Results

The final riboregulator system was assembled by co-transformation of the two necessary system parts into E.coli. For the FACS-measurements we transformed a pSB1A2-plasmid containing RRkey together with a pSB1C3-plasmid containing the BioBrick RRGFP, which was created by exchanging the hokD-sequence of RRlocked_site with a GFP flanked by BamHI and HindIII via restriction cloning. The transformed cells were selected on a plate containing Chloramphenicol and Ampicillin and were verified via colony PCR. Positive selected clones contained the complete riboregulator system controlling GFP-expression and were grown in LB-media containing Chloramphenicol, Ampicillin and 20mM Glucose over night. The same procedure was done with pSB1A2-RRK3 and pSB1C3-RRL3G to assemble the second riboregulator system. As a control for the GFP-expression levels we also transformed E.coli with araC-pBAD-GFP (BBa_K1602045) which is GFP cloned downstream of the araC-regulated pBAD-promoter. This control culture should only express GFP in presence of arabinose.

After incubating for 16 hours at 32°C 10µl of the cultures were inoculated in two separate flasks of LB-media, one containing 20mM glucose and the other 2mM arabinose, and were incubated over night. On the next day 1ml of each culture was pelleted and resuspended in PBS in order to measure the GFP-expression in the cells via FACS. A culture of untransformed E.coli (TOP10) was used as negative control.

In the two control cultures containing araC-pBAD-GFP (Fig.1 B) the fluorescence could be detected as expected: The culture grown in LB containing glucose showed a very low fluorescence while the fluorescence levels of the culture grown in LB containing arabinose were much higher. Suprisingly we were not able to detect any fluorescence in all of the cultures with the riboregulator systems (Fig.1 C+D) whether they were grown with arabinose or glucose. The levels of detected fluorescence were very similar to the levels of the negative control (Fig.1 A) which leads us to the conclusion that no GFP was expressed at all.

Parallel to the FACS-measurements we performed spread plate assays to determine if the riboregulator systems controlling hokD-expression indeed work as a functional killswitch. The riboregulator systems were assembled by co-transforming RRlocked together with araC-pBAD-RRkey and RRL3H together with RRK3 as described above. As control for each riboregulator we used E.coli containing only the the cis-repressed part of the riboregulator without the key to open it. As positive control E.coli transformed with araC-pBAD-hokD. After growing each culture separately in LB-medium with glucose and arabinose over night at 37°C we took 100µl of each culture, diluted it several times and spread the dilutions on LB-agar-plates containing the appropriate antibiotics and 20mM glucose. The plates were incubated over night at 37°C until single colonies were visible. By counting the colonies we were able to calculate the colony forming units (CFU) per ml culture in order to determine if the bacteria only survived in the media containing glucose.

The positive control expressing hokD under control of the araC-pBAD-promoter showed showed an expecting behavior of the cells. The amount of CFU/ml was much higher in the culture grown with glucose than in the culture containing arabinose. This indicates that in the culture grown with arabinose hokD was expressed and the cells died. The negative controls only containing half of the riboregulator system showed similar amounts of living cells as expected. But again the results for the complete riboregulators were not as expected. It was not possible to detect a significant reduction in CFU/ml in the culture grown in arabinose compared to the culture grown in glucose. This indicates that hokD is not expressed as we hoped for and that our designed killswitches do not work as expected.

From these results we have to conclude that the gene expression controlled by our riboregulators is not activated by the addition of the trans-activating RNA-Sequence.