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 (a href="http://parts.igem.org/Part:BBa_K1602045">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 killswitches. In order to assemble the final riboregulator-systems the parts RRL3H (BBa_K1602044) and RRK3 (BBa_K1602046) on two seperate plasmids (pSB1C3 and pSB1A2) were co-transformed into E.coli(Top10) for the first riboregulator. For the second riboregulator containing hokD the part RRlocked (BBa_K1602050) was co-transformed with araC-pBAD-RRkey (BBa_K1602051). Positive transformants were selected by using two antibiotics, Chloramphenicol and Ampicillin, and verified via colony-PCR. A strain of E.coli (TOP10) transformed with araCpBAD-hokD (BBa_K1602043) was used as positive control. As negative control for each riboregulator Top10 were transformed only with the cis-repressed part (RRL3H and RRlocked) of each system. All five cultures were grown in LB-medium with the respective antibiotics containing 20mM glucose at 37°C over night. Afterwards 10µl of each culture were inoculated in two seperate flasks of LB-medium (with the respective antibiotics), one containing 20mM Glucose, the other one 2mM arabinose. After 16 hours of incubation at 37°C an aliquot of each culture was diluted several times and different dillutions were spread on LB-agar plates containing 20mM glucose. The plates were incubated at 37°C over night until single colonies were distinquishable. The amount of colonies was counted in order to determine the colony forming units per ml culture (CFU/ml) to compare the amount of living bacteria in the cultures grown with glucose and arabinose.
The positive control (araC-pBAD-hokD) showed a significant reduction of CFU/ml in the culture grown with arabinose compared to the culture grown with glucose indicating that the presence of glucose repressed hokD-expression while the addition of arabinose leads to production of HokD resulting in increased cell death (Fig.2). The results for the riboregulators however did not turn out as expected. There was no significant reduction in the CFU/ml of the culture grown with arabinose compared to the culture grown with glucose observable for both riboregulator-systems (RRK3+RRL3H and araC-pBAD-RRkey+RRlocked). To validate the gathered data a second experiment was conducted. Therefore four new co-transformed riboregulator-systems were selected as mentioned above and grown in LB-medium containing 20mM glucose for 16 hours at 37°C. After incubation the cultures were diluted and the same volume of several dilutions was plated on two different LB-agar-plates: one contained 20mM glucose and the other one 2mM arabinose. The plates were incubated at 37°C until seperate colonies were distinquisable and the CFU/ml were calculated. As controls served again Top10 with araC-pBAD-hokD (positive) and Top10 containing only RRlocked (negative).
As before we were not able to observe the anticipated behaviour of the riboregulator. There was no significant reduction in CFU/ml observable when growing the bacteria in the presence of arabinose compared to glucose(Fig.3). The riboregulator-systems 1 and 3 even showed a slight increase in the amount of CFU/ml on the plates containing arabinose. These results could also indicate that the repression of hokD via the cis-repressing sequence is not as tight as we hoped for. If this would be the case hokD might be expressed no matter what environment the bacteria are growing in leading to unwanted cell death.
To gain further insight into the stability of our crRNA construct and to simulate it's complex behaviour we used Molecular Dynamics (MD) to study possible interactions. Therefore we predicted the three dimensional structure with an harmonic potential and various constrains (Fig.4).
After we got the 3D-structure we were able to analyze the dynamic nature of our crRNA construct. In order to test the locking abillity of the construct we quantified the motion within this construct surrounded by its native environment at 600 K. The simulation was carried out with gromacs using Amber03 force field. To access stability we used the all atom root mean square deviation (RMSD) as well as the root mean square fluctuation (RMSF) to gain insight into the molecular movement and flexibility (Fig.5).
Both plots point out only small structural changes as well as atomistic rigidity of our crRNA. We observed only small fluctuations within our construct. The whole structure altered only ~3 Angström from its predicted position and the atomic fluctuation ranged from 1-2 Angström per atom indicating that the regulation of gene expression should be tight and that no unwanted cell death should occure.
Based on all our results it seems most likely that the malfunction of the riboregulator might be because the interaction between the taRNA and the crRNA does not occure as predicted resulting in a constant repression of hokD even when induced with arabinose. It also could be a problem that both parts were located on seperate plasmids which were co-transformed into the cells. It is possible that this results in an unfavorable situation for the bacteria to produce enough of both parts necessary for the riboregulator to work.
To further investigate this hypothesis we cloned both parts of the riboregulator next to each other on one plasmid resulting in the BioBricks RRhoK (BBa_K1602052) and RRK3-RRL3G (BBa_K1602047) but we were not able to repeat the experiment so far due to time constraints.