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 OD[sub]600[/sub]. 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.