Adhesion Module
Adhesion Module:
Cycle1 Assessment of the OppA Protein
Design (D):
Objective: To verify whether the OppA protein from Lactobacillus plantarum can bind to NaHS on the surface of olfactory epithelial cells. Using Autodock and HDOCK software, molecular docking will be performed between the OppA protein and NaHS, Mucin, as well as other potentially interacting small molecules or proteins.
The binding affinities of seven different OppA proteins will be compared by evaluating their binding free energy (ΔG).
Additional Safety Consideration: Since the engineered bacteria may reach the intestinal tract, a Mucin binding test has been incorporated into the design to assess potential risks.
Build (B):
A molecular docking model was constructed to perform virtual docking of OppA with NaHS and Mucin, yielding binding energy and confidence scores.
The docking results were visualized, and a heatmap was generated to illustrate the binding capabilities of the different OppA variants.
Test (T):
The experimental results demonstrated that the binding energies between NaHS and all OppA variants were less than -6 kcal/mol, indicating significant binding interactions.
Among them, Lp_0018 exhibited the strongest binding with NaHS, which is consistent with the findings reported in the literature.
Furthermore, flexible docking results indicated a potential interaction between OppA and mucin, suggesting a possible risk of side effects.
Learn (L):
The strong and specific binding between OppA and NaHS was confirmed, supporting its role as a key adhesion factor.
Furthermore, the identified risk of Mucin binding highlights the necessity of incorporating a hypoxia-induced suicide switch into the engineered bacterial design.
These findings thereby provide a rationale for selecting specific OppA proteins (e.g., LP_0018) for subsequent overexpression.
Cycle2 Overexpression of Adhesion Factors
As the expression of adhesion factors is regulated by the Control Module, their construction and characterization are described in the corresponding section below.
Cycle3 Adhesin Expression and Optimization
Design (D):
Objective: To achieve specific and orthogonal adhesion in a multi-strain system using the Intercellular Adhesion Toolkit.
Strategy:
- Overexpress the Ag2 adhesin in Lactobacillus plantarum.
- Construct four adhesin pathways (Ag2, Ag3, Nb2, Nb3) in Escherichia coli.
Design an orthogonal validation experiment and an IPTG induction concentration optimization assay.
Build (B):
In L. plantarum: The plasmid pSIP403-LysM-Ag2 was constructed, electroporated into L. plantarum, and positive clones were screened.
In E. coli: Using the pDest backbone, plasmids pDest-LysM-Ag3, pDest-LysM-Nb3, pDest-LysM-Ag2, and pDest-LysM-Nb2 were assembled via seamless cloning.
These constructs were transformed into E. coli and screened on ampicillin-containing medium.
Test (T):
Optimal Induction Condition: A concentration gradient assay determined that the optimal IPTG concentration is 0.8 mM.
Orthogonality Test: The Nb3--Ag3 and Nb2--Ag2 pairs demonstrated the strongest specific binding, successfully validating orthogonality.
Effect of IPTG on Adhesion: Compared to the uninduced control, IPTG addition significantly enhanced adhesion.
Fluorescence Microscopy: Strains expressing RFP and GFP enabled clear visualization of enhanced adhesion.
Co-culture Experiment: In a mixed culture of E. coli and L. plantarum, the introduction of sppip and IPTG induction further strengthened the adhesion effect.
Learn (L):
The optimal working concentration of IPTG was confirmed to be 0.8 mM.
Orthogonality and enhanced adhesion were successfully validated, supporting the initial design rationale.
Results from fluorescence microscopy and OD600 measurements were consistent, reinforcing experimental reliability.
Co-culture results demonstrated that multi-strain interactions can be effectively controlled.
Future Optimization: Explore systems that do not require costly inducers and improve overall system stability.
Therapeutic Module
Therapeutic Module
Cycle1 L-Dopa production module
Design (D):
To establish L-DOPA production capability in Escherichia coli, we plan to implement metabolic engineering in this host. We aim to first verify its biosynthesis capacity and then regulate it via a control module. To this end, we initially cloned the hpaBC gene cluster into a pET-28a vector, constructing a T7 promoter-driven expression cassette (as shown in the figure below). The resulting recombinant plasmid was then introduced into E. coli to enable heterologous expression of 4-hydroxyphenylacetate 3-hydroxylase under induction conditions. Ultimately, the engineered strain can utilize endogenous L-tyrosine---generated through its own metabolic pathways---as the substrate for the catalytic production of L-DOPA.
