Results

Objective

Riboregulators are essential to the E. calculus project. We designed 4 different riboregulators that would have been used for the whole project. 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_K11320042. We then added RFP (BBa_E1010) and Bxb1 (BBa_K907000) to BBa_K11320042.

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 (BIOBRICK NAME) 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 the 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 β-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.

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

Trop mega top!!

Titre 3

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