Part:BBa_K1602044
RRL3H
Composite part consisting of a constitutive promoter (BBa_J23100), Lock3 (BBa_J01080) and hokD (BBa_K1497008). It belongs to a two-part killswitch-system for E.coli utillizing a riboregulator for posttransciptional regulation of hokD. Upon transcription the sequence of Lock3 forms a hairpin-secondary-structure masking the ribosome binding site (RBS) and therefore prevents translation of the following hokD-sequence. If the corresponding trans-activating RNA-sequence (taRNA) (Key3 BBa_J01086) is present the two sequences form a RNA-RNA-complex. This leads to a helix shift and the release of the RBS enabling the expression of hokD resulting in cell death.
Functional Parameters
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 and as negative control for each riboregulator Top10 transformed with only 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). This might indicate that 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.
One possible reason for the malfunction of the riboregulator could be the fact 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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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