Build (B):
The hpaBC gene cluster was assembled into the pET-28a plasmid using seamless cloning. The resulting recombinant plasmid was then transformed into Escherichia coli competent cells. Primary screening was performed on ampicillin-resistant plates, followed by further verification of positive clones via colony PCR. Ultimately, engineered strains harboring the correct recombinant plasmid were successfully obtained.
Test (T):
The verified engineered strain was activated and subsequently inoculated at a 1:100 ratio into 20 mL of LB medium for fermentation culture at 37°C and 220 rpm. After 6 hours of cultivation (corresponding to the logarithmic growth phase), isopropyl-β-D-thiogalactoside (IPTG) was added to the induction group to a final concentration of 1 mM to induce protein expression. Post-induction, cultivation continued until 32 hours. To monitor the kinetics of cell growth and product synthesis, samples were taken at regular intervals for OD600 measurement. Additionally, samples were collected every 8 hours, centrifuged, and appropriately processed. The supernatant was analyzed via high-performance liquid chromatography to quantify L-DOPA content, while the melanin content generated from L-DOPA oxidation was assessed by measuring the OD400 of the samples. The color changes observed during the fermentation process are shown in Figure 2, and detailed experimental results are provided in the Results section.
Learn (L):
We demonstrate the successful construction of an L-DOPA-producing E. coli strain. This strain operates via a heterologous pathway encoded by the hpaBC gene cluster, which centers on the 4-hydroxyphenylacetate 3-hydroxylase enzyme to drive the single-step conversion of L-tyrosine to L-DOPA.
Cycle2 Glutathione production module
Design (D):
To establish glutathione production capability in Escherichia coli, we plan to implement metabolic engineering in this host. We aim to first verify its biosynthetic capacity and subsequently regulate it via a control module. To this end, the gshAB gene cluster was inserted into the pET-28a vector, constructing a T7 promoter-driven expression unit, as illustrated in Figure 3. The resulting recombinant plasmid was then introduced into E. coli to enable heterologous expression of a bifunctional glutathione synthetase under induction conditions. Ultimately, the engineered strain can catalyze the condensation of glutamate, cysteine, and glycine, thereby achieving glutathione synthesis.
Build (B):
The gshAB gene cluster was assembled into the pET-28a plasmid using seamless cloning. The resulting recombinant plasmid was transformed into Escherichia coli competent cells. Primary screening was conducted on kanamycin-resistant plates, followed by further verification of positive clones via colony PCR. Ultimately, engineered strains harboring the correct recombinant plasmid were successfully obtained.
Test (T):
The verified engineered strain was activated and inoculated at a 1:100 ratio into 25 mL of LB medium for fermentation culture at 37°C and 220 rpm. After 6 hours of cultivation (corresponding to the logarithmic growth phase), IPTG was added to the induction group to a final concentration of 1 mM to induce protein expression. At 8 hours of cultivation, glutamate, cysteine, and glycine were supplemented to the amino acid supplementation group to a final concentration of 25 mM each. Post-induction, cultivation continued until 22 hours. To monitor the kinetics of cell growth and product synthesis, samples were taken at regular intervals for OD₆₀₀ measurement. During the later stages of fermentation, samples were collected every 8 hours, centrifuged, and appropriately processed. The supernatant was analyzed via high-performance liquid chromatography to quantify the contents of reduced glutathione (GSH) and oxidized glutathione (GSSG). Detailed experimental results are provided in the Results section.
Learn (L):
We demonstrate the successful construction of a glutathione-producing E. coli strain. This strain operates via a heterologous pathway encoded by the gshAB gene cluster, which centers on a bifunctional glutathione synthetase enzyme to drive the sequential one-step conversion of the three substrate amino acids into glutathione.
Control Module
Control Module
Version1 Communication based on AHLs
In our first-round design, we envisioned establishing bidirectional communication between Lactiplantibacillus plantarum and Escherichia coli through AHL signaling, so that each species could activate the functional genes of the other and thereby achieve coordinated regulation of the consortium.
As illustrated in the figure, we planned to introduce an exogenous RhlI synthase into L. plantarum to serve as the AHL sender module, while simultaneously incorporating LuxR and the Plux promoter as the AHL receiver module. The same design logic was applied to E. coli, enabling mutual signal exchange and synchronized gene expression across both strains.
Cycle 1 --- Testing the native Plux promoter in L. plantarum
Design (D):
To explore whether the native Plux promoter could function in Lactiplantibacillus plantarum, we constructed the plasmid pNZ8148-P23-LuxR-Plux-RFP. In this design, the P23 promoter constitutively expresses the LuxR protein. Upon binding to 3OC6-HSL, the LuxR--AHL complex interacts with the Lux box region of Plux, thereby activating the downstream mRFP1 reporter gene. This setup allows us to qualitatively and quantitatively measure promoter activity through fluorescence detection with a microplate reader.
