Module 3: OR-gate Biosafety
Live bacterial therapeutics require rigorous biosafety review before entering the human body. Thus, we prioritized the suicide regulation of bacterial strains at the initial design stage. To this end, we constructed an “OR-gate dual-input responsive” suicide system using two signals including “active arabinose administration” and “passive temperature sensing”, enabling intelligent control over the survival of engineered E. coli. This system helps to prevent horizontal gene transfer (HGT) in our external environment while endowing the live bacterial therapy with sufficient flexibility on the premise of ensuring biosafety.
Our biosafety genetic circuit leverages a temperature-sensitive operon, modified to incorporate a self-repressive regulatory system. By tuning the expression intensity of repressor proteins and assembling the requisite promoter elements, we successfully constructed the OR-gate genetic circuit and characterized the suicidal effect of the toxin protein CcdB (a natural DNA gyrase inhibitor), ensuring that the survival of the engineered bacteria remains under strict control.
1.Assessment of Toxin-Antitoxin System Efficacy
We obtained Salmonella haroring the plasmid with ccdA/ccdB toxin-antitoxin genes from Prof. Wang's lab. Since the genes amplified by PCR were not optimized for the codon preference of E. coli, preliminary characterization of their properties was required to achieve suicide regulation of our strains. To this end, we expressed CcdB alone and co-expressed the toxin-antitoxin system, respectively, and verified whether they could normally kill E. coliby examining the survival status of transformed bacteria.
We first amplified the ccdA and ccdB genes from the extracted Salmonella plasmid by PCR and ligated them with the pSB3K3 plasmid backbone via Gibson Assembly. The homologous arms of the specific PCR primers incorporated a strong constitutive promoter and a strong RBS sequence to enhance gene expression. The assembled products were transformed into E. coli TOP10 and spread on kanamycin-containing LB plates to screen for positive clones. Single colonies on the plates were sent to a company for sequencing verification.
The results showed that only 2 single colonies grew on the LB plate after transforming the Gibson Assembly product of J23101-RBS34-ccdB into TOP10 competent cells, and both were confirmed to be empty pSB3K3 plasmids lacking the ccdB fragment by sequencing. In contrast, strains co-expressing the toxin-antitoxin genes grew normally on LB plates, with correct sequencing results. These results basically verified that ccdA and ccdB from Salmonella can exert normal functions in E. coli.
Figure 1. Construction and characterization of the toxin-antitoxin system in E. coli. A. Results of electrophoretic bands of pSB3K3 plasmid vector (~2.7 kb), ccdA-ccdB fragment (541 bp) and ccdB fragment (348 bp) after PCR. B. Plasmid map of pSB3K3-J23115-ccdA-ccdB. C. Plasmid map of pSB3K3-J23101-RBS34-ccdB. D. Only two single colonies grew on the LB plate after transforming TOP10 with the plasmid J23101-RBS34-ccdB. E. Sequencing results of the J115-ccdA-ccdB plasmid. F. Sequencing results of the two colonies grown on the LB plate for toxicity validation.
2.Construction and characterization of an self-repressive system based on a temperature-responsive operon
Following verification of the functions of ccdA and ccdB, we planned to construct an OR-gate kill-switch with dual active-passive regulation. Through literature research, we employed two input signals: the temperature difference between in vivo and in vitro environments, and arabinose, which is present at low levels in common foods, by leveraging the widely used arabinose operon and sequence-optimized temperature-responsive operon elements to build the OR-gate circuit. The OR-gate logic was constructed based on a self-repressive system, which regulated the expression of repressor proteins and the activity of corresponding promoters. Therefore, it was necessary to construct and characterize this self-repressive system based on the temperature-responsive operon.
Figure 2. Schematic diagram of the temperature-responsive self-repressive circuit
(1)Construction of temperature-controlled self-repressive systems
The DNA sequence encoding the temperature-responsive self-repressive element was commercially synthesized and amplified by PCR. The resulting DNA fragment was then assembled with a GFP reporter gene and the pSB3K3 plasmid backbone via Gibson Assembly. The constructed plasmid was transformed into E. coli TOP10 competent cells, which were plated on LB agar containing kanamycin for selection. Positive clones were then screened and verified by DNA sequencing. To fine-tune the expression level of this system, we incorporated RBS sequences of varying strengths including RBS30, RBS32, RBS34 (strength: RBS34 > RBS30 > RBS32) via primer design to regulate the expression of the downstream GFP. Ultimately, we successfully constructed a set of temperature-controlled self-repressive systems with different expression strengths for subsequent characterization.
