iGEM 2025 — Team scu-china

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Notebook

## A detailed notebook is provided.

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Engineering

### Contents

[1. *sinR*/*tasA* Knock-out](#sinR-tasA-knock-out)

[2. Adhesion Module](#adhesion-module)

[2.1 Plasmid Selection](#plasmid-selection)
[2.2 Fluorescence Detection](#protein-expression)
[2.3 Adhesion Force Test](#adhesion-force)

[3. Function Test Module](#absorption-module)
[4. Safety Module](#suicide-system)

## 1. *sinR*/*tasA* Knock-out

#### Cycle I - Design

In order to knock out the original *sinR*-*tasA* gene in the genome of *Bacillus subtilis*, we applied CRISPR-Cas9 techniques. After designing and synthesizing the corresponding sgRNA, we managed its ligation to a linearized vector, and then linked the plasmid with the upstream and downstream fragments of the *sinR*-*tasA* gene by homologous recombination, in the purpose of constructing two knockout plasmids that met the goal.

#### Cycle I - Build

We obtained CDS sequences of *tasA* and *sinR* genes, and designed and selected two sgRNAs using the online platform CHOPCHOP. The single stranded oligonucleotides synthesized by the company (including the protruding end and 20 nt target specific sequence) were annealed to form dsDNA, which was then ligated to the pJOE8999 plasmid utilizing Golden Gate Assembly.

#### Cycle I - Test

We extracted successfully ligated pJOE-sg*tasA*-donor plasmids and transferred them into the chemocompetent *Bacillus subtilis* 168 cells. After spreading and culturing, we performed colony PCR and found that all of them were positive. Due to the abnormal positive rate, we suspected that the culture of *Bacillus subtilis* was contaminated with Escherichia coli DH5α containing the target plasmid. Therefore, we re PCR using primers on the *Bacillus subtilis* genome, and only one lane showed a positive result.

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| ![5](https://static.igem.wiki/teams/5810/engineering/knockout/fig1.webp) |

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| ![5](https://static.igem.wiki/teams/5810/engineering/knockout/fig2.webp) |

Fig1-1,2 Colony PCR validation transfer results, M: DL5000 Marker. Target gene produced an amplicon of ~1,000 bp

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| ![5](https://static.igem.wiki/teams/5810/engineering/knockout/fig3.avif) |

Fig1-3 Colony PCR validation after primer replacement, M: DL2000 Marker. Target gene produced an amplicon of ~1,000 bp

#### Cycle I - Learn

Based on the two validation results of the transformed *Bacillus subtilis*, we believed that the competent cells of *Bacillus subtilis* were likely previously contaminated by pJOE-sg*tasA*-donor plasmid positive Escherichia coli DH5α during the cultivation process. We planned to re-extract the plasmid and prepare new competent cells for transformation. Afterwards, the plasmid extracted from E.coli were transformed into *Bacillus subtilis*. In addition to verifying whether the transformed strain possessed the target segment, it was also necessary to verify whether the strain was *Bacillus subtilis*.

#### Cycle II - Design

In order to solve the problem of *E. coli*contamination and obtain the KO *Bacillus subtilis* strains, we re-extracted the knockout plasmid and transferred it into *Bacillus subtilis* for further validation.

#### Cycle II - Build

Extract the pJOE8999-sg*tasA*-donor plasmid and transfer it into the chemocompetent *Bacillus subtilis* 6051. Two different sets of primers were applied to PCR of positive colonies: the first set used primers that amplified the upstream fragment of *sinR*-*tasA* to test whether the *tasA* and *sinR* genes were knocked out;The second group used primers from another site on the genome of *Bacillus subtilis* to test whether the selected colony was *Bacillus subtilis*. Colonies that were positive in both PCR groups were selected for further expansion.

#### Cycle II - Test

Performing colony PCR, firstly, knocking out validation was performed, and it was found that the target length fragment was not obtained

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| ![5](https://static.igem.wiki/teams/5810/engineering/knockout/fig4.avif) |

Fig1-4 Colony PCR validation of knockout results. Target gene produced an amplicon of ~1,100 bp, M: DL5000 Marker

Meanwhile, verify whether the colonies on the plate are *Bacillus subtilis*. No expected band appears, the results are completely negative. However, the appearance of a single colony is similar to that of *Bacillus subtilis*. Thus, we extracted the genome of putative positive colonies for PCR and found that there were bands meeting expected size, indicating the colonies were *Bacillus subtilis*.

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| ![5](https://static.igem.wiki/teams/5810/engineering/knockout/fig5.avif) |

Fig1-5 Colony PCR verification to determine if it is *Bacillus subtilis*. Target gene produced an amplicon of ~ 2300 bp, M: DL5000 Marker

#### Cycle II - Learn

We speculated that the CRISPR KO efficiency was low in strain 6051, so we attempted to replace the strain before attempting knockout.

#### Cycle III - Design

We planned to replace the *Bacillus subtilis* strain 6051 with 168 for plasmid knockout transformation, and re-extract the plasmid for knockout.

#### Cycle III - Build

Extract the pJOE8999-sg*tasA*-donor plasmid, configure chemocompetent *Bacillus subtilis* 168 cells and transfer it into the plasmid. Perform colony PCR to confirm successful transfer of the knockout plasmid.

#### Cycle III - Test

Colony PCR was performed on the single colony of *Bacillus subtilis* 168 that had been transferred into the knockout plasmid, and electrophoresis confirmed the successful knockout of the *Bacillus subtilis* 168 strain and the successful elimination of the knockout plasmid.