Build (B):
The plasmid was first assembled in E. coli MC1061 and validated by colony PCR and sequencing.
The verified construct was then transformed into L. plantarum WCFS1, and positive colonies were confirmed by colony PCR.
Test (T):
We induced the L. plantarum culture at mid-log phase with 100 nM 3OC6-HSL at 30°C. After 12 hours, fluorescence intensity was measured using a microplate reader. The results are shown below.
Learn (L):
We observed that the native Plux promoter did not exhibit a detectable response in L. plantarum. Literature review suggested that this may be due to differences in host sigma factors, which affect promoter recognition and activity across species. Therefore, we concluded that host-adaptive promoter engineering would be required to achieve functional AHL responsiveness in L. plantarum.
Cycle 2 --- Adaptive modification of Plux in L. plantarum
Design (D):
From Cycle 1, we learned that the native Plux promoter did not function in L. plantarum, indicating the need for host-adaptive modification. In this iteration, we aimed to preserve the Lux box region while systematically replacing the −10 region, −35 region, and spacer sequences of Plux, thereby generating a panel of Plux variants with potentially improved compatibility.
Build (B):
Using seamless DNA assembly, we introduced targeted mutations based on the Cycle 1 construct pNZ8148-P23-LuxR-Plux-RFP. The plasmid was first assembled in E. coli MC1061 and validated by colony PCR and sequencing.
This yielded a series of variant plasmids:
- pNZ8148-P23-LuxR-Plux0101-RFP
- pNZ8148-P23-LuxR-Plux0201-RFP
- pNZ8148-P23-LuxR-Plux0210-RFP
- pNZ8148-P23-LuxR-Plux0300-RFP
- pNZ8148-P23-LuxR-Plux0220-RFP
Test (T):
The constructs were introduced into L. plantarum WCFS1, and positive colonies were confirmed by colony PCR.
We induced the L. plantarum culture at mid-log phase with 100 nM 3OC6-HSL at 30°C. After 12 hours, fluorescence intensity was measured using a microplate reader. The results are shown below.
The results are summarized below.
Learn (L):
Despite these modifications, none of the Plux variants showed significant promoter activity in L. plantarum. After discussion with our PI, we concluded that further large-scale or high-throughput promoter engineering would be time- and resource-intensive and still carry high uncertainty in outcome. Therefore, we decided to shift strategy: instead of forcing AHL-based communication in L. plantarum, we turned to literature and identified that this strain naturally relies on AIP-mediated quorum sensing (typical in Gram-positive bacteria). From this point, we planned to redesign our system so that L. plantarum would utilize AIP-based communication, while E. coli would be engineered to interface through AIP to achieve interspecies signaling.
Version2 Communication based on AHL and AIP
In this version of our design, we decided to retain the AHL-based communication from L. plantarum to E. coli, while enabling E. coli to communicate back with L. plantarum through AIP signaling.
Moving forward, we will focus on the engineering construction of the four core modules:
AHL receiver, AHL sender, AIP sender, AIP receiver
AHL sensor:
Cycle1:
Design (D):
To evaluate the activity of the quorum-sensing promoter Plux and determine its appropriate induction concentration, we constructed the plasmid pETDuet-1-J23110-LuxR-Plux-RFP. In this design, the J23110 promoter constitutively expresses LuxR. When LuxR binds to 3OC6-HSL, the complex interacts with the Lux box of Plux, thereby activating the downstream mRFP1 reporter gene. This system allowed us to monitor promoter activity both qualitatively and quantitatively using a microplate reader.
Build (B):
The functional fragment was synthesized by Tsingke, and the assembled plasmid was introduced into E. coli. Colony PCR confirmed the successful construction.
Test (T):
Fluorescence was measured after induction with 3OC6-HSL. However, no detectable fluorescence was observed under the tested conditions.
Learn (L):
We investigated possible causes, including plasmid instability and inducer degradation, but these did not explain the failure. During discussions at the Conference of China iGEMer Community (CCiC) with Xiaoyao Zhou from iGEM WHU, we learned that both 3OC6-HSL and LuxR require sufficient intracellular concentrations to activate Plux. Since J23110 is a relatively weak promoter, it was suggested that we replace it with the stronger J23100 promoter to achieve adequate LuxR expression.
