μL MQ
Figure 1.Wells containing lysed cells that had been transformed with TEVp and iGal. The wells also contained X-gal and MilliQ as buffer. The pictures are how the experiment looks after 1, 2, 3, and 6 days since its start. The experimental setup is described in table 1.
Table 2. Experimental setup of mixing TEVp, B-gal and X-gal in PBS as a buffer.
| Date: 3/9 | TEV5.4.2 | TEV5.4.5 | TEV5.4.6 | NO TEV |
|---|---|---|---|---|
| iGal_1.2 | Y | Y | Y | Y - TEV |
| iGal_3.1 | Y | Y | Y | Y - TEV |
| iGal_3.3 | Y | Y | Y | Y - TEV |
| iGal_3.4 | Y | Y | Y | Y - TEV |
| iGal_4.1 | Y | Y | Y | Y - TEV |
| iGal_4.2 | Y | Y | Y | Y - TEV |
| iGal_4.3 | Y | Y | Y | Y - TEV |
| No iGal | Y - iGal | Y - iGal | Y - iGal | Only X-gal and PBS |
Y: 5 μL X-gal stock (20 mg/ml), 30 μL iGal lysed cell slurry, 10 μL TEV lysed cell slurry, 100 μL PBS
Figure 2. Wells containing lysed cells that had been transformed with TEVp and iGal. The wells also contained X-gal and PBS as buffer. The pictures are how the experiment looks after 1, 2, and 5 days since its start. The experimental setup is described in table 2.
Experiment description
Our designed Inhibited Beta-galactosidase (iGal) should become active when it comes into contact with TEV protease (TEVp). Only an activated iGal would turn X-gal into a blue pigment, indicating a positive response. The experiment was designed to express these proteins inside different S. cerevisiae cells, and then to lyse the cells to get the extracted proteins into contact. The cells were lysed and added into wells with X-gal with MilliQ water as buffer. Three types of negative control were also included. The first negative control was TEVp + X-gal which checked that the lysed cells didn’t naturally break down X-gal. The second negative control was iGal + X-gal, which checked that iGal didn’t break down X-gal before it got into contact with TEVp. The third negative control being only X-gal was to check that X-gal didn’t naturally break down in the buffer or other environmental conditions. It was observed that some of the wells containing the lysed cell mixtures turned blue, but so did the first and second negative controls. The experiment was run again in a similar fashion again, but changing the buffer from MilliQ to PBS. The result was similar, with some of the tests turning blue and the first and second negative controls also turning blue.Discussion
As the first negative control turned blue, but not the third, and since TEVp does not degrade X-gal, this result indicates that there is something within lysed cell slurry of S. cerevisiae that can degrade X-gal, without any iGal or Beta-galactosidase needing to be present.There’s a risk that iGal would not function as intended, that it would always be active rather than only becoming active when it comes into contact with TEVp. This would cause every test to become blue as well as the second negative control. Although, with Occam's razor as a principle, there already is the explanation above for why the first negative control turns blue. If the first negative control is blue, then it is because the lysed cells are breaking down X-gal, which would mean that the second negative control would also turn blue. This is because the cells used in the tests came from the same S. cerevisiae cultivation. This means that there is no evidence to confirm that iGal is not functional as both controls were blue and not only the second control.
No obvious pattern alongside any column or row (eg. any particular version of TEVp or iGal) could be observed. The effect of mixing the cell lysates did not consistently make the test more blue than the controls, indicating there is no obvious effect from mixing these. If it is assumed that the second-order effect of adding TEVp-lysate and iGal-lysate is zero, the effect of each addition should give a linear response. Although, no obvious linear effect could be observed. If the effect was linear, the tests would simply be a combination of the “blue-ness” of the controls, but in some cases, the controls are more blue than the tests. This indicates that there is some other unaccounted factor that caused the X-gal to degrade and the assumption that only TEVp, iGal, X-gal and a cell-lysate was added doesn’t explain the results. It is as if different things are pipetted up from the same tube, despite it only containing one type of cell-lysate, TEVp or iGal.
One possible explanation for the results is that the content within the tubes containing the lysed cells had sedimented into different layers, with some layers that contained something that could degrade X-gal. If this was the case, the depth that one would pipette at would determine if the compound that could degrade X-gal would get transferred to the well or not. Meaning if one would pipette from the bottom of the tube, this might cause the well to have turned blue. This is not too unreasonable of an explanation, as the cell lysate was never centrifuged and the supernatant wasn’t separated from what would be the pellet.
In either way, there was no obvious effect from mixing the cell lysates, indicating that the tests did not work as desired. This could be because the genes had failed to be expressed, had mutated and were non-functional, had been degraded by other proteases, the buffer not allowing it to function properly, misfolding of iGal such that the cut-site for TEVp wasn’t exposed or misfolding inducing loss of function. Or simply being that even though TEVp had cut iGal it is unable to form the tetramer, or that iGal is enzymatically non-functional in the first place. As the tests didn’t show any effect from mixing the cell lysates, the combination of errors and time constraints didn’t allow us to troubleshoot and test the system further.
Replication of the experiment
If one were to replicate this experiment, one would ideally use protein purification to separate TEVp and iGal from the cell lysate. If that option isn’t available, it would be good to use protease inhibitors that don't affect TEVp, to keep the TEVp and iGal from being degraded. It would be recommended to centrifuge the cell lysate to potentially exclude the compound in Saccaremyseas cerevisiae that degrades the X-gal. TEVp requires a reducing buffer to be able to function, so it may be necessary to add that to the test. It would also be good to use larger volumes of liquid as the water evaporates and changes the volume significantly. Some wells almost dried out for the test that started on 2/9, as the volume was 90 μL. X-gal is light sensitive, so storing it in a dark room or encased in aluminium foil is advised. Heat could degrade X-gal, but in the timespan of a few days in 30 degrees and in MilliQ water or PBS, no noticeable degradation of X-gal could be seen by eye in this test as it didn’t turn blue. This test used X-gal, although it was simply suspended in MilliQ and not fully dissolved. It would be better if the X-gal would be dissolved in something and this solvent or buffer should be selected such that it does not denature the proteins.With the results from AlphaFold on the structure of iGal, there exists a risk that it is always active. The reason for this is that a linker is possibly too long and could allow for the Alpha and Omega fragments to complement each other despite the linker not being cleaved. If this would be the case, iGal would have to be redesigned, possibly by just shortening the linker. So if the test would be performed in the future and it would display positive results with just iGal and X-gal, this could be a solution. It would be a good idea to use AlphaFold to see what length of the linker would not result in the Alpha-fragment reaching the spot on the Omega-fragment that would result in alpha-complementation.
If the test would display negative results it would be good to test if TEVp is functional. With fluorescent proteins and the right tools, it could be easy to see if TEVp functions or not by measuring if the FRET phenomena is happening. It would be relatively easy to know if the fluorescent proteins are expressed and possible to quantify it with a plate reader. If the addition of TEVp would decrease the FRET-phenomena more than a control, it’s a clear sign that the TEVp functions. If TEVp functions, but the test is negative, it could imply that the problem lies with iGal and not TEVp. All this means that using fluorescent proteins is a good way of error-searching as it is relatively easy to know if they function or not with the right tools.