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| ![5](https://static.igem.wiki/teams/5810/engineering/knockout/fig6.avif) |

Fig1-6 PCR validation of the genome of *Bacillus subtilis* 6051, M: DL5000 Marker,1-5: PCR product of 6051 genome, 6: positive control, 7: negative control. Target gene produced an amplicon of ~1,000 bp

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| ![5](https://static.igem.wiki/teams/5810/engineering/knockout/fig7.avif) |

Fig1-7 Colony PCR validation of knockout and plasmid elimination. Lane 2-5: Using plasmid specific primers, and it was found that there was no fragment size corresponding to the plasmid, proving the successful elimination of pJOE8999 plasmids; Lane 6-9: Use primers upstream and downstream of the target gene to obtain fragments of the expected size, target gene produced an amplicon of ~1,000 bp, proving successful knockout. M: DL5000 Marke

#### Cycle III - Learn

After the first two rounds of improvement, we successfully avoided the problems of *E. coli*contamination and low strain knockout efficiency, and constructed *Bacillus subtilis* 168 with *sinR*-*tasA* knockout, providing the original strain for subsequent gene transfer.

## 2. Adhesion Module

### 2.1. Plasmid selection

#### Cycle I - Design

At the beginning of the project design, since we expected the engineered bacteria to express the TasA protein and other binding proteins we introduced in large quantities, we first chose to use the pMA5 plasmid with a relatively high copy number. Its promoter is HpaⅡ, which is a constitutive expression We used this plasmid as the backbone to construct a variety of fusion plasmids and transformed them into the engineered bacteria.

#### Cycle I - Build

The fusion plasmids pMA5-*tasA*, pMA5-*tasA*-spy tag, pMA5-*tasA*-mcherry, pMA5-*tasA*-mfp5, pMA5-PS tag1 and pMA5-PS tag2 were constructed using pMA5 as the plasmid backbone and transformed into the engineered bacteria respectively.

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| ![5](https://static.igem.wiki/teams/5810/engineering/adhesion/fig-1-1-the-atlas-of-six-plasmids-constructed-with-pma5-as-the-backbone.webp) |

Fig 2.1-1 The atlas of six plasmids constructed with pMA5 as the backbone

#### Cycle I - Test

We found that the engineered bacteria expressing TasA or TasA fusion protein grew very slowly and were not easy to form biofilms and conduct functional tests. The growth curve results showed that compared with the wild-type and the control group with *tasA* and *sinR* knockout, the growth of the six groups of engineered bacteria transformed with plasmids was inhibited to varying degrees.

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| ![5](https://static.igem.wiki/teams/5810/engineering/adhesion/fig-1-2-the-growth-curves-of-six-engineered-bacteria-transformed-different-plasmids.webp) |

Fig 2.1-2 The growth curves of six engineered bacteria transformed different plasmids

#### Cycle I - Learn

Ⅰ. Because the engineered bacteria carrying TasA or TasA fusion proteins grow very slowly and are difficult to form biofilms, subsequent functional tests cannot be conducted.

II．We speculated that the overexpression of TasA or TasA fusion protein may be toxic to the engineered bacteria themselves, so we decided to replace the plasmid with Low-copy induced expression plasmid pHT01 to avoid overexpression of TasA or TasA fusion protein to inhibit the growth of engineered bacteria.

#### Cycle II - Design

We replaced the plasmid backbone with pHT01 and changed the promoter to lac operator, which is an induced expression pathway. We used this plasmid as the backbone to construct the fusion plasmid and applied it to subsequent experiments.

#### Cycle II - Build

pHT01-*tasA*, pHT01-*tasA*-spy tag, pHT01-*tasA*-mcherry, pHT01-*tasA*-mfp5, pHT01-PS tag1 and pHT01-PS tag2 fusion plasmids were constructed using pHT01 as the plasmid backbone and were used in subsequent experiments

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| ![5](https://static.igem.wiki/teams/5810/engineering/adhesion/fig-1-3-the-atlas-of-six-plasmids-constructed-with-pma5-as-the-backbone.webp) |

Fig 2.1-3 The atlas of six plasmids constructed with pMA5 as the backbone.

#### Cycle II - Test

The results showed that after the plasmid constructed with pHT01 as the backbone was transformed into the engineered bacteria, there was no obvious growth inhibition effect. The engineered bacteria could grow normally and form biofilms normally.

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| ![5](https://static.igem.wiki/teams/5810/engineering/adhesion/fig-1-4-the-membrane-forming-effect-of-the-engineered-bacteria-transfected-with-the-plasmid-constructed-using-pht01-as-the-backbone.webp) |

Fig 2.1-4 The membrane-forming effect of the engineered bacteria transfected with the plasmid constructed using pHT01 as the backbone.

#### Cycle II - Learn

We finally decided to use the inducible promoter plasmid pHT01. The fusion plasmid constructed with it as the backbone, when introduced into the engineered bacteria for induced expression, showed no obvious toxicity. The engineered bacteria were able to grow normally and could also form cell membranes normally after induction.

### 2.2. Fluorescence Detection of Fusion Protein Expression

#### Cycle I - Design

In our experiment, we need to verify the expression of target proteins (such as TasA, spycatcher, etc.) on the biological membrane. We will couple the protein to be tested with the red fluorescent protein mcherry for expression. By observing the fluorescence of the bacterial liquid and the fluorescence of the extracted fusion protein, we can indicate whether the target protein is expressed

#### Cycle I - Build

The plasmids pHT01-*tasA*-mCherry and pET-28a-mCherry-spycatcher were constructed via homologous recombination, and then transformed into *Bacillus subtilis*  168 and Escherichia coli BL21, respectively.

In a biological safety cabinet, pick the successfully transformed single colonies into 15 ml culture tubes containing 5 ml LB liquid medium, and incubate at 37°C with shaking at 220 rpm. When the OD value reaches approximately 0.8-1.1, take 40 μl of the bacterial culture and add it to 50 ml centrifuge tubes containing 40 ml LB liquid medium. Add 0.5-1 mM IPTG for induction, then incubate at 24°C with shaking at 220 rpm for 6-18 hours.

After induction, observe the bacterial culture under an ultraviolet lamp to check for fluorescence. Subsequently, centrifuge the bacterial culture, resuspend the pellet, lyse the cells, extract the proteins, and observe for fluorescence under an ultraviolet lamp again. Additionally, a portion of the bacterial culture can be observed under a fluorescence microscope.