Cycle2:
Design (D):
In the previous design cycle, we observed that the J23110 promoter exhibited relatively weak activity, necessitating a stronger promoter for the expression of the LuxR protein. Consequently, the constitutive J23100 promoter was adopted to drive LuxR expression. When LuxR binds to the autoinducer 3OC6-HSL, the resulting complex activates transcription by binding to the Lux Box of the Plux promoter, thereby initiating expression of the mRFP1 reporter gene. This design enables qualitative and quantitative detection of fluorescence using a microplate reader.
Build (B):
Building upon the Cycle 1 plasmid pET-J23110-LuxR-Plux-RFP, we successfully replaced the promoter. The correct construction of the resulting plasmid, pET-J23100-LuxR-Plux-RFP, was confirmed by colony PCR and sequencing.
Test (T):
Fluorescence intensity following induction was measured using a microplate reader, and a dose-response assay was conducted across a range of concentrations. Detailed results are presented in the Results section.
Learn (L):
Our findings confirm the hypothesis that a sufficient intracellular concentration of LuxR protein is required for its binding with 3OC6-HSL and subsequent activation of transcription from the Plux promoter. The J23110 promoter demonstrated insufficient strength for this purpose, whereas the J23100 promoter effectively met the requirement. Furthermore, the Plux promoter exhibited clear concentration-dependent inducibility, with its activity increasing in response to higher concentrations of 3OC6-HSL.
This observation also led us to reconsider whether the previously observed non-responsiveness of Plux in Lactobacillus plantarum could similarly be attributed to inadequate LuxR expression. However, after reviewing the literature and consulting with our advisor, we learned that P23 is already considered a strong promoter in lactic acid bacteria, making insufficient protein expression an unlikely explanation. The earlier lack of response is more likely due to promoter incompatibility in that specific host context.
AHL producer:
Cycle1:
Design (D):
To enable the synthesis of 3OC6-HSL in Lactiplantibacillus plantarum WCFS1 for the subsequent activation of the Plux promoter in Escherichia coli, we constructed the plasmid pNZ8148-P23-LuxI. The core objective of this design is to achieve stable production of 3OC6-HSL in L. plantarum. The vector pNZ8148 is a commonly used expression plasmid for lactic acid bacteria, featuring a lactococcal replicon suitable for this host. In our design, a codon-optimized LuxI gene was inserted downstream of the strong P23 promoter to enable sustained, high-level constitutive expression.
Build (B):
The functional P23-LuxI sequence was synthesized by Tsingke Biotechnology Co. Following its acquisition, we first constructed the pNZ8148-P23-RFP plasmid in E. coli MC1061, with correct assembly confirmed by colony PCR and sequencing.
The verified plasmid was then electroporated into Lactiplantibacillus plantarum, yielding correct transformants identified through colony PCR.
Test (T):
The 12-hour cultured Lactobacillus plantarum was processed and co-cultured with Escherichia coli harboring the plasmid pET-J23100-LuxR-Plux-RFP. After 6 hours of induction, fluorescence was quantified using a microplate reader and visualized by fluorescence microscopy. Detailed results are presented in the Results section.
Learn (L):
It can be concluded that the engineered AHL-sending module in Lactiplantibacillus plantarum was capable of eliciting a response in the engineered Escherichia coli receiver strain, though the induced signal intensity fell short of that achieved by the external addition of 100 nM 3OC6-HSL. To enhance the system performance, the following optimization strategies will be implemented: (1) Optimizing the expression level of LuxI or improving precursor substrate availability in L. plantarum to enhance AHL synthesis. (2) Modifying the RBS of the RFP gene or employing a stronger Plux promoter variant to amplify the response intensity.
AIP Receiver:
Design (D):
For the AIP receiver module in L. plantarum, we selected the sppip AIP signal and co-expressed the sppK and sppR genes to form a membrane-associated two-component sensor system. Upon activation by AIP binding, the sensor kinase SppK undergoes autophosphorylation at a conserved histidine residue. The phosphoryl group is then transferred to the cognate response regulator SppR in the cytoplasm. Phosphorylated SppR binds specifically to the Pspp promoter region, thereby enhancing the transcription of downstream genes. To quantitatively characterize this system, we used mRFP1 as a reporter gene and performed detailed measurements of its expression.
Build (B):
The plasmid pSIP403-Pspp-RFP was constructed using the pSIP403 backbone, and its correct assembly was verified by colony PCR and sequencing.
The confirmed plasmid was subsequently electroporated into Lactiplantibacillus plantarum WCFS1, with successful transformation validated again by colony PCR.
