Difference between revisions of "Team:Tuebingen/Results"

Mutagenisation of the RFC10 restriction sites and the ApaI restriction site in the backbone of the pRS plasmids were performed by mutagenesis PCR. Positive clones were screened by restriction analysis with the respective restriction enzymes (compare plasmid maps: link [project description pRS vectors]).

Then, the old multiple cloning site (MCS) was replaced by restriction digest (ApaI, SacI) and subsequent ligation of annealed oligonucleotides containing matching overhangs. Verification of successful replacement of the old MCS was performed by using a restriction site that was only present in the initial pRS MCS (SalI).

Figure 1: Schematic agarose gel showing the migration pattern of various pRS plasmids incubated with different restriction enzymes. Figure 2: Restriction analysis of various pRS plasmid constructs.

In figure 1, a schematic agarose gel showing the migration pattern of the three pRS vectors when digested with different restriction enzymes is depicted. Figure 2 shows the restriction analysis of various pRS plasmid constructs. The used pRS vectors show the expected migration behaviour. Lane 3 in figure 2c shows an additional fragment at around 5 kb which indicates an incomplete digestion of the pRS316 plasmid. In summary, every RFC10 restriction site in the pRS plasmid backbones was removed and the MCS was replaced. Unfortunately, due to time constraints we were not able to insert a terminator.

To check whether the RFP (BBa_E1010) we want to implement in our Cre reporter cassette works as expected, the RFP was cloned into the pTUM104 vector under the control of the pGAL1 promoter. Yeast cells were transformed with this construct and grown in medium with 2% galactose. The RFP fluorescence intensity was then determined in the plate reader. We used different media to resuspend the cells after centrifugation (as indicated in figure 1).

Figure 1: Relative fluorescence units normalised to OD₆₀₀. Data were obtained from 3 independent biological replicates.

In figure 1 one can see that the RFP-construct containing cells show a significantly higher RFP fluorescence than the wild type cells. In addition to that, it shows that the media for resuspension does not strongly influence the fluorescence.

In order to check fluorescence of the Cre reporter cassette we expressed the complete construct (BBa_K1680025) in Saccharomyces cerevisiae in the pTUM100 plasmid (BBa_K801000). Microscopy images show that RFP is expressed in these cells (compare figure 2).

Figure 2: Fluorescence and brightfield pictures of cells expressing the Cre reporter stop cassette. The left picture shows the bright field channel, the middle picture the RFP channel and the right picture the overlay of both channels.

In order to assess the function of our Cre reporter regarding recombination, a plasmid containing the stop cassette was digested with EcoRI and PstI to get linearised backbone DNA and the stop cassette DNA. Then, purified Cre recombinase was added and incubated at 37°C. At certain time points samples were taken, heat inactivated and loaded on an agarose gel.

We expected that the backbone DNA (around 5 kb) stays unaffected (except possible DNAse contaminations in the purified Cre recombinase) in all samples, while the stop cassette (around 3 kb) should show a shift of approximately 1 kb (DNA size between the two loxP sites). In addition to that, a 1kb circularized DNA fragment should be visible. Figure 3 shows the experimental results.

Figure 3: Agarose gel showing the Cre reporter cassette incubated with Cre recombinase

It can be seen that the backbone DNA fragment stays unaffected during the experiment, while the stop cassette DNA fragment intensity slightly decreases. Furthermore, after 60 and 90 minutes an additional fragment can be seen at around 2 kb, which represents the stop cassette after recombination. Unfortunately, the small circularized fragment is not visible. Therefore, we conclude that the recombination of our stop cassette by Cre recombinase works as expected.

To check for leaking NanoLuc expression from the Cre reporter cassette, we measured the luciferase activity in overnight cultures of S. cerevisiae carrying the cre reporter construct but no additional plasmid with a Cre recombinase. As shown in figure 3, the luciferase within the cre reporter cassette does not show significant activity compared to wild-type and positive control (pADH-nanoluc).

Figure 4: Cells with Cre reporter cassette do not show significant luciferase activity. RLU=relative luminescence units, positive control = pADH-nanoluc.

In conclusion, we managed to design and transform a working Cre reporter cassette which would be suitable to work with a co-transformed Cre recombinase to form the memory unit of the sensor system.

First, Dronpa and NLS-Dronpa were cloned under the control of the pGAL1 promoter and yeast cells were transformed with these constructs. Cells were grown in galactose-containing media and fluorescence levels were determined in the plate reader. Different media were used to resuspend the cells to determine whether this has an effect on the fluorescence levels.

Figure 1: Relative fluorescence units normalised to OD₆₀₀. Data were obtained from 3 independent biological replicates.

In figure 1 it can be seen that the Dronpa construct-containing cells show a significantly higher fluorescence signal than the wild type cells. In addition to that, cells expressing NLS-Dronpa show a higher fluorescence intensity than the NLS-Dronpa expressing cells. Possibly, the Dronpa proteins are localized to the nucleus and cannot be degraded as efficiently as the Dronpa proteins located in the cytoplasm. Furthermore, one can see that the resuspension media does not have an effect.

In order to characterise Dronpa we tried to express it in E.coli and afterwards purify it. Therefore, we cloned the coding sequence of Dronpa in the pETue, previously known as pETblue (https://2014.igem.org/Team:Tuebingen/Project/Plasmids) vector and transformed the vector in BL21(DE3) E.colis. The pETue vector introduces an N-terminal 6xHis-tag followed by a thrombin cleavage site to the inserted DNA fragment.