#### Cycle I - Test

The results showed that we successfully constructed the plasmid and transformed it into *Bacillus subtilis*  168. However, neither the cultured bacterial suspension nor the proteins extracted after lysis exhibited fluorescence. In contrast, the pET-28a-mCherry-spycatcher plasmid introduced into Escherichia coli BL21 was able to express and produce fluorescence.

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| ![Fig3 Underwater adhesion mechanism of Mfp5 protein](https://static.igem.wiki/teams/5810/engineering/adhesion/fig-2-1-protein-solution-extracted-after-cultivation-of-bacillus-subtilis-168-with-the-plasmid-introduced-left.webp) |

Fig 2.2-1 Protein solution extracted after cultivation of *Bacillus subtilis*  168 (with the plasmid introduced) (Left)
Protein solution extracted after cultivation of Escherichia coli BL21 (with the plasmid introduced) (Right)

#### Cycle I - Learn

It is speculated that during the experiment, the reasons why *Bacillus subtilis*  168 did not exhibit red fluorescence may be as follows:

Ⅰ. *Bacillus subtilis*  expresses endogenous TasA protein, which may have interfered with the expression of the exogenously introduced TasA fusion protein, resulting in the failure to observe fluorescence.

Ⅱ. The induction temperature or time conditions we used may not be suitable for the expression of this inducible expression pathway in *Bacillus subtilis*  168, and we will optimize the conditions for protein induction and expression.

#### Cycle II - Design

Due to the poor effect of liquid induction, we decided to try plate induction to promote the expression of the exogenous TasA-mCherry protein. Meanwhile, since the expression of endogenous TasA protein may interfere with the expression of the exogenously introduced TasA fusion protein, we chose to use *Bacillus subtilis*  with the *tasA* gene knocked out. Additionally, we extended the induction time.

#### Cycle II - Build

Take 5 μL of the overnight-cultured bacterial suspension of TasA-mCherry *tasA*⁻ *Bacillus subtilis*  168, inoculate it onto SYG solid medium containing IPTG, and incubate statically at 37°C overnight.

#### Cycle II - Test

We observed that the biofilm formed on the plate by the engineered bacteria harboring the TasA-mCherry plasmid exhibited weak red fluorescence compared to the control group of bacteria without the plasmid introduced.

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| ![5](https://static.igem.wiki/teams/5810/engineering/adhesion/fig-2-2-the-biofilm-formed-after-film-forming-induction.webp) |

Fig 2.2-2 The biofilm formed after film-forming induction

#### Cycle II - Learn

I. Plate induction enabled the pHT01-*tasA*-mcherry engineered bacteria to produce weak fluorescence; however, the fluorescence we observed was relatively faint, and further optimization of the induction conditions may be required in subsequent experiments.

II. In subsequent experiments, we still need to induce the expression of the TasA fusion protein under liquid conditions; therefore, we will continue to attempt liquid induction.

#### Cycle III - Design

We plan to use *Bacillus subtilis*  with the *tasA* gene knocked out for liquid induction experiments, and correspondingly extend the induction duration.

#### Cycle III - Build

Based on the protocol of Cycle Ⅰ, the experimental strain was replaced with *tasA*⁻ *Bacillus subtilis*  168, and the IPTG induction time was extended to 48 hours.

#### Cycle III - Test

We were pleased to observe that after 48 hours of induction, the centrifuged bacterial suspension of *tasA*⁻ *Bacillus subtilis*  168 exhibited fluorescence. Additionally, the *tasA*⁻ *Bacillus subtilis*  168 strain expressing red fluorescence was observed under a fluorescence microscope.

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| ![5](https://static.igem.wiki/teams/5810/engineering/adhesion/fig-2-3-tasa-b-subtilis-168-with-pht01-tasa-mcherry-observed-under-a-fluorescence-microscope.webp) |

Fig 2.2-3 *tasA*- *B. subtilis* 168 with pHT01-*tasA*-mcherry observed under a fluorescence microscope

#### Cycle III - Learn

I. An analysis of why we had failed to induce fluorescence previously suggests the following possibilities: the fusion protein may have been poorly expressed under low-concentration IPTG induction, or it may not express during the logarithmic growth phase but only in the stationary phase. Additionally, the maximum induction time we used previously was only about 24 hours, whereas in this experiment, the induction time was extended to nearly 48 hours, which ultimately allowed us to observe fluorescence.

II. The knockout of *tasA* significantly enhanced the ability of *Bacillus subtilis*  168 to express the exogenous fusion protein, enabling TasA-mcherry to produce fluorescence.

### 2.3. Adhesion Force Test

#### Cycle I - Design

In our experiment, we need to verify the adhesion effect of the proteins. We have designed two adhesion systems, namely the TasA-binding protein fusion protein system and the SpyTag-SpyCatcher system. A total of 4 experimental groups are set up, which are *tasA*-spytag (mfp5-spycatcher), *tasA*-mfp5, *tasA*-PS tag1, and *tasA*-PS tag2. We will incubate the engineered bacteria carrying the above plasmids with polystyrene beads respectively. After processing the polystyrene beads, we will observe them using a scanning electron microscope (SEM) to indicate the adhesion effect of the adhesive proteins.

#### Cycle I - Build

Soak the polystyrene beads in the film-forming bacterial suspensions expressing TasA-mfp5, TasA-PS tag1, and TasA-PS tag2 proteins respectively. After 48 hours of incubation, retrieve the treated beads. The bacterial attachment by our genetic engineering bacteria on the treated surface was observed by SEM after performing the bacterial anti-adhesive experiments. Samples were treated before testing as follows. Firstly, the samples were washed thrice with sterile PBS, then fixed with 4 wt% glutaraldehyde solution for 2 h, and washed twice with phosphate buffer and once again with distilled water. Then the fixed bacteria were dehydrated with a series of gradient ethanol solution (50, 75, 90, and 100 wt%) for fifteen minutes. Afterwards, all the samples were dried completely in a ventilation, then pre-coated with platinum at 30 mA for 2 min and further observed by FESEM.

