Results

Objective

Riboregulators are essential to the E. calculus project. We designed 4 different riboregulators that were tailored to be used with the four different logic gates. However, characterization of the riboregulator principle was only performed with the R1 design, due to time limitation. The two objectives were: Characterize riboregulators with an RFP as output and two inducible promoters. Characterize riboregulators with Bxb1 recombinase as output and two inducible promoters; the characterization of the expression is possible with our XOR gate containing RFP as output (BBa_K1132032).

Constructions

The design of the riboregulator for characterization purposes was: a pTet promoter with two terminators followed by the pLac promoter. Both promoters have the RNA regulatory sequences (see Logic Gates Module). This part is BBa_K1132042. We then added RFP (BBa_E1010) and Bxb1 (BBa_K907000) to BBa_K1132042.

For both systems, the expression of the TetR repressor is needed. We (unsuccessfully) tried to clone the tetracycline resistance under the control of a constitutive promoter, a ribosome binding site and terminator on both constructs. Lack of time prompted us to concentrate our efforts to a simpler test, by transformation of the RFP construct (BBa_E1010) into various strains expressing TetR.
Expression was measured either by visual inspection of small cultures after centrifugation or fluorescence analysis.

Results

Riboregulator with RFP

DH5-1 strain

When the RFP riboregulator plasmid is transformed into the E. coli DH5-1 strain, RFP is produced. Due to the relatively weak effect of LacI, the repressor of the pLac promoter (BBa_R0011) the pLac promoter is not blocked and we can see some RFP production. Our control containing pLac-RFP (BBa_J04450) can give the level of leakiness of the promoter. Furthermore, TetR, the repressor of the pTetR promoter (BBa_R0065), is not expressed in this strain hence the first promoter of the riboregulator system cannot be down regulated and then behaves as a non-controllable promoter in this strain.

XL1-Blue

We then used the RFP riboregulator in 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.

We tried different concentration of aTc in order to determine the maximum concentration usable to have the highest while avoiding growth inhibition. The graph below measures the growth inhibition effect with different aTc concentrations.

Strain growth is not inhibited at a concentration of 100 ng/mL of aTc. For the next characterizations, we choose to use two aTc concentrations (50 and 200 ng/mL) and 1mM IPTG.

XL1-Blue was transformed with i)the riboregulator (R1.Plac with RFP); ii) a positive control (Plac with RFP) and 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 trnascriptional 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 at 30°C or at 37°C.

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 sequestered.

The leak of the Plac promoter is lower with the riboregulator than without (comparison with the positive control). This conclusion has been confirmed with induction by IPTG.

With aTc and without IPTG, in theory, the leak of the Plac promoter is revelate, because with aTc the ribosome binding site is released. But, the expression of RFP is still higher with the positive control. Nevertheless, the different with the positive control is smaller with aTc.

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. By doing so, transcription is restored at the pTetR promoter. Hence, this promoter, although not directly transcribing the RFP gene, can augment the RFP content. Therefore, the pLac riboregulator cannot sequester 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.

Riboregulator with Bxb1 integrase

For the riboregulator with the Bxb1 integrase, the objective was to compare the switch of the XOR gate with/without IPTG and aTc. The riboregulator with RFP was characterized the last day before the freeze of the wiki, and we did not have enough time to prove the influence of the riboregulator to the switch of the gate.

Discussion

The riboregulator has been characterized a few hours before the Wiki freeze. This is why our results may seem a bit preliminary. We only characterized one riboregulator structure. The level of RFP with te maximal concentration of IPTG and aTc confirm 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 P1 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.

Riboregulation was an essential module of our E. calculus design. Results obtained with the recombinases show that low levels of some of the tested recombinases might be sufficient to promote the logic gates switches. Even if our initial design may not be the most effective riboregulator (see Riboregulators), many different riboregulators can be designed and probably tailored to one's needs. Clearly, in our case, the strength of the second promoter is of utmost importance. As recombinases will work at very low concentrations, even a weak and constitutive promoter could be used. Although we have only characterized one riboregulator, we have deposited in the registry a series of different regulators with various changes in the intervening RNA sequences and that should hopefully create various regulatory controls. All of them can be tested with any reporter gene, the second promoter can be changed at will for other promoters.