Survival Module

In the Survival module, we introduced the salt-tolerant genes, gspM and echM to arm our biofilm with the necessary adaptability against inhospitable conditions.

Bacterial growth curve analysis

We aim to quantitatively compare the growth curves of the engineered bacteria and wild-type strains carrying the [echM-gspM]-pET28a(+) plasmid under high-salt (600 mM NaCl) and salt-free conditions, in order to validate the function of the salt-tolerance gene. The OD600 values of each strain group in different media were measured at regular intervals from 0 to 27 hours, and a Logistic model was applied for curve fitting and analysis.

We found that the engineered bacteria in 600mM NaCl medium showed normal growth conditions similar to those in LB medium without NaCl, while the growth of Escherichia coli in the negative control group was significantly inhibited in a high-salt environment. These results proves that salt-tolerant genes do indeed make engineered bacteria more adaptable to high-salt environments. However, further analysis reveals that the salt-free LB medium is slightly more suitable for the growth of engineered bacteria compared to the 600mM NaCl salt-containing LB medium. This indicates that the engineered bacteria are still to some extent inhibited by the stress of a high-salt environment.

Light suicide module

By fusing the LOVdeg tag to TetR, the engineered  E. coli gains the ability to sense blue light (450–490 nm) and thereby regulate activation of the suicide mechanism downstream of the Tet operon. Consequently, upon entering open marine environments and encountering blue light, the engineered bacteria will autonomously initiate their suicide program, thereby mitigating the risk of engineered bacteria contamination.

Fluorescence quantification system

The fluorescence quantification system was established to simulate experimental conditions and qualitatively detect three key indicators: the proper characterization of the TetR-LOVdeg protein, the normal inducible expression of sfGFP of gene circuits, and the leakage of the light-induced suicide system. Additionally,  we used this system to semi-quantitatively determine the leakage level of gene circuits via fluorescence quantification.

Protein expression of sfGFP and TetR-LOVdeg

Bacteria were lysed under both dark and light conditions, followed by protein extraction and subsequent Western blot (WB) assay.

Normal expression of sfGFP and TetR-LOVdeg monomer (Dimer would be degraded by SDS treatment due to non-covalent bond connection.) was detected in the supernatant. Furthermore, following SDS treatment, the LOVdeg moiety of the TetR-LOVdeg fusion protein—harboring a 6×His-tag—was successfully detected. Notably, besides the band corresponding to the target protein, we always observed additional "non-specific bands" even after optimizing the blocking step to its optimal state. We propose that this phenomenon arises from the successful unfolding of the Jα domain in LOVdeg: this unfolding exposes the EAA-containing SsrA tag, which subsequently mediates the recruitment of endogenous ClpX and ClpA enzymes, thereby triggering N-terminal degradation.

Leakage rate measurement

Bacterial suspension was diluted and uniformly spread on LK plates; following a certain period of light-protected incubation, color-based colony screening was performed using ImageJ software, enabling the extraction and counting of green colonies.

In this experiment, the leakage rate of the fluorescence quantification system was determined to be 84.92% for the BL21(DE3) strain and 82.51% for the JM109(DE3) strain. It was indeed observed that the system exhibits severe leakage; yet we remain undeterred, as a high leakage rate is not equivalent to a high leakage magnitude and does not necessarily imply constitutive overexpression of the toxic MazF protein in the light-induced suicide system, nor does it indicate a failure to exert the intended light-induced lethal function.

Suicide system (L.U.C.I.A.)

A suicide system was established, which serves as the light-induced suicide module we aim to ultimately implement. Additionally, we qualitatively detected the proper characterization of the TetR-LOVdeg protein in the gene circuit and the normal inducible expression of the toxic MazF protein in the same circuit. Meanwhile, the suicide rate was qualitatively reflected via bacterial liquid dilution and spreading, and growth curve determination was used to assess whether the strains failed to grow as expected under inducing conditions.

Protein expression of MazF and TetR-LOVdeg

Bacteria were lysed under both dark and light conditions, followed by protein extraction and subsequent Western blot (WB) assay.

We detected sfGFP and TetR-LOVdeg at normal levels in the supernatant. Furthermore, after SDS treatment, the LOVdeg moiety of the TetR-LOVdeg fusion protein—carrying a 6×His-tag—was successfully detected. In the light-treated experimental group, a significant amount of the toxic protein MazF was synthesized, confirming the correctness of the gene pathway. However, in lane 5 (JM109(DE3) under dark conditions), a small amount of MazF was also observed, indicating that the gene circuit still exhibits leakage. Meanwhile, we clearly observed that the target protein content in the pellet fraction was significantly higher than in the supernatant fraction. We therefore speculate that the leakage of the gene circuit may be related to the improper folding of the TetR-LOVdeg fusion protein.

Suicide strain growth curve measurement

We measured the mazF genotype of E.coli JM109(DE3) growth curve under lighting conditions

It was observed that the growth of the JM109(DE3) strain was indeed severely affected by the toxic expression of MazF: growth was temporarily arrested at one stage, yet it eventually resumed normal growth and failed to achieve the desired permanent growth arrest.

Programmable biofilm

Ag-Nb System

To enable functional cooperation, we engineered E. coli to express outer membrane-anchored nanobody–antigen (Nb–Ag) pairs. This modification not only facilitates spontaneous cell adhesion but also allows the generation of programmable assembly patterns within the living biofilm.

To verify that biofilm formation is mediated by the intimin–antigen/nanobody (intimin–Ag3/Nb3) interaction, we performed both aggregation assays and fluorescence microscopy imaging to confirm its capacity to induce biofilm formation.