#### Cycle I - Test

Under the SEM, some *Bacillus subtilis*  adhering to the polystyrene beads can be observed. The bacterial cells are rod-shaped, with a single cell size of 2–3

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| ![5](https://static.igem.wiki/teams/5810/engineering/adhesion/fig-3-1-adherent-strains-on-the-surface-of-polystyrene-beads-observed-under-sem.webp) |

micrometers, and no capsule is present. However, the number of these adherent cells is far lower than our expectations.
Fig 2.3-1 Adherent strains on the surface of polystyrene beads observed under SEM

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| ![5](https://static.igem.wiki/teams/5810/engineering/adhesion/fig-3-2-adherent-strains-on-the-surface-of-polystyrene-beads-observed-under-sem-magnified.webp) |

Fig 2.3.2 Adherent strains on the surface of polystyrene beads observed under SEM (Magnified)

#### Cycle I - Learn

The number of strains observed via SEM was significantly lower than our expectations. We speculate that this may be due to the fact that when processing the polystyrene beads, soaking and fixing them with glutaraldehyde caused the biofilm to detach, preventing bacteria from adhering to the surface of the polystyrene beads any longer. Since SEM requires glutaraldehyde treatment, it is difficult to obtain ideal results; therefore, we need to design additional experiments to indicate the adhesion force of the binding protein.

#### Cycle II - Design

Since SEM observation requires glutaraldehyde treatment, which causes biofilm detachment and thus fails to observe the adhesion of engineered bacteria, we have chosen to verify the adhesion force of the binding protein through the polystyrene bead-loaded CFU counting assay, and quantitatively express the adhesion force using the plate counting method.

#### Cycle II - Build

Immerse the polystyrene beads in the SYG film-forming bacterial suspensions of the engineered bacteria pHT01-*tasA*-mfp5, pHT01-*tasA*-PS tag1, and pHT01-*tasA*-PS tag2. Meanwhile, set up the *tasA* group and the control group (*tasA*⁻ *sinR*⁻ 168) as controls. For the SpyTag-SpyCatcher system, co-incubate the beads with the pHT01-*tasA*-spytag engineered bacterial suspension and the mfp5-spycatcher protein; use the beads incubated solely with the pHT01-*tasA*-spytag engineered bacterial suspension as the control. After 48 hours of incubation, retrieve the treated beads. Subsequently, rinse the beads 3 times with 1 mL of sterile water, add 1 mL of PBS, and perform ultrasonic oscillation for 2 minutes. Prepare serial dilutions of the eluted bacterial suspension at 100×, 1000×, and 10000×, then spread the diluted suspensions onto LB solid plates. Place the plates in a 37°C incubator for 1 day of cultivation, after which take them out for colony counting.

#### Cycle II - Test

The results showed that the spytag (mfp5-spycatcher) system exhibited the strongest adhesion force, followed by mfp5, PS-tag1, and PS-tag2 in descending order. In contrast, the control group, TasA group, and spytag group all had relatively weak adhesion force.

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| ![5](https://static.igem.wiki/teams/5810/engineering/adhesion/fig3-3-cfu-counting-results-of-tasa-fusion-proteins.webp) |

Fig 2.3-3 CFU Counting Results of TasA Fusion Proteins

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| ![5](https://static.igem.wiki/teams/5810/engineering/adhesion/fig3-4-cfu-counting-results-of-the-spytag-spycatcher-system.webp) |

Fig 2.3-4 CFU Counting Results of the SpyTag/SpyCatcher System

#### Cycle II - Learn

Ⅰ. This experiment has proven to a certain extent that our adhesion systems are effective, as all experimental scheme demonstrated better adhesion effects compared to the control group. Among the binding proteins, mfp5 exhibited the best adhesion effect; meanwhile, the adhesion effect of the spytag-spycatcher system was superior to that of the TasA-binding protein fusion protein system.

II. This experiment only counted CFUs (Colony-Forming Units), which cannot reflect the biofilm formation quantity—an indicator we care most about in practical applications. Therefore, we need to design additional experiments to characterize the adhesion force of the binding protein by directly detecting biofilm-related data.

#### Cycle III - Design

We further conducted a crystal violet staining experiment on the aforementioned polystyrene beads to directly detect biofilm-related data, which serves as supplementary evidence for the results of the polystyrene bead colony counting experiment.

#### Cycle III - Build

Immerse the polystyrene beads in the SYG film-forming bacterial suspensions of the engineered bacteria pHT01-*tasA*-mfp5, pHT01-*tasA*-PS tag1, and pHT01-*tasA*-PS tag2. Meanwhile, set up the *tasA* group and the control group (*tasA*⁻ *sinR*⁻ 168) as controls. For the SpyTag-SpyCatcher system, co-incubate the beads with both the pHT01-*tasA*-spytag engineered bacterial suspension and the mfp5-spycatcher protein; use the beads incubated solely with the pHT01-*tasA*-spytag engineered bacterial suspension as the control.

After 48 hours of incubation, retrieve the treated beads. Add 1.4 mL of 0.1% crystal violet to a 1.5 mL centrifuge tube for staining, then invert the tube and incubate for 20 minutes. Subsequently, take out the beads and rinse them 2–3 times with sterile PBS. After allowing them to air-dry, decolorize the beads with 95% ethanol for 15 minutes. Collect the decolorized solution and measure its absorbance at 570 nm.

#### Cycle III - Test

The results of the crystal violet assay showed that the SpyTag-SpyCatcher system, TasA-mfp5, TasA-tag1, and TasA-tag2 all exhibited good adhesion effects, while the TasA group and the control group had poor adhesion effects. Consistently, the SpyTag-SpyCatcher system showed the strongest adhesion effect, and mfp5 remained the binding protein with the strongest adhesion effect.

