Difference between revisions of "Part:BBa K1132043"

Latest revision as of 16:52, 6 October 2013

R1-pLac-RFP

Assembly between BBa_K1132042 and BBa_E1010

Usage and Biology

This riboregulator part limits leak of a promoter introducing a translation regulation, supplemental to the common transcription regulation. It allows a more precise regulation of the downstream gene, which is RFP in this case.

Information about the riboreguator design and usage can be found on the R1-pLac page.

Sequence and Features

Characterizations

The RFP riboregulator was transformed into the XL1-Blue E. coli strain that expresses TetR (with the Tn10) and a modified version of LacI, LacIq, a better repressor of the pLac promoter.
For the following experiments, we used IPTG (Isopropyl ß-D-1-thiogalactopyranoside) that induces expression of the pLac promoter and aTc (AnhydroTetraCycline) that, when bound to TetR inhibits its action on the pTet promoter and thus allowing its transcription. The following table describes the expected results. Reminder: IPTG will control the level of production of the second promoter. In the absence of transcription at the first promoter, the RNA produced will not be translated (RBS blocked). With aTc, TetR does not block transcription of the first promoter, the second RNA is produced and can bin to the pLac RNA thus releasing the RBS. Translation occurs and RFP is produced.

XL1-Blue was transformed with:
i)the riboregulator (R1.Plac with RFP);
ii) a positive control (Plac with RFP);
iii) a negative control (without RFP).
The three transformations were plated on LBC plates without IPTG and aTc. Comparison of the three plates leads to encouraging results. Compared to the Plac.RFP construct, the riboregulator expresses a small amount of RFP. This result shows that the riboregulator is completely capable of repressing the transcriptional leak of the Plac promoter.

We then cultivated the three strains in liquid LBC for fluorescence and visual inspection of the cell pellet. Different tests wer performed: with or without aTc, with or without IPTG for the negative control (no RFP in the strain), the positive control (Plac with RFP) and the riboregulator with RFP at 30°C or at 37°C.

Phenotype observations

The samples shown on this picture are all the present biobrick, except the blank on the left.

At 37°C, with aTc and IPTG, the promoter with the riboregulator is induced. In comparaison to 37°C, at 30°C, the phenotype of the induction is not clear; it can be because the secondary structure is to strong, then the ribosome binding site of the RFP is blocked.

The leak of the Plac promoter is lower with the riboregulator than without (comparison with the positive control). Our riboregulation system works, limiting basal leak of the pLac promoter.


This conclusion has been confirmed with induction by IPTG : the difference is still present, in particular at 30°C where secondary structures seem to be more stable.

With aTc and without IPTG, in theory, the leak of the Plac promoter is important, because with aTc the ribosome binding site of the RFP is unlocked. But the expression of RFP is still higher with the positive control (pLac-RFP induced by IPTG).

Fluorescence measurement

The graph above describes the results obtained. Fluorescence signals of each strain was normalized to the level of fluorescence obtained with pLac-RFP. Careful examination of the graph shows that:
- aTc can really inhibit TetR repression. By doing so, transcription is restored at the pTetR promoter. Hence, this promoter, although not directly transcribing the RFP gene, can increase the RFP content. Therefore, the pLac riboregulator cannot block the RBS anymore.
- IPTG, in the absence of aTc induces expression of RFP. This means that the TetR protein is not sufficiently produced in this strain to fully repress the pTetR promoter. In consequence, there is probably enough transcripts from the pTetR promoter and IPTG induction can still occur.
- aTc induction produces a sharp increase in fluorescence, meaning that the RFP level is really higher.

The level of RFP with te maximal concentration of IPTG and aTc confirms the usefulness of our riboregulator to limit the leaky transcription of any promoter. In our system, depending of the level of aTc and IPTG, the expression of the output gene can be controled. Temperature is also an important parameter, because of the different annealing temperature effects of the intervening RNA sequence.
Transcription without aTc and without IPTG is not null. The structure of the R1 riboregulator may need some ameliorations to restrict the leaky transcription to almost zero transcript. Furthermore, in order to control the strength of the final construct, the interfering RNA and the blocking ribosome binding site sequence can be redesigned and modulated at will.