Difference between revisions of "Part:BBa K1680025"

Revision as of 01:54, 26 September 2015

RFP-luciferase Cre reporter

This part encodes a reporter cassette for the Cre Recombinase part:BBa_K1680006 in yeast. An active Cre protein will switch expression from RFP to NanoLuc luciferase.

In order to check fluorescence of the Cre reporter cassette Team Tübingen 2015 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 1).

Figure 1: 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.

Team Tübingen 2015 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 2 shows the experimental results.

Figure 2: 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, Team Tübingen 2015 concluded that the recombination of the stop cassette by Cre recombinase works as expected.

To check for leaking NanoLuc expression from the Cre reporter cassette, Team Tübingen 2015 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 3: Cells with Cre reporter cassette do not show significant luciferase activity. RLU=relative luminescence units, positive control = pADH-nanoluc.

In conclusion, Team Tübingen 2015 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.

Sequence and Features