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| ![5](https://static.igem.wiki/teams/5810/engineering/adhesion/figure-3-5-statistical-results-of-crystal-violet-staining-for-tasa-fusion-proteins.webp) |

Fig2.3-5 Statistical Results of Crystal Violet Staining for TasA Fusion Proteins

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| ![5](https://static.igem.wiki/teams/5810/engineering/adhesion/figure-3-6-statistical-results-of-crystal-violet-staining-for-the-spytag-spycatcher-system.webp) |

Fig2.3-6 Statistical Results of Crystal Violet Staining for the SpyTag/SpyCatcher System

#### Cycle III - Learn

Ⅰ. The results of the crystal violet assay are quite similar to those of the polystyrene bead-loaded CFU counting experiment. Both experiments confirm that the fusion proteins we designed can be expressed and exhibit strong adhesion force, among which mfp5 shows the best adhesion effect. Additionally, our spytag-spycatcher system can also effectively generate adhesion force, and its effect is better than that of the individual binding proteins.

Ⅱ. Building on the aforementioned experiments, we still need to verify whether our adhesion systems can work properly under the complex conditions of sewage treatment in practical applications. This verification will provide data support for the subsequent functional testing module.

#### Cycle IV - Design

Since our engineered bacteria will be applied in sewage treatment, they may be exposed to different pH values and Cd²⁺ concentrations. Therefore, we also need to design experiments to verify whether our engineered bacteria can adhere under different pH values and different Cd²⁺ concentrations.

#### Cycle IV - Build

We set up pH gradients of 3, 4, 5, 6, and 7 for the incubation of TasA-mfp5, with 5 μg/mL chloramphenicol and 1 mM IPTG added to the SYG medium for induction.

Additionally, we established Cd²⁺ concentration gradients of 0, 0.25, 0.5, 1, and 2 mol/L for the incubation of TasA-mfp5. Similarly, 5 μg/mL chloramphenicol and 1 mM IPTG were added to the SYG medium for induction.

#### Cycle IV - Test

The experimental results showed that the engineered bacteria could grow normally within the range of Cd²⁺ concentration from 0 to 2 mol/L, and also grow normally within the range of pH value from 3 to 7.

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| ![5](https://static.igem.wiki/teams/5810/engineering/adhesion/figure-3-7-colony-count-statistics-under-different-cd-concentrations.webp) |

Fig2.3-7 Colony Count Statistics Under Different Cd²⁺ Concentrations

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| ![5](https://static.igem.wiki/teams/5810/engineering/adhesion/figure-3-8-colony-count-statistics-under-different-ph-values.webp) |

Fig2.3-8 Colony Count Statistics Under Different pH Values

#### Cycle IV - Learn

Ⅰ. The engineered bacteria can grow normally within the specified Cd²⁺ concentration range (0–2 mol/L), indicating that they can adapt to the high Cd²⁺ concentrations present in sewage treatment environments.

Ⅱ. The engineered bacteria can grow normally within the specified pH range (3–7), indicating that they can adapt to the weakly acidic environment present in sewage treatment scenarios.

## 3. Function Test Module

#### Cycle I - Design

To investigate the growth curves of *Bacillus subtilis,* 168 Δ*tasA*Δ*sinR* harboring the recombinant plasmid pHT01-*epcadR-PcadR-mCherry* under different Cd²⁺ concentrations, we prepared bacterial cultures at 0, 0.5, 1.0, and 2.0 mmol/L Cd²⁺ concentrations. Optical density (OD) values were measured at 0, 2, 5, 18, and 30 hours using a microplate reader. The data were plotted as line graphs to investigate the inhibitory effects of different Cd²⁺ concentrations on bacterial growth and to analyze the growth curves.

#### Cycle I - Build

Prepare LB liquid medium with Cd²⁺ concentrations of 0, 1.0, 2.0, and 4.0 mmol/L in a laminar flow hood. Pipette 100 μL of each into a 96-well plate. Then, add 100 μL of wild-type *Bacillus subtilis,* or Δ*tasA*Δ*sinR* *Bacillus subtilis,* 168 containing recombinant plasmid pHT01-*epcadR-PcadR-mCherry*, or LB liquid medium as a blank control. Incubate at 37°C on a shaking incubator at 200 rpm. Measure the OD values at 0, 2, 5, 18, 30 hours using a microplate reader. The data are plotted as a line graph.

#### Cycle I - Test

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| ![5](https://static.igem.wiki/teams/5810/engineering/function/figure1.avif) |

Figure 3-1. Growth Curves of Wild-type *Bacillus subtilis,* and Strains Harboring Recombinant Plasmid pHT01-*epcadR-PcadR-mCherry*

As shown in the Figure.3-1 , the growth of nearly all bacterial cultures containing Cd²⁺ was severely inhibited, remaining at their initial OD values. We believe this is because the Cd²⁺ concentrations set exceeded the tolerance range of the bacteria, causing them to be poisoned and unable to grow or reproduce within the measurement period. In contrast, the bacterial cultures without added Cd²⁺ exhibited a typical bacterial growth curve, divided into the lag phase, log phase, stationary phase, and death phase.

#### Cycle I - Learn

The Cd²⁺ concentration in this experiment was excessively high. After reviewing literature on the cadmium tolerance range of *Bacillus subtilis,*, we redesigned the Cd²⁺ solution gradient and appropriately extended the growth curve measurement time to reflect the strain's maximum cadmium tolerance concentration. Additionally, when adding the LB blank control, strict aseptic technique must be maintained to prevent bacterial contamination that could invalidate the control. For the next experiment, we will also appropriately reduce the shaker speed to prevent bacterial culture spillage and cross-contamination. Furthermore, we will add an appropriate amount of PBS to the gaps between sample wells on the plate to minimize medium evaporation.

#### Cycle II - Design

After reviewing literature on the Cd²⁺ growth concentration range for *Bacillus subtilis,*, we redesigned and significantly reduced the Cd²⁺ concentration. Bacterial cultures were prepared at concentrations of 1.5, 0.5, 0.25, and 0 mg/L Cd²⁺. OD values were measured at 0, 3, 16, and 48 hours, and the optimal Cd²⁺ concentration for bacterial growth was selected for subsequent fluorescent protein induction experiments.

#### Cycle II - Build

In a laminar flow hood, prepare Cd²⁺ solutions with concentrations of 1.5, 0.5, 0.25, and 0 mg/L. 0 mg/L, along with bacterial cultures containing the recombinant plasmid pHT01-*epcadR-pcadR-mcherry*. LB liquid medium at the same concentrations was prepared as a blank control. Cultures were incubated at 37°C on a shaking incubator at 200 rpm. OD values were measured at 0, 3, 16, and 48 hours using a microplate reader. The obtained data were plotted as a line graph.

