Part:BBa_K4888001
IPTG+Theophylline double expression control system (+RBS)
This component is an inducible regulatory system, that can be used fused upstream to the protein of interest.
To produce the protein, both IPTG and theophylline must be present simultaneously.
Interest of the system
In certain cases, it is crucial to precisely regulate protein expression and, more importantly, prevent unintended expression.
While the IPTG inducible Lac operator is a reliable inducible system, it can exhibit leakage. Moreover, our intention is to employ this design for protein expression within the gastrointestinal tract, where lactose might be present.
The theophylline riboswitch introduces a hairpin structure in mRNA, thereby obstructing translation and providing an additional layer of security to prevent undesired protein production. Alone, it can also be leaky starting from 30 degrees.
By using the two systems combined, we ensure no unwanted expression.
Functionnement
This component combines two inducible systems, a T7 promoter and a robust ribosome binding site (RBS).
The T7 promoter is a constitutive promoter used in E. coli.
The IPTG induction system operates in conjunction with the lac operon and the lacI system. LacI proteins are synthesized and inhibit the lac operon, thus preventing translation.
The theophylline induction system employs a riboswitch sequence that induces mRNA to form a hairpin structure in the absence of the target molecule. The ribosome binding site (RBS) is embedded within this folded structure, rendering it inaccessible to ribosomal machinery. The presence of theophylline leads to a conformational change in the hairpin structure, freeing the RBS and enabling transcription to proceed.
Composition and Functionnement of the part
Proof of concept
Expression of SPAC - western blot
We used this double expression control system to express SPAC, a mucus-binding protein. See BBa_K4888002.
In our experiments, the negative control consisted of transformed bacteria that had not been exposed to a specific molecule. We conducted incubation experiments using IPTG only, theophylline only, and a combination of both. These experiments were conducted under two distinct conditions: early induction and late induction.
In the early induction scenario, we introduced theophylline followed immediately by IPTG. In the late induction scenario, theophylline was initially introduced, allowing time for the hairpin structure to change, after which IPTG was added. Additionally, we conducted these experiments at two different temperatures, with a specific emphasis on 37 degrees Celsius, as it is crucial for our goal of expressing the protein within the human body.
Western experiment - expression of Spac using the double expression control system
Western blot analysis of SDS-boiled bacteria. The left panel displays bacteria subjected to protein induction at 18°C overnight, while the right panel shows induction at 37°C for 2 hours. 'Early induction' refers to the addition of Theophylline (Th) to the medium during the launch of the secondary culture, while 'late induction' indicates the simultaneous addition of Th and IPTG. The remaining bands were induced using either Th, IPTG, or no inducer.
Bands were detected at approximately 130 kDa, slightly above the expected 122 kDa molecular weight of SpaC, confirming successful protein expression at both 37°C and 18°C. Conversely, no signal was observed in the absence of Theophylline (Th) or Isopropyl-β-D-thiogalactopyranosid (IPTG), indicating no protein expression.
As previously reported, there was some leakiness in the lac operon, as evidenced by the signal in the "Th only" condition. Furthermore, consistent with the reduced riboswitch tightness at lower temperatures [2], a signal was observed in the IPTG condition at 18°C, whereas translation was suppressed at 37°C. Notably, initiating the secondary culture with Theophylline (Th) rather than adding it during IPTG induction resulted in an increased protein yield.
We show that the part works as expected.
Expression of D1/D2 SPAC - western blot
We truncated SPAC to allow better transport to the surface and did another western plot to verify the expression, checking again the function of the part. See part See BBa_K4888003 and BBa_K4888004
Western experiment - expression of truncated SPAC using the double expression control system
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left: Western Blot analysis of boiled bacteria hosting the D2 construct, induced with Theophylline (Th), IPTG, both, or neither.
right: Western Blot analysis of boiled bacteria hosting the D1D2 construct, induced with Theophylline (Th), IPTG, both, or neither.
Both D1D2 and D2 showed the highest protein expression levels when induced with both Theophylline and IPTG. The observed bands corresponded to the expected sizes of approximately 80 kDa for D1D2 and 57 kDa for D2, respectively.
Notably, IPTG or Theophylline alone still led to a modest level of protein expression, indicating that both expression systems have some degree of leakiness. However, when both inducers were absent, D2 expression was completely prevented, and D1D2 expression was minimal.
Interestingly, when compared to the initial construct, SpaC, only the lac promoter displayed leakiness, while the Theophylline switch remained tightly regulated. SpaC expression was observed only in the presence of Theophylline, with minimal expression under other conditions.
We have confirmed that the proposed expression system is effective in minimizing the leakiness of both promoters when they are combined. We used this system to express our truncated protein of interest, see part BBa_K4888005
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 1206
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 1206
- 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 1206
- 1000COMPATIBLE WITH RFC[1000]
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