Part:BBa_K1602054
RRGFP
RRGFP is part of a two-part riboregulator-system for posttransciptional regulation of GFP-expression. The part was created by inserting a GFP-gene flanked by the restriction sites BamHI and HindIII into RRlocked_site via restriction cloning. It consists of the fused sequences of a constitutive promoter (BBa_J23100), a cis-repressing sequence (cr), GFP (BBa_E0040) and a terminator (BBa_B0015). The restriction sites upstream and downstream of the GFP sequence allow for an easy exchange of the GFP gene with another gene of interest.
Upon transcription the cis-repressing sequence forms a hairpin-secondary-structure masking the ribosome binding site (RBS) and therefore prevents translation of the following GFP-sequence. If the corresponding trans-activating RNA-sequence (taRNA) (RRKey-BBa_K1602049) 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 GFP as the gene of interest (GOI).
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 it would be necessary to clone both parts of the riboregulator next to each other on one plasmid in order to test if the riboregulator-system then works as expected.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 91
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 737
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