Template:BOKU-Vienna/Results

Ethanol responsive ubGFP expression

Experimental Setup

We incubated 100 mL each of a 0.1 OD600 culture of our Pichia pastoris strain transformed with our Ethanol responsive construct expressing our ubiquitin tagged GFP in YPD medium with 0%, 1%, 2% and 4% v/v Ethanol for several days and followed the amount of GFP produced by doing plate reader measurements of Fluorescence and Abs600.

Results

The growth rate depended heavily on the ethanol concentration, especially the culture grown with 4% ethanol in the medium displayed much slower growth. Therefore we used the ratio of cell density and fluorescence for the evaluation of our data to get a better idea of how much the individual cells were producing.

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           <img src="" alt="Results EtOH construct">
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As expected, the cultures incubated with higher ethanol concentrations produced more GFP during the first two days of measurements. One thing that surprised us, however, was that the fluorescence dropped significantly on the third day across all cultures. We suspect that this was probably either due to the ethanol being taken up and metabolised by the cells or evaporating during the incubation at 30°C.

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           <img src="" alt="Results EtOH construct">
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If we were to repeat this test, which we unfortunately did not have the time to do it would be important to verify whether the reduction in fluorescence in the end actually correlates with a reduction in ethanol concentration, for example by measuring ethanol concentration at each sampling point with HPLC.

Testing our toggle switch in yeast

With this experiment we wanted to test many hypotheses that are linked to the functionality of our system to fully or partially prove our concept of a working dCas9 toggle switch in yeast.

<a href="" data-toggle="lightbox"> <img src="" alt="Results Liposomen construct"> </a>

This is a diagram presenting our results as the ratios of cells showing green fluorescence when excited at 488 nm. The data was collected by randomly counting cells under the fluorecsence microscope with . Bars show the wilson score with the wilson score interval (red) at an error-probability of 5 %. The upper bar represents cells that were inoculated and incubated with gRNA-lipoplexes, or without respectively, for ~24 h. The lower bar shows the same preparation after ~48 h of incubation.

Disproven Hypotheses:

1. UGFP is not being expressed due to a malfunction of our designed regulatory region.
2. UGFP mRNA can not be translated into fully functional UGFP. Since we see green fluorescence when exposing our cells with a laser at the absorption maximum (488 nm) of UGFP hypotheses 1. and 2. can not be confirmed.
   2. Liposomes do not interact with yeast cells at all. All experiments conducted with the toggle switch were made with the same yeast culture and breed in exactly the same conditions, which means they should show an equal proportion of fluorescent cells if this hypothesis should held.
3. Liposomes are not taken up by yeast cells. When mixing negatively-charged gRNA with positively-charged liposomes resulting lipoplexes should mainly have gRNA bound to their outer membrane. This means gRNA can get into the rel, when the outer membrane of the liposome is exposed to the yeast cytosol. This happens when it is taken up by the cell. However, the toggle switch tested with these lipoplexes show a highly significant difference in fluoresence disproving this hypothesis.
4. Liposomes do not fuse with yeast cell membranes. Liposomes that were prepared in a solution containing gRNA and washed from afterwards should mostly have gRNA included in them or at their inner membrane. These lipoplexes spill out their gRNA into the cytosol when fusing with cell membranes. Still they show different levels of fluorescence, disproving this hypothesis.
5. Liposomes themselves have a direct influence to our system. If this hypothesis could stand its ground, fluorescence values should not be different in lipoplexes with gRNA OFF and gRNA ON.
6. gRNA is degraded before or unable to reach the DNA in the nucleus.
7. Our designed gRNAs do not interact with our regulatory regions.
8. dCas9 is not expressed or shows any malfunction.
9. dCas9s can not repress our genes at all. Since hypothesis 6 is disproven only gRNA bound to dCas9 is left to cause significant differences in UGFP-production in the first place this further disproves hypothesis 7, 8, 9 and 10.
10. Our designed ribozymes are not able to produce free gRNA.
    12. Our designed gRNA is exported out of the nucleus or degraded before it can become active. According to Ma et al. (2016) gRNA half lives in living cells are between 15---120 minutes, which means there should not be any gRNAs left from the initial addition after 24 or 48 hours. Furthermore if they were to persist that period in lipoplexes we would see a decline in UGFP-expression in all preparations rather than an increase over time, since lipoplex concentration is also decreasing with their consumption. This is why a lower fluorescence value in some of our preparations has to come from gRNA interactions that were produced from the cell. This disproves hypothesis 11 and further 12.
11. Our designed gRNA targets stop our constitutive promoters from working. Disapproval of hypothesis 1 and 2 together with the fact that UGFP is expressed under the control of a constitutive GAP promoter with gRNA targets cloned into it lets us refute this hypothesis.
12. Fluorescein-tagged gRNA does not show any activity in presence of dCas9.

Hypotheses not disproven

15. Our system is not generating correctly functioning gRNA ON. If gRNA ON was working correctly we would expect fluorescence valot being expressed dues to be high and stay high when the system is initiated with gRNA ON. This is not the case for Lipoplexes with tagged-gRNA ON and when bound to the outer membrane. However tagged-gRNA could show lower activity or dysfunction due to the fluorescein-tag, but it still shows a difference disproving 14 a malfunction of our designed regulatory region.
16. Our system is not generating correctly functioning gRNA OFF. If gRNA OFF works as intended we should see no or constant lower levels of UGFP being expressed from the toggle switch when initiated with gRNA OFF. However we see rather the same or even higher levels. Another clue this hypothesis might be correct is that our negative control (UGFP controlled by gRNA OFF, together with gRNA OFF controlled by gRNA ON, which was not incubated with gRNA ON) shows a similar result to our positive control (only UGFP controlled by gRNA OFF).
    17. Our whole toggle switch is not functioning as intended. This hypothesis cannot be falsified, because it would require hypotheses 15 and 16 to be disproven. Our toggle switch would show the right behaviour if we assume that our gRNA "ON" was indeed gRNA "OFF" and vice versa. This could have some reasons, the first and most likely is that they were interchanged after their production or in the process of testing the switch. A rather less problem is that they both somehow show a higher affinity to the target sites of the other. Alignments of their sequences do not support this.

References:

Ma, Hanhui; Tu, Li-Chun; Naseri, Ardalan; Huisman, Maximiliaan; Zhang, Shaojie; Grunwald, David; Pederson, Thoru (2016): CRISPR-Cas9 nuclear dynamics and target recognition in living cells. In: The Journal of cell biology 214 (5), S. 529–537. DOI: 10.1083/jcb.201604115 gRNA OFF construct is not being expressed due to a malfunction of our designed regulatory region.

18. Fluorescein-tagged gRNA does not show any activity in presence of dCas9.