Test (T):
The temporal and dose-response profiles of the spp system were characterized, with detailed data presented in the Results section.
Learn (L):
The robust performance of the spp system in L. plantarum validates it as a reliable and effective receiver component.
AIP Sender:
Design (D):
We designed an AIP secretion system in E. coli utilizing its type I secretion pathway:
First, the plasmid pRSFDuet-T7-CvaAB was constructed to express the CvaA and CvaB proteins, which assemble with the endogenous TolC protein to form a transmembrane complex.
Then, the sppip sequence was fused to the CvaC15 signal peptide, yielding the plasmid pBAD24-CvaC15-SppIP.
Upon induction with arabinose and IPTG, the CvaC15 signal peptide directs the SppIP precursor to the CvaAB complex. Following cleavage by CvaB, mature SppIP is transported extracellularly through the channel formed by CvaA and TolC.
Build (B):
We constructed two plasmids: pRSFDuet-T7-CvaAB for expressing CvaA and CvaB, and pBAD24-CvaC15-SppIP encoding the fusion of the CvaC15 signal peptide with SppIP. Both plasmids were co-transformed into E. coli, and successful strain construction was verified by colony PCR.
Test (T):
Subsequently, the co-transformed E. coli culture was induced and then subjected to centrifugation. The supernatant was collected, filter-sterilized, and added to Lactiplantibacillus plantarum harboring the reporter plasmid pSIP403-PsppA-RFP to test whether the AIP signal produced by the sender module could be detected by the receiver module. Detailed results are presented in the Results section.
Learn (L):
The strategy of utilizing the type I secretion system in E. coli for SppIP secretion proved effective, generating a sufficient quantity of the AIP to elicit a robust response in Lactiplantibacillus plantarum.
Application
HpaBC Production Regulation
Design (D):
We designed the plasmid pET-J23100-LuxR-Plux-HpaBC to test the control of L-DOPA synthesis by exogenous and endogenous 3OC6-HSL signals through the Plux-HpaBC module in E. coli. In this construct, the constitutive J23100 promoter ensures sustained LuxR expression, maintaining adequate intracellular regulator levels. The resulting LuxR-3OC6-HSL complex binds specifically to the Lux Box of the Plux promoter, activating transcription of the downstream HpaBC enzyme system. This enables the oxidation of tyrosine to L-DOPA, thereby establishing a direct link between quorum-sensing signals and functional metabolic output.
Build (B):
The plasmid was successfully assembled, with correct construction verified by colony PCR and DNA sequencing.
Test (T):
We performed exogenous and endogenous induction experiments as follows:
cultures of E. coli harboring pET-J23100-LuxR-Plux-HpaBC were supplemented with either 100 nM 3OC6-HSL (exogenous) or 500 μL of supernatant from Lactiplantibacillus plantarum containing pNZ8148-P23-LuxI (endogenous). Following 24 hours of incubation, the bacterial cultures were collected for subsequent measurements. Detailed results are provided in the Results section.
Learn (L):
The experimental results demonstrate that the Plux-HpaBC module can be successfully activated under both exogenous and endogenous induction conditions, exhibiting functional output (L-DOPA production). However, the signal strength and product titer under endogenous induction were lower than those achieved with the addition of 100 nM AHL. This suggests the following potential directions for future optimization:
- Enhance AHL synthesis in L. plantarum by optimizing LuxI expression levels or improving precursor substrate supply.
- Increase the response intensity by modifying the RBS of HpaBC or employing stronger Plux promoter variants.
- Boost the final product titer through metabolic engineering strategies to enhance the host's supply of the L-DOPA precursor, L-tyrosine.
Regulation of the Lp_0018 Adhesin
Design (D):
In the adhesion module, we aim to enhance the colonization capacity of Lactiplantibacillus plantarum on the olfactory epithelium by overexpressing the OppA protein variant LP_0018. To achieve this, we constructed an overexpression vector in which the expression of LP_0018 is controlled by the PsppA promoter.
Build (B):
Based on the existing pSIP403 plasmid (containing the PsppA promoter and a dual-gene expression framework), pSIP403-LP_0018 was assembled using seamless cloning. The constructed plasmid was first transformed into Escherichia coli for propagation, and subsequently electroporated into Lactiplantibacillus plantarum. Positive transformants were selected on erythromycin-containing medium.
Test (T):
An engineered strain of Lactiplantibacillus plantarum stably maintaining Lp_0018 (OppA) was successfully obtained. The successful plasmid integration and functional expression in L. plantarum were verified, with detailed results presented in the Results section.