#### Cycle II - Test

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| ![5](https://static.igem.wiki/teams/5810/engineering/function/figure2.avif) |

Figure 3-2. Growth Curves of the *Bacillus subtilis,* 168 Knockout Strain and the Strain Harboring the Recombinant Plasmid pHT01-*epcadR-pcadR-mcherry*

As shown in the Figure. 3-2, at the tested concentrations (in mg/L), all three bacterial strains exhibited a pronounced growth trend and proliferated rapidly, indicating that Cd²⁺ inhibition was relatively mild at the current concentrations. The strongest inhibitory effect on bacterial growth was observed at a Cd²⁺ concentration of 1.5 mg/L, while at concentrations of 0.5 and 0.25 mg/L, the inhibitory effects were relatively mild with minimal differences. However, due to insufficient measurement time, the death phase of the bacterial community was not observed.

#### Cycle II - Learn

Bacterial growth was robust at this concentration and the group of pHT01-*epcadR-pcadR-mcherry* exhibited a pronounced growth trend across all Cd²⁺ concentrations, indicating that the Δ*tasA*Δ*sinR* *Bacillus subtilis,* 168 is capable of growing under Cd2+ with the experimental concentration and plasmid pHT01-*epcadR-pcadR-mcherry* didn’t influence the tolerance ability.

#### Cycle III - Design

Based on the previous determination of the growth curves of engineered bacteria under cadmium ion concentrations of 1.5, 0.5, 0.25, and 0 mg/L, we selected the cadmium ion concentration that yielded the optimal growth of the engineered bacteria to further verify the differential expression of fluorescent proteins. Thus, we designed an experiment to verify the variation pattern of fluorescent protein expression intensity with cadmium ion concentration and time under a cadmium ion concentration of 0.5 mg/L.

#### Cycle III - Build

Single colony of Δ*tasA*Δ*sinR* *Bacillus subtilis,* 168 containing the recombinant plasmid pHT01-*epcadR-PcadR-mCherry* was picked. Bacterial suspensions with a cadmium ion concentration of 0.5 mg/L were prepared in a clean bench, added to sample well plates, and their fluorescence values were measured at 0, 4, and 8 hours using a fluorescence spectrophotometer. The data were recorded and subjected to statistical analysis.

#### Cycle III - Test

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| ![5](https://static.igem.wiki/teams/5810/engineering/function/figure3.avif) |

Figure 3-3. Fluorescent Protein Expression of Strains Containing Recombinant Plasmids pHT01-*ePcadR-PcadR-mCherry*

Under induction with a cadmium ion concentration of 0.5 mg/L, strain containing the recombinant plasmid pHT01-*ePcadR-PcadR-mCherry* could be induced by cadmium ions to express fluorescent proteins (Figure 3). This indicates that *PcadR* in the strains can specifically recognize cadmium ions and activate the expression of downstream fluorescent proteins, further verifying the responsive function of *PcadR* to cadmium ions.

#### Cycle III - Learn

Notably, the knockout strain without plasmid introduction also exhibited a certain degree of fluorescent expression. This is presumably due to the leaky expression of the plasmid promoter. In addition to the potential leaky expression of the plasmid promoter, the fluorescent expression level of pHT01-*ePcadR-PcadR-mCherry* was relatively low. We guessed the induction time was short and there was lack of gradient concentration of Cd2+. Consequently, we planed to extend testing time and set a series of Cd2+ concentration to further verify sensing function.

#### Cycle IV - Design

Due to the abnormal fluorescence expression data in the previous round, we redesigned another set of cadmium ion concentration gradients, namely 0, 1.0, and 2.0 mmol/L, to verify the variation law of the expression intensity of fluorescent proteins with cadmium ion concentration and time.

#### Cycle IV - Build

Single colonies of Δ*tasA*Δ*sinR* *Bacillus subtilis,* 168 containing the recombinant plasmid pHT01-*epcadR-PcadR-mCherry* was picked. Bacterial solutions with cadmium ion concentrations of 0, 1.0, and 2.0 mmol/L were prepared in a clean bench, added to sample well plates, and the fluorescence values were measured with a fluorescence spectrophotometer within a specified time, followed by recording and statistical analysis.

#### Cycle IV - Test

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| ![5](https://static.igem.wiki/teams/5810/engineering/function/figure4.avif) |

Figure 3-4. Fluorescence intensity statistics of pHT01-*epcadR-PcadR-mCherry*

As shown in the Figure 3-4, under cadmium concentrations of 0, 1, and 2 mmol/L, the fluorescence expression intensity of pHT01-*epcadR-PcadR-mCherry* showed a significant positive correlation with both culture time and cadmium ion concentration. During the 25-hour culture process, the fluorescence intensity gradually increased and reached the maximum at 25 hours. In contrast, the fluorescence intensity of the control group without cadmium addition remained at a low level throughout the culture period.

#### Cycle IV - Learn

After conducting a significant difference analysis on the 0 mmol/L group in Figure 3 as the control group, it was found that the fluorescence expression intensity of the pHT01-*epcadR-PcadR-mCherry* strain had a strong correlation with the cadmium ion concentration. This result preliminarily verified that the recombinant strain has a responsive ability to cadmium ions, laying a foundation for subsequent studies such as constructing a cadmium ion biosensor based on this strain.

## 4. Safety Module

The suicide system is an important part of this project and is related to the biosafety of the entire project. Due to the toxicity of *mazF* to prokaryotes, it has become quite difficult to ensure the stable expression of the *mazF* gene on the plasmid. In the early stage of the experiment, we were puzzled by the weak killing effect of *mazF*. Later, we verified our speculation through a series of experiments and ensured the stable expression of *mazF* to facilitate the further progress of the experiment.