Figure 3. Construction of expression plasmids for the self-repressive circuit. A. Results of electrophoretic bands of PtlpA-TlpA* with various RBS sequences (~1.2 kb) and the pSB3K3 plasmid backbone fragments harboring GFP (~3.6 kb) after PCR. B. Plasmid map of pSB3K3- PtlpA-TlpA*-RBS30-GFP. C. Sequencing results of target plasmids harboring various RBS sequences.
(2)Characterization and optimization of temperature-controlled self-repressive systems
After constructing the aforementioned plasmids, we used 37 °C and 30 °C to represent the in vivo and in vitro temperatures, respectively. The corresponding E. coli TOP10 strains were cultured in constant-temperature shakers at the respective temperatures for 9 hours. Samples were collected at regular intervals to plot the curves of unit fluorescence intensity versus culture time under different temperature conditions. This was to determine the optimal temperature-induced expression time while evaluating the leakage expression level of this temperature-regulated composite elements under low-temperature conditions.
The results showed that the unit fluorescence intensity at 37 ℃ first increased and then decreased, reaching a peak at approximately 4 hours. Moreover, the level of fluorescent leakage expression at 30 ℃ was generally very low, thus we subsequently selected 4 hours as the culture time for the temperature-regulated signal. Furthermore, we compared the 37 ℃ and 30 ℃ switching signals corresponding to the three RBS sequences at 4 h, and found that they could all achieve a switching effect of one order of magnitude. Comprehensively considering the gene leakage level at 30 ℃ and the expression effect of downstream genes, we ultimately chose the composite element containing RBS30 and 4-h induction time to optimize the expression of the OR-gate genetic circuit.
Figure 4. Characterization and optimization of expression plasmids for the self-repressive circuit. A. Measurement results of unit fluorescence intensity over 9 hours at different temperatures for TOP10 harboring plasmid pSB3K3- PtlpA-TlpA*-RBS30-GFP. B. Temperature-regulated switching effects of three circuits with various RBS sequences at 4 h.
3.Construction and Optimization of the Biosafety OR-Gate Genetic Circuit
Building upon the temperature-responsive self-repressive system and through multiple rounds of design iteration (turn to our Engineering page for detailed information on biosafety circuit cycles), we successfully constructed an OR-gate kill-switch. However, the complex regulation of the arabinose operon made it unsuitable for direct use in OR-gate logic regulation. To address this, we employed a tandem dual-promoter strategy. This design ensures the expression of the target gene downstream of the circuit in response to the temperature signals even without arabinose induction, thereby achieving dual-input control over bacterial survival.
(1)A strategy to bypass the arabinose operon constraint for OR-gate circuit construction
Preliminary experiments confirmed that activation of the PBAD promoter requires the simultaneous presence of arabinose and the regulatory protein AraC. In short, the promoter remains inactive in the absence of AraC. To circumvent this limitation, we introduced a strong constitutive promoter J23101 upstream of PBAD, creating a tandem dual-promoter system. This allows for expression of the downstream gene even in the absence of AraC. To prevent potential negative effects on transcription and translation efficiency caused by the extended upstream sequence, we incorporated the self-cleaving ribozyme insulator RiboJ (BBa_25XUNXCE) between the composite promoter and the reporter gene. RiboJ can undergo self-catalyzed cleavage to separate the transcript from RNA leader sequences, thereby enhancing downstream gene expression.
Accordingly, we constructed a verification plasmid, pSB3K3-J23101-PBAD-RiboJ-GFP. The RiboJ-GFP gene fragment was obtained via PCR using a plasmid provided by Prof. Wang’s lab as the template. J23101 promoter sequence was introduced via PCR primers designed with homologous arms, and these fragments were assembled into the pSB3K3 backbone via Gibson Assembly. The constructed plasmid was then transformed into E. coli TOP10 cells, and positive clones were screened on KanR LB agar plates and verified by sequencing.
After confirming the plasmid sequence, TOP10 cells harboring the verification plasmid were cultured overnight at 37 °C, and their fluorescence intensity was measured. Cells transformed with the empty pSB3K3 plasmid served as the negative control. The results demonstrated significant fluorescence expression in the engineered strain which lacks the araC gene, confirming that the tandem dual-promoter strategy effectively overcame the inherent constraint of the arabinose operon, enabling the intended circuit function.
