Part:BBa_K1602051
araCpBad-RRkey
Composite part consisting of an araC-regulated pBAD-promoter (BBa_K808000) cloned upstream of RRkey (BBa_K1602049). It is half of a two-part riboregulator-system for E.coli for posttransciptional regulation of gene expression. Upon transcription the produced trans-activating RNA-sequence (taRNA) forms a RNA-RNA-complex with corresponding cis-repressing sequences (crRNA). This leads to a helix shift and the release of the formerly masked ribosome binding site (RBS) enabling the expression of the regulated gene of interest (GOI). The corresponding crRNAs are RRlocked, RRlocked_site and RRGFP.
Because of the araC-regulated pBAD-promoter production of the taRNA is induced by the addition of arabniose. In the presence of glucose araCpBAD shows a very low basal expression level. In combination with the corresponding crRNA-parts (RRlocked and RRlocked_site) this results in an induceable riboregulator-system which represses gene expression in the presence of glucose but is inducable through the addition of arabinose.
Functional Parameters
In order to assemble the final riboregulator the parts RRGFP (BBa_K1602054) and araCpBAD-RRkey (BBa_K1602051) on two seperate plasmids (pSB1C3 and pSB1A2) were co-transformed into E.coli(Top10). Positive transformants were selected by using two antibiotics, Chloramphenicol and Ampicillin, and verified via colony-PCR.
As controls served a culture of TOP10 without plasmid, one transformed with araCpBad-GFP (BBa_K1602055) as positive control and one transformed only with the cis-repressed part of the riboregulator (RRGFP - BBa_K1602054) as negative control. All four 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 1 ml of each culture was pelleted by centrifugation and resuspended in PBS for subsequent FACS-measurements.
The positive control (Fig.3 B) showed a significant difference in the detected fluorescence-levels between the culture grown with glucose and the culture grown with arabinose indicating that the addition of 20mM glucose to the medium is sufficient to repress GFP-expression through the araC-regulated pBAD-promoter. Surprisingly we were not able to detect the same difference in the cultures containing the assembled riboregulator (Fig.3 D). The measured fluorescence for the induced culture grown with arabinose was the same as for the culture grown with glucose. Furthermore was the detected GFP-Signal very simmilar to the results of the negativ controls (Fig.3 A+C) what leads to the conclusion that no GFP was expressed in the culture containing the riboregulator at all.
We have to assume that the interaction between the two parts of the riboregulator does not happen as anticipated, leaving the riboregulator-system constantly "locked" and preventing GFP expression, even after induction.
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.
To further investigate this hypothesis we cloned both parts of the riboregulator next to each other on one plasmid without the araC-pBAD resulting in the BioBrick RRhoK (BBa_K1602052). But due to time constraints we were not able to characterize this part yet.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1144
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 979
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1215
Illegal SapI site found at 961
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