Escherichia coli self aggregation assay

We measured the OD600 of the supernatant at 0, 2,4, 6 hours to reflect the bacteria quantity remaining in the supernatant.

We observed that at 6 hours, in the IPTG-induced E. coli samples, bacteria percentage remaining in the supernatant was significantly lower compared to the uninduced samples. This indicates that the intimin-Ag/Nb pairs can effectively promote the binding between E. coli.

Fluorescence microscopy imaging

To make the experimental results more intuitive, we respectively used mCherry and sfGFP to characterize the expressions of Ag and Nb.

1. Insert the intimin-Ag3 fusion protein and characterize it with mCherr
2. Insert the intimin-Nb3 fusion protein and characterize it with sfGFP

In the culture medium, the alternating distribution of red- and green-labeled  Escherichia coli is clearly observable. Additionally, large bacterial clusters are present, within which individual red and green bacterial cells are difficult to distinguish.

Biofilm Enhancement

Verification of Ag43-OmpA expression

T hrough literature research, we identified the key proteins Ag43 and OmpA that influence biofilm formation. Ag43 specifically mediates cell aggregation, adhesion, and biofilm development; OmpA, as a key protein for bacterial colonization and biofilm formation, significantly promotes biofilm formation on hydrophobic surfaces.

The wet experiment verified the expression of the target protein by Western blot and detected the biofilm formation ability by crystal violet staining.

Exploration of the optimal induction concentration of IPTG

We utilize lactose operons to regulate the expression of these two exogenous genes. Determining the optimal IPTG induction concentration ensures sufficient expression of the target proteins while minimizing toxicity to host cells. To reduce experimental workload, we referred to the study by XJTLU-CHINA (2023) and employed a predictive model to estimate the optimal IPTG induction concentration range, thereby guiding experimental design and improving efficiency.

During the experiment of inducing protein expression, we used the prediction graph of the optimal concentration of mRNA synthesis induced by IPTG in cycle 1 and conducted gradient attempts near the 400μM IPTG concentration. The final experiment found that the optimal induction concentration of IPTG was approximately 600μM.

Crystal violet staining of biofilm

Crystal violet staining is a foundational, widely used method for visualizing and semi-quantifying microbial biofilms. Its principle relies on electrostatic binding between the cationic crystal violet dye and anionic components of biofilms (e.g., extracellular polysaccharides, peptidoglycan) in the extracellular polymeric substance (EPS) matrix, resulting in visible purple staining; bound dye can also be solubilized with acetic acid for absorbance-based biomass quantification. This method is favored for its simplicity, cost-effectiveness, and utility as a preliminary screening tool for biofilm research.

It was found that the biofilm formation ability of the engineered bacteria in the experimental group introduced with [ag43-ompA]-pET28a(+) plasmid was significantly enhanced compared with that of the control group introduced with pET28a(+) empty plasmid.

Arginine-Inducible Sedimentation

We aim to fuse arginine-rich oligopeptides to the common  E. coli extracellular platform CsgA, thereby inducing diverse modes of spore settlement and enhancing the efficiency and stability of spore adhesion through a biologically mediated mechanism.

Protein expression of CsgA-GSGGSG-RYRYRYR

We introduced a recombinant plasmid encoding the CsgA–arginine oligopeptide fusion gene into Escherichia coli.

Western blot analysis was performed to verify expression of the target protein, while a spore release assay was used to compare the efficacy of the sedimentation function. We tested gradient induction with IPTG concentrations ranging from 0.6 to 1.2 mM and varied induction temperatures (16 °C, 30 °C, and 37 °C). However, the target protein was not detected in either the supernatant or the pellet following cell lysis.

Sanger sequencing of the [CsgA-GSGGSG-RYRYRYR]-pET28a(+) plasmid

To identify the cause of failed protein expression, we performed Ni-NTA column purification in parallel with Sanger sequencing of the original recombinant plasmid and the mini-prep plasmid from the engineered E. coli strain. This approach enabled us to distinguish between expression-related issues and genetic problems such as mutations or rearrangements in the fusion gene.

Based on the Sanger sequencing results, we identified the presence of a premature stop codon within the coding sequence of the gene in JM109(DE3), which truncates the polypeptide chain and prevents full-length protein synthesis. This finding directly explains the lack of detectable protein expression in our previous experiments. To address this issue, we plan to retransformation the plasmid, ensuring the integrity of the open reading frame and restoring the intended protein expression.

Proof of the concept

Ulva spores diffusion

Spore release and cultivation of Ulva prolifera  is also a critical component of the wet experiments. Using a constant-temperature light incubator, we successfully cultured Ulva prolifera from nutrient-supplemented spore solution, obtaining both its juvenile and adult stages. Furthermore, via stimulation with fresh seawater, a large number of motile spores were successfully released from sporophyte-derived Ulva prolifera fragments, which were then used for the spore attachment rate assay.

Determination of adsorption rate of Ulva spores

To verify the ability of engineered bacteria to adsorb spores, we conducted spore adsorption experiments. Currently, we selected BL21(DE3) engineered bacteria for experimental verification. After spore release, the spore solution was diluted to 10⁷ spores/ml. Subsequently, 15 ml of the diluted spore solution was added to Petri dishes containing engineered bacterial biofilm in LK (LB + kanamycin) medium, dishes containing only LK medium, and blank dishes without any medium. In the experiment, we periodically (0 min, 5 min, 30 min,60 min, 120 min, 240 min, 480 min) used a hemocytometer to observe the spore concentration in the supernatant, taking the supernatant concentration at 0 min as the total concentration, and calculated the adsorption rate using the formula (total concentration - supernatant concentration) / total concentration. The results are shown in the figure below, indicating that the engineered bacteria have a significant adsorption effect on spores.