Figure 5. A tandem dual-promoter strategy adopted to construct the OR-gate circuit. A. Schematic diagram of the tandem dual-promoter system. B. Results of electrophoretic bands of plasmid backbone vector (~2.7 kb) and the RiboJ-GFP fragment harboring the J23101 promoter sequence (~1 kb) after PCR. C. Plasmid map of pSB3K3-J23101-PBAD-RiboJ-GFP. D. Sequencing results of the target plasmid harboring the dual-promoter element. E. Functional validation of the tandem dual-promoter circuit.
(2)Construction of the biosafety OR-gate genetic circuit
Building on the characterization of the basic and composite elements, we proceeded to construct the complete biosafety OR-gate circuit by integrating the arabinose repressor protein AraC and the temperature-regulated self-repressive system. Following the aforementioned procedure, the plasmid backbone, araC gene fragment, self-repressive composite element, and dual-promoter expression unit were amplified via PCR using specific primers. These fragments were then assembled using Gibson Assembly, transformed into E. coli TOP10, and positive clones were selected with verification by DNA sequencing. Ultimately, we successfully assembled the biosafety OR-gate circuit with GFP as the reporter gene.
Figure 6. Construction of the biosafety OR-gate genetic circuit. A. Results of electrophoretic bands of the pSB3K3 plasmid backbone fragment harboring J23101- PBAD-RiboJ-GFP (~3.7 kb) and PtlpA-tlpA*-araC fragments with different RBS sequences (~2.2 kb) after PCR. B. Plasmid map of pSB3K3- PtlpA-tlpA*-araC-J23101- PBAD-RiboJ-GFP. C. Sequencing results of the target OR-gate biosafety plasmid.
(3)Characterization and optimization of the biosafety OR-gate genetic circuit
The sequence-verified plasmid was then transformed into TOP10 for functional characterization. Bacterial cultures were incubated in shakers at 37 °C and 30 °C. A gradient of arabinose concentrations including 0, 0.01, 0.039, 0.153 and 0.6 mM was established based on literature references. The moment of inducer addition was defined as time zero, followed by a 4-hour cultivation period. We then sampled the cultures simultaneously to measure the specific fluorescence intensity, and draw the induce-response curves.
The results indicated that a significant level of leaky expression persisted at 37 °C even in the absence of arabinose. However, the overall circuit response aligned with our design principles. The unit fluorescence intensity increased with higher arabinose concentrations, and the expression level was substantially greater at 30 °C compared to 37 °C. The switching behavior of the circuit was assessed under four conditions representing the different input logic states of the OR gate, and there was an approximately 5-fold difference in signal intensity between the 'ON' and 'OFF' states, demonstrating that the OR-gate circuit is sufficient to mediate dual-input control over bacterial survival.
Figure 7. Characterization of the biosafety OR-gate genetic circuit. A. Results of temperature and arabinose response curves of the biosafety circuit. B. Switching effects of the biosafety circuit under four input states.
Conclusions
By integrating the aforementioned elements and designs, we have constructed a temperature-arabinose dual-input OR-gate genetic circuit, which successfully achieved an ON/OFF signal amplitude of approximately 5-fold. Furthermore, the response threshold can be dynamically adjusted by regulating the expression intensity of subsequent toxin-antitoxin genes, thereby preventing unintended cell death of the strain in the "OFF" state and ensuring its ability to produce GLP-1 in the human intestine.
However, during actual construction, we found that ccdA/ccdB elements were difficult to effectively integrate into this biosafety circuit, possibly due to mismatched signal intensities. Subsequent work should involve sequence optimization of this toxin-antitoxin system or selection of natural toxin-antitoxin systems from E. coli. In addition, if ccdB is successfully incorporated into the circuit, the performance will be affected by transformation efficiency and it will impose extremely high demands on temperature control during operation. Due to time constraints, we may not be able to conduct subsequent experiments on optimizing culture and characterization conditions, but we have preliminarily verified its feasibility.
In the future, we plan to further develop more intelligently regulated biosafety switches, such as optimizing recently reported fucose operons, utilizing specifically targeted lysogenic phages, and integrating bacterial quorum sensing systems or other intestinal signal molecule-sensing modules. This will ensure that the strain maintains adequate activity both during pre-administration storage and in the intestine, advancing efforts to achieve the industrialization of oral bacterial agents.