#### Cycle I - Design

In the early stage of the experiment, we used liquid-induced culture to verify the toxic effect of *mazF* on *Bacillus subtilis*. The experimental group with IPTG concentration of 0.4mg/mL and the control group without IPTG were designed. The OD600 was measured every two hours. According to our experimental expectation, as time elapsed, the absorbance of the experimental group should gradually be lower than that of the control group, indicating that the growth of the engineered bacteria was inhibited.

#### Cycle I - Build

i. Use the chemical transformation method to transfer the pHT01 - *mazF* plasmid into *Bacillus subtilis*. After verification, inoculate the successfully transformed *Bacillus subtilis* into a new LB medium, taking 10 microliters each.

ii. After culturing for 18 hours, take 500 microliters of the bacterial solution and inoculate it into 50 milliliters of LB medium with an IPTG concentration of 0.4 mg/mL and 50 milliliters of LB medium without IPTG respectively. Measure the OD600 absorbance value every 2 hours.

iii. Draw a line graph based on the experimental results.

#### Cycle I - Test

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| ![5](https://static.igem.wiki/teams/5810/engineering/safety/figure1.webp) |

Fig4-1 Liquid culture absorbance curve

Compared with the control group without IPTG addition, only a very weak inhibitory effect was shown in the first six hours. As time passed, the inhibitory effect weakened, which was very inconsistent with our expectations.

#### Cycle I - Learn

Since *mazF* did not show an obvious inhibitory effect, we speculated that the low expression of *mazF* without IPTG imposed stress on *Bacillus subtilis*. Bacteria that could resist the low expression of *mazF* were selected and over - grew, resulting in insensitivity to IPTG induction. Since this result failed to prove the function of the *mazF* gene, the experiment needs further improvement.

#### Cycle II - Design

To prevent the continuously selection of resistant bacteria during the expansion culture of successfully transformed *Bacillus subtilis*, we directly induced the newly transformed *Bacillus subtilis*. Referring to the experimental methods of Wageningen\_UR in 2021, we added the bacterial solution to the culture medium containing different concentrations of IPTG and measured the OD600 absorbance after culturing for a period of time. We expect that as the concentration of IPTG increases, the absorbance of the bacterial solution gradually decreases, indicating that the newly transformed *Bacillus subtilis* is highly sensitive to IPTG.

#### Cycle II - Build

i. Inoculating newly transformed *Bacillus subtilis* in LB culture medium and growing overnight.

ii. inoculating the culture in LB medium supplemented with different concentrations of IPTG, so that the OD600=0.1, and detecting the absorbance value of OD600 after a period of time.

iii. Draw a line chart according to the experimental results.

#### Cycle II - Test

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| ![5](https://static.igem.wiki/teams/5810/engineering/safety/figure2.webp) |

Fig4-2 Changes in OD600 absorbance values with varying IPTG concentrations

We can find that in liquid culture, a low concentration of IPTG has a good inhibitory effect on *Bacillus subtilis*, indicating that this strain of *Bacillus subtilis* is highly sensitive to IPTG, which is consistent with our hypothesis.

#### Cycle II - Learn

However, under liquid culture conditions, the dead bacteria still remain in the culture medium, which will affect the OD600 absorbance value. Therefore, the absorbance value under this condition cannot accurately reflect the killing effect of *mazF*. So, we plan to conduct the same experiment on solid medium. By physically isolating the bacteria, the killing effect of the toxin can be more intuitively shown.

#### Cycle III - Design

Based on the above experimental results, we referred to the experimental methods of SCU-China in 2023 and used an IPTG gradient plate for verification (the IPTG concentration shows a gradient distribution on the medium from one side to the other). First, we verified through the IPTG gradient plate that the newly transformed *Bacillus subtilis* could correctly express *mazF* and had good sensitivity to IPTG. After transforming the pHT01-*mazF* plasmid into *Bacillus subtilis*, the cells were immediately spread on the IPTG gradient plate for induction to form single colonies of the transformed colonies. We expect that as the IPTG concentration gradually increases, the number of colonies on the solid medium will gradually decrease.

#### Cycle III - Build

i. Prepare LB solid medium, autoclave it at 121°C for 20 minutes for sterilization, and then cool it to about 50°C. After adding kanamycin solution, pour it into a plate supported at one end by 5 mm. After solidification, add LB K medium containing IPTG solutions of different concentrations to make an IPTG gradient plate.

ii. Transform the competent cells of *Bacillus subtilis* with the pHT01-*mazF* plasmid. Take 50 μL of the diluted culture and spread it evenly on the IPTG gradient plate. Incubate the plate in a constant - temperature incubator. Observe after 24 hours and count the number of single colonies.

#### Cycle III - Test

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| ![5](https://static.igem.wiki/teams/5810/results/add01.avif) |

Fig4-3 Results of IPTG gradient plate induction experiment

As the concentration of IPTG increased, the number of single colonies continued to decrease. This is consistent with our hypothesis. However, since the concentration of IPTG could not be quantitatively analyzed, this result can only preliminarily verify our speculation, and more rigorous experiments are needed for confirmation.

#### Cycle III - Learn

Compared with the initial IPTG gradient liquid culture, we successfully obtained the expected results, preliminarily verified our hypothesis, and proved the effectiveness of this part.

#### Cycle IV - Design

For a more quantitative analysis, plates containing different concentrations of IPTG were used for induction experiments. We expect that the number of single colonies will continuously decrease as the concentration of IPTG increases.

#### Cycle IV - Build

i. Transform *Bacillus subtilis* competent cells with the pHT01-*mazF* plasmid, and then evenly spread 50 μL of the diluted culture onto a series of plates with different IPTG concentrations. Incubate the plates in a constant - temperature incubator.

ii. After 24 hours, conduct observations, count the number of single colonies, and draw a line graph.

#### Cycle IV - Test

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| ![5](https://static.igem.wiki/teams/5810/results/add02.avif) |

Fig4-4 Results of induction experiments with different concentrations of IPTG plates

As the concentration of IPTG increased, the number of single colonies on the plate showed a significant downward trend. When the IPTG concentration reached 0.04 mg/mL, the low-expression *mazF* began to exhibit a relatively obvious bactericidal effect.

