Difference between revisions of "Part:BBa K2329000"

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A non-reversible switch

The promotor TEF1 within two mutated LoxP-sites. The mutation in the LoxP-sites introduce a non-reversible switch. This switch-system make it possible to switch the direction of the promotor, and so on encoding different genes.

Characterization

A study of only the flipping of the promotor was made, by adding an inducible Cre-recombinase plasmid. The idea behind the construction of the system plasmid (Cre-Cas9) was that if the switch system worked, the pTEF1 would flipped and switch direction. So, instead of coding GFP it would code for Cas9. Therefor losing theirs GFP expression. The inducible Cre-recombinase plasmid was added due to the consider of the possible weakness of our systems original promotor FUS1 and the activation of it, so the Cre-recombinase wouldn’t be enough. The encoding of Cre-recombinase in the plasmid is activated in present of galactose. So, the cell culture, with the flipping system and Cre-recombinase plasmid, was grown in a Delf media, containing galactose. By watch under fluorescence microscope under three days, it would be possible to draw conclusion if the switch is non-reversible or not, at least after three days.

Three replicates were done, and all gave a similar result. The three days are presented in Figure 1

A clear pattern is seen. The expression of fluorescence appears to decrease over the days. During the two-last day, some fluorescence was still visible in some few cells but not catch on picture. This tell that the flip perhaps isn’t 100% efficient.

The fluorescent picture indicate that the flip of the promotor might worked, and stopped the encoding of GFP. To really make sure of it, and get an easy look on sequence level of the construct a colony-PCR was run. This will also make sure that the whole promotor flanked with loxP-sites haven’t been cut out as well, which is unlikely due to the LoxP-site is designed in opposite direction. Figure 2 show the gel electrophoresis picture. The upper run show only a band if the flip succeeded and the lower run show both a band if it succeeded, at 1300 bp, and if not succeeded, at 500 bp. A negative control was run as well, which showed no band at the upper run and a band at 500 bp in the lower run, which it supposed to do. In the gel picture, it is visible that one of three seems to have flipped correct. The other two seems to have a problem in the PCR, because no band was shown in the lower run as well, there it should appear a band regardless the direction of the promotor,

With this the conclusion can be draw that the non-reversible system worked.

Sequence and Features