Learn (L):
The Pspp induction system was successfully validated and can be utilized to control the expression of the Lp_0018 (OppA) protein.
Safety Module
Safety Module
Construction of a Hypoxia-Induced Suicide Module
Cycle 1
Design (D):
Given the significant risk of engineered bacteria---designed for colonization on the olfactory epithelium---detaching and translocating to the intestinal tract, we designed a hypoxia-responsive suicide element based on comparative analysis of oxygen concentrations in the nasal cavity and gut. This element consists of a hypoxia-inducible promoter (e.g., phyb) and the toxin gene MazF. We initially constructed this circuit in Escherichia coli using the pETDuet plasmid, into which an expression cassette containing the phyb promoter and MazF gene was inserted.
Build (B):
The plasmid pETDuet-phyb-MazF was constructed using the laboratory's existing pETDuet backbone and gene sequences for phyb and MazF obtained from the iGEM parts registry, with assembly accomplished via homologous recombination.
Test (T):
The constructed pETDuet-phyb-MazF plasmid was transformed into E. coli, and transformants were selected on ampicillin-resistant plates. Positive clones were subcultured and streaked on fresh plates, followed by static incubation at 37°C for 48 hours under both aerobic and anaerobic conditions. Anaerobic conditions were generated using commercial anaerobic gas packs and sachets. Three parallel experimental replicates were included for each condition. Detailed results are provided in the Results section.
Learn (L):
The experimental results demonstrate that, compared to cultivation under aerobic conditions, our engineered hypoxia-responsive suicide element significantly retarded the growth of E. coli under anaerobic conditions, thereby validating the design objective.
Cycle 2
Design (D):
Based on the initial tests from the first cycle, we have preliminarily validated the functionality of the designed hypoxia-responsive suicide element in E. coli. The next step involves verifying its feasibility in Lactiplantibacillus plantarum (Lp). To this end, we selected the pNZ8148 plasmid and inserted an expression cassette containing the hypoxia-inducible promoter phyb and the toxin gene MazF.
Build (B):
Using the laboratory's existing pNZ8148 backbone and amplifying the phyb-MazF fragment from the previously constructed pET-phyb-MazF plasmid, we assembled the pNZ8148-phyb-MazF plasmid via homologous recombination.
Test (T):
The constructed plasmid was first transformed into E. coli and screened on ampicillin-resistant plates to isolate correct transformants. Subsequently, the plasmid was electroporated into L. plantarum.
Learn (L):
During validation, we observed severely impaired growth of L. plantarum after the introduction of the suicide element, suggesting that it may impose a substantial metabolic burden on the host. For future work, alternative suicide elements should be designed.
Construction of a Thermosensitive Suicide Module
Cycle 1
Design (D):
Considering that the nasal cavity is an open environment and our engineered bacteria designed for olfactory epithelium colonization may detach, there is a risk of the bacteria escaping to the external environment. Based on the idealized assumption that the nasal cavity maintains a constant temperature of 37°C while the external environment remains at 30°C or below, we designed a thermosensitive suicide element composed of the temperature-sensitive Cl857 protein and the toxin protein MazF.
To preliminarily validate the functionality of the Cl857 protein, we selected the pETDuet plasmid and inserted an expression cassette containing Cl857 and the red fluorescent protein (RFP) gene. This construct was initially tested in E. coli.
Build (B):
Using the laboratory's existing pETDuet backbone and sequences for Cl857 and other required genes obtained from the iGEM parts registry, we assembled the plasmid pETDuet-J23110-Cl857-pR-phIF-pPhIF-RFP via homologous recombination.
Test (T):
The constructed plasmid was transformed into E. coli, and transformants were selected on ampicillin-resistant plates. Positive clones were subcultured and subjected to the following experiments: Liquid cultures of the transformants were grown at 30°C and 37°C for 16 hours. Subsequently, the cultures were subcultured at a 1:200 inoculation ratio and incubated for 24 hours, after which fluorescence intensity and OD600 were measured.
Learn (L):
No significant difference in specific fluorescence intensity was observed between the engineered bacteria cultured at 30°C and 37°C, indicating that the results did not meet expectations.
Comparison with the original literature revealed two key differences:
- Promoter type for Cl857: We used the constitutive promoter J23110, while the original study employed the pRM promoter, which is positively regulated by cI857.
- Plasmid configuration: We integrated all genetic elements into a single plasmid, whereas the original study distributed the Cl857 expression circuit and the downstream phIF/pPhIF modules across two compatible plasmids.
