Difference between revisions of "Part:BBa K1680025"

Revision as of 00:35, 26 September 2015

RFP-luciferase Cre reporter

This part encodes a reporter cassette for the Cre Recombinase (e.g parts XYZ) in Yeast. An active Cre protein will switch expression from RFP to NanoLuc luciferase.

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).

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.

<img id="resultsprs1" style="max-height: 250px;display: block; margin-left: auto;margin-right: auto;"

src=""/> 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).

<img id="resultsprs1" style="max-height: 250px;display: block; margin-left: auto;margin-right: auto;"

src=""/> 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.

Sequence and Features