A liquid culture was inoculated with BL21(DE) bacteria containing the pETue-Dronpa construct. The culture was grown to an OD₆₀₀ of 0.8 and the cells were then induced by adding 0.5 mM IPTG and shifted to 25°C overnight.

Induced and uninduced cells were pelleted, lysed and loaded on a 10% acrylamide gel (see figure 2).

Figure 2: Test expression of E. coli containing the pETue-Dronpa construct.

The induced cells show an additional band at around 25 kDa, which cannot be seen in the non-induced cells.

We then tried to purify 6xHis-Dronpa in a larger scale experiment. We used the same expression conditions as before. Then, the cells were pelleted and lysis was performed using the sonicator. After that, the lysate was centrifuged to remove the insoluble parts and incubated was incubated with Ni-NTA beads at 4°C. We eluted the proteins from the beads by using buffer supplemented with 200 mM imidazole (compare figure 3).

Figure 3: Purification of 6xHis tagged Dronpa.

It can be observed that the His-tagged Dronpa protein is clearly visible in the soluble, flow through and wash fractions. Unfortunately, almost all protein is lost during the washing procedure. Milder washing conditions could probably improve the protein yield. Due to time constraints, the purification could not be repeated.

GFP-trap

As the GFP-trap offers an easy purification method for all GFP-tagged proteins, we wondered whether it would also bind to Dronpa, as it has a high structural similarity to GFP. We also tested the RFP-trap to see whether this might bind if the GFP-trap did not. Yeast cells were lysed and the lysate was added to the GFP-trap, as well as an RFP-trap and blocked beads as a negative control using the protocol provided by ChromoTek GmbH (http://www.chromotek.com/fileadmin/Redaktion/Products/Nano-Traps/Protocols/GFP-Trap_A__manual_13-12-10.pdf).

Figure 4 shows the resulting SDS-PAGE gel, stained with coomassie, of the positive control, Sec61-GFP. As we could not see any bound GFP after elution of all bound proteins from the beads, the lysis of the yeast cells may not have been efficient enough to retain the structure of the GFP or since the SEC61 is located in the ER it might be that the fusion protein is hyperglycosylated and cannot be recognized by the beads. As we did not have enough time to optimize the lysis protocol, we could not do any further experiments.

GFP-Trap.png Figure 4: SDS-PAGE gel after GFP-trap purification. ()

Microscopy

In order to assess Dronpa fluorescence we expressed Dronpa and NLS-Dronpa in Saccharomyces cerevisiae in the pTUM104 plasmid (BBa_K801004). Expression of the normal Dronpa protein and an NLS-Dronpa fusion showed different distribution of the protein throughout the cell (Figure 5). The circular arrangement of the fluorescence signal in the cells expressing NLS-Dronpa indicates that the protein accumulates in the nucleus and that the nuclear location sequence (BBa_K1680004) works as intended.

pTUM104-Dronpa

pTUM104-NLS-Dronpa

Wild type cells

Figure 5: Fluorescence and brightfield pictures of cells expressing Dronpa, NLS-dronpa and wild type cells. The left pictures shows the Dronpa fluorescence channel, the middle picture the brightfield channel and the right picture the overlay of both channels.

Dronpa photoswitching

In addition to simple pictures of Dronpa expressing cells, we were also able to show photoswitching of Dronpa under the microscope. Figure 6 shows both switching off and switching on of NLS-Dronpa. Off switching was facilitated by illuminating the cells with a 488 nm laser during each frame. Respectively on switching was facilitated by illuminating cells with a 405 nm laser during each frame. The starting and end points of both photoswitching transitions are also shown in Figure 7.

Figure 6: Animated graphic showing consecutive off and on photoswitching of Dronpa. Each frame of the video corresponds to 286 ms. Off switching of dronpa was achieved by illumination with 488 nm in 2.5 min. Follow-up switching on by illumination with 405 nm yielded immediate recovery of ground state fluorescence.

Figure 7: Overlay pictures of Dronpa fluorescence and brightfield channel. The scale bar (left picture) corresponds to a size of 3 µm. From left to right: Dronpa before illumination, Dronpa after 1 min 488 nm illumination, Dronpa after 2 min (total) 488 nm illumination, Dronpa after 0.3 sec (follow up) 405 nm illumination.

In addition to the Dronpa photoswitching at the microscope we also illuminated cells expressing Dronpa or NLS-dronpa directly in a 96 well plate. For this setup (Figure 8 and figure 9) we used 50W halogen lamp powered with 16V and 4.8A as source for white light and used a monochromator to single out a specific wavelength.

Figure 8: Schematic of the setup we used to illuminate Dronpa expressing cells directly in a 96 well plate. Figure 9: Photograph of our Dronpa illumination setup for 96 well plates.

With the monochromator set to 488 nm one Dronpa and one NLS-Dronpa expressing sample were illuminated for 10 min. Afterwards Dronpa fluorescence of the illuminated samples, non illuminated samples grown in the same batch and wild type cells were measured in a Tecan plate reader (Figure 10). Unfortunately, we could not detect any effect on the Dronpa fluorescence using our own illumination setup. Probably, the intensity of the light source was not high enough to activate photoswitching.

Figure 10: Relative fluorescence measured in wild type cells, Dronpa and NLS-Dronpa expressing cells. One sample of each cell type was measured before and after illumination with 488 nm light from the monochromator setup for 10 min.