The pRM promoter is inherently weak and highly dependent on Cl857 protein levels. When cI857 accumulates sufficiently, it forms dimers that bind to the OR2 operator site near pRM, physically interacting with RNA polymerase to enhance transcription of Cl857---creating a positive feedback loop. In contrast, the constitutive J23110 promoter drives transcription at a constant rate, resulting in lower regulatory precision.
Additionally, the dual-plasmid system used in the literature, though imposing a higher metabolic burden and slower growth on the host, facilitated better product expression.
These differences may account for the limited efficacy observed in our current design. Corresponding adjustments will be made in the next cycle.
Cycle 2
Design (D):
Based on the outcomes of the previous DBTL cycle, we constructed a dual-plasmid system for this round: one plasmid, pETDuet, carrying the expression cassette with *pRM-Cl857*; the other, pJUMP, carrying the pR-phIF-pPhIF-RFP cassette. The system was validated in E. coli.
Build (B):
Using homologous recombination, we successfully constructed the two plasmids: pETDuet-pRM-Cl857 and pJUMP-pR-phIF-pPhIF-RFP.
Test (T):
The constructed plasmids were co-transformed into E. coli, and positive transformants were selected on double-antibiotic plates containing ampicillin and kanamycin.
Validated transformants were cultured in liquid medium at 30°C and 37°C for 16 hours, followed by subculturing at a 1:200 inoculation ratio for another 24 hours. Fluorescence intensity and OD600 were measured for each condition.
Learn (L):
Our experimental results preliminarily validated the functionality of the thermosensitive regulatory system. The next step will involve replacing the fluorescent protein gene with the toxin gene MazF for subsequent suicide module verification.
Cycle 3
Design (D):
We continued to employ the dual-plasmid system, replacing the red fluorescent protein (RFP) coding sequence with that of the toxin protein MazF, and validated the construct in Escherichia coli.
Build (B):
Using homologous recombination, we successfully constructed the plasmids pETDuet-pRM-Cl857 and pJUMP-pR-phIF-pPhIF-MazF.
Test (T):
The two plasmids were co-transformed into E. coli, and positive transformants were selected on double-antibiotic plates containing ampicillin and kanamycin.
Learn (L):
Experimental results indicated a suboptimal response of E. coli to the constructed thermosensitive suicide system, as a considerable number of engineered cells remained viable at 30°C. Subsequent efforts should focus on enhancing the expression level of the toxin protein to achieve the desired growth inhibition effect.
Emergency Containment Module Construction
Cycle 1
Design (D):
To address potential biosafety risks arising from unexpected incidents, we designed an emergency containment system for rapid system shutdown in Escherichia coli Nissle 1917. The initial pathway was constructed in E. coli using the pETDuet plasmid, into which an expression cassette containing the L-arabinose-inducible pBAD promoter and the toxin gene MazF was inserted.
Build (B):
The plasmid pETDuet-pBAD-MazF was successfully assembled via homologous recombination.
Test (T):
The constructed plasmid was transformed into E. coli, and positive transformants were selected on chloramphenicol-resistant plates. Growth curves were subsequently determined for validation.
Learn (L):
Experimental results demonstrated marked growth retardation in the engineered E. coli upon L-arabinose induction, confirming the functional success of the emergency containment module.
Cycle 2
Design (D):
Following the successful validation of the L-arabinose-inducible suicide system in E. coli, we subsequently designed a nisin-inducible suicide system tailored for Lactiplantibacillus plantarum. We selected the pNZ8148 plasmid and inserted an expression cassette consisting of the nisin-inducible promoter PnisA and the toxin gene MazF.
Build (B):
The plasmid pNZ8148-PnisA-MazF was constructed via homologous recombination.
Test (T):
The recombinant plasmid was transformed into L. plantarum, and positive transformants were selected on chloramphenicol-containing plates. Growth curves were subsequently determined to assess system functionality.
Learn (L):
Experimental results showed that nisin induction significantly slowed the growth of the engineered L. plantarum, confirming the effectiveness of the constructed genetic module. With this step, we have successfully built and validated inducible suicide systems for both E. coli and L. plantarum, establishing an emergency containment mechanism that substantially reduces potential biosafety risks in case of unexpected incidents.
Fluorescent characterization of microbiota
Fluorescent characterization of microbiota:
Cycle 1
Design (D):
In order to be able to characterise the coexistence of EcN and Lp more intuitively, we intend to construct four fluorescent plasmids: pET-J23119-YFP, pET-PBAD-YFP, pNZ8148-P23-RFP, and pSIP403-Pspp-RFP, as shown in Fig. 1, and introduce the yellow fluorescent plasmid into EcN, and introduce the red fluorescent plasmid into Lp, by observing the fluorescence of the bacteriophage to characterise the coexistence of the two colonies.
Build (B):
The fluorescent protein gene was constructed into the commonly used plasmid by seamless cloning, and the resulting recombinant plasmid was transformed into EcN and Lp. Finally, the engineered strains containing the correct recombinant plasmids were successfully obtained by preliminary screening with the corresponding resistance plates and further verifying the positive clones by colony PCR.
Test (T):
We tested the fluorescence intensity of the constitutive fluorescent protein-expressing strains, and also tested the fluorescence intensity of the inducible fluorescent protein-expressing strains under different culture conditions. The fluorescence intensity of EcN, which constitutively expresses yellow fluorescent protein, was significant, and the fluorescence intensity of inducible expression was not as significant as that of the constitutive type; the fluorescence intensity of Lp, which constitutively expresses red fluorescent protein, was significant, but not as significant as that of the inducible type, and the fluorescence intensity was not as significant as that of EcN due to the fact that Lp is a gram-positive bacterium with a thicker cell wall. In addition, we also came up with suitable culture conditions for our experiments, and at the same time, we mapped out the suitable medium conditions for co-culturing EcN and Lp, and adopted them in our subsequent experiments.
Learn (L):
We tested the expression intensity of a number of fluorescent protein-related components in the iGEM kit and, in the course of our experiments, discovered the influence of cell wall thickness in Gram-positive bacteria on the observation of the fluorescence intensity of their fluorescent proteins.
Cycle 2
Design (D):
In order to quantitatively analyse the subpopulations of bacteria expressing different fluorescent proteins in the co-culture system, we planned to use flow cytometry. By performing dual-channel detection of yellow fluorescence and red fluorescence on the bacterial population, we can accurately count and characterise the number and proportion of YFP- and RFP-expressing bacteria in the population at different times of the co-culture.
Build (B):
For initial co-culture pre-experiments, we explicitly set up the following three groups: blank control (unengineered wild-type strains), single-positive control (strains expressing only a single fluorescent protein, respectively), and experimental group (co-culture system).
Test (T):
However, preliminary flow cytometry results (Fig. 2) showed poor fluorescence fractionation of cell populations. The problem was mainly attributed to two technical limitations: first, the emission spectral range of the selected EYFP protein was too broad, which led to its severe spectral crosstalk among detection channels; second, the flow cytometer was equipped with a laser whose excitation wavelength did not exactly match with the optimal absorption wavelength of the mRFP protein that we used, which led to the low excitation efficiency.
Learn (L):
In response to the fluorescence detection problems in the pre-experiment, we reviewed and optimised the experimental design. Firstly, we decided to use a protein with a narrower emission spectrum and a better match with the laser wavelength of the instrument in order to minimise spectral crosstalk and improve excitation efficiency. Based on this, we chose surperfolder GFP (sfGFP), which has better spectral characteristics, as an alternative, and plan to supplement it with more intuitive characterisation methods for validation.
Cycle 3
Design (D):
In the next step, we will construct the pJUMP-J23100-sfGFP plasmid (see Figure 3) and transform EcN, and switch to fluorescence microscopy to characterise the spatio-temporal dynamics of the bacterial population in the co-culture system by direct observation of the two-colour fluorescence distribution.
Build (B):
The GFP fluorescent protein gene was constructed into a common plasmid by seamless cloning, and the resulting recombinant plasmid was transformed into EcN. Finally, the engineered strain containing the correct recombinant plasmid was successfully obtained by initial screening through canna-resistant plates and further verification of the positive clones by colony PCR.
Test (T):
After the activation of the correctly validated engineering strains, they were transferred to 20 mL of LB medium at an inoculum of 1:100, and the fermentation culture was carried out at 37°C and 220 rpm. To monitor the growth of the bacteria, samples were taken at regular intervals to determine the OD600 value; meanwhile, samples were taken periodically at predetermined time points during the incubation process. The obtained samples were washed with PBS buffer, resuspended and fixed on slides for fluorescence microscopy. Detailed experimental results are shown in Results.
Learn (L):
Based on the experimental results, we confirmed that sfGFP and mRFP are suitable for the two-colour fluorescent labelling system in this study. In addition, EcN and Lp were confirmed to be able to coexist stably for a long period of time in the specific medium obtained from the exploration, which meets the basic requirements for co-culture experiments.