#### Cycle IV - Learn

The experimental results prove the bactericidal function of *mazF*. However, this system cannot exist stably. As the growth time of the successfully transformed *Bacillus subtilis* increases and the number of sub - cultures increases, the number of bacteria resistant to the low - expressed *mazF* toxin protein will gradually increase and gain a growth advantage, resulting in a gradual decrease in sensitivity to IPTG. Therefore, we need to find a way to make this system stable and sustainable.

#### Cycle V - Design

To improve the original suicide system, the *mazE* antitoxin gene controlled by the constitutive p43 promoter was introduced into the pHT01-*mazF* plasmid to construct the pHT01-*mazF*-*mazE* plasmid.

#### Cycle V - Build

i. Obtain the linearized vector and target fragment by PCR, and then construct the pHT01-*mazF*-*mazE* plasmid using Gibson assembly.

ii. Transform *Bacillus subtilis* competent cells with the pHT01-*mazF*-*mazE* plasmid. After several days of expansion culture, spread 50 μL of the diluted culture evenly onto plates with a series of IPTG concentrations. Incubate the plates in a constant - temperature incubator.

iii. After 24 hours, conduct observations, count the number of single colonies, and draw a line graph.

#### Cycle V - Test

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| ![5](https://static.igem.wiki/teams/5810/engineering/safety/figure5.webp) |

Fig4-5 Experimental results of inducing the *mazF*&*mazE* system on plates with different IPTG concentrations

Our experimental results show that even after many generations of expansion and cultivation, this bacterium still exhibits high sensitivity to IPTG. Moreover, the constitutively expressed *mazE* protein can neutralize the *mazF* toxin protein, endowing our system with the characteristic of long - term stability.

#### Cycle V - Learn

The experimental results show that the *mazE* antitoxin protein weakens the stress effect of the *mazF* toxin protein on prokaryotes to a certain extent, making it difficult to screen out strains resistant to *mazF*.

![Loading Image](https://static.igem.wiki/teams/5810/scu/logo.webp)

![Heavy metal pollution background](https://static.igem.wiki/teams/4817/wiki/wiki/results/part1/19-1.jpg)

Experiments

### Contents

[Molecular Cloning](#molecular-cloning)
[*sinR-tasA* Knock Out](#knock-out)
[Adhesion Module](#adhesive-module)
[Function Test Module](#adsorption-module)
[Safety Module](#suicide-module)

## Molecular Cloning

Molecular cloning chapter describes all of the construction of plasmid in our program This chapter includes:

1. Plasmid extraction
2. Agarose gel electrophoresis
3. Gel extraction
4. Preparation of *Bacillus subtilis* Competent Cells
5. Transformation
6. Plasmids Construction by homologous recombination

## *sinR-tasA* Knock Out

## Adhesion Module

This chapter includes:

1. Protein Expression
2. Biofilm Induction

## Function Test Module

This chapter includes:

1. Verification of the *epcadR-pcadR-mcherry sensing module*
2. Verification of *tasA-smtA* adsorption module
3. Verification of the *epcadR-pcadR-mcherry-tasA-mfp5* sensing-adhesion module
4. Verification of *tasA-smtA* adsorption-adhesion module

## Safety Module

This chapter includes:

1. Operon efficiency verification

![Loading Image](https://static.igem.wiki/teams/5810/scu/logo.webp)

![Heavy metal pollution background](https://static.igem.wiki/teams/4817/wiki/wiki/results/part1/19-1.jpg)

Results

### Contents

[1. *sinR*/*tasA* Knock-out](#sinR-tasA-knock-out)

[2. Adhesion Module](#adhesion-module)

[2.1 *tasA* Fusion Protein](#tasA-fusion-protein)
[2.2 SpyTag-SpyCatcher System](#spytag-spycatcher-system)

[3. Function Test Module](#absorption-module)

[3.1 *epcadR-pcadR-mcherry* Sensing Functional Module](#sensing)
[3.2 *tasA-smtA* Adsorption Functional Module](#adsorption)
[3.3 *epcadR-pcadR-mcherry-tasA-mfp5* Sensing-adhesion Functional Module](#sensing-adhesion)
[3.4 Verification of Adsorption-adhesion Function Module](#adsorption-adhesion)

[4. Safety Module](#suicide-system)

[4.1 *ComQXPA* Induction System](#introduction)
[4.2 MazF Toxin Proteins](#toxin)
[4.3 MazE Antitoxin Proteins](#antitoxin)
[4.4 Overall Biosafety Part](#overall)

[5. Outlook](#outlook)

## 1. *sinR*/*tasA* Knock-out

### Knockout Plasmid ConstructionTargeted knockout of *B. subtilis* 168

We visited the National Center for Biotechnology Information ( https://www.ncbi.nlm.nih.gov) to download the CDS sequences of the target genes *tasA* and *sinR*. Subsequently, we used the online software CHOPCHOP ( https://chopchop.cbu.uib.no) for sgRNA design. We selected the two most efficient sgRNAs as experimental sgRNA sequences and handed them over to the professional company to synthesize oligonucleotide chains.

Table 1-1:sgRNA for gene knockout

| sgRNA | Sequence |
| --- | --- |
| sgRNA-*tasA* | TCTCATTCGAAGCTACACAG |
| sgRNA-*sinR* | AACCGAGACGTCCAGAACAG |

The sgRNA was ligated by Golden Gate, and the upstream and downstream homologous arms of *sinR*-*tasA* gene were ligated by Gibson homologous recombination.

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| ![5](https://static.igem.wiki/teams/5810/results/sinr-tasa-knock-out/figure1-1.webp) |

Fig 1-1: Constructed Knockout Plasmid

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| ![5](https://static.igem.wiki/teams/5810/results/sinr-tasa-knock-out/figure1-2-a.webp)  ![5](https://static.igem.wiki/teams/5810/results/sinr-tasa-knock-out/figure1-2-b.webp) |

Fig 1-2: pJOE8999-sgRNA Sequence Alignment Output

The sequencing results showed that we successfully constructed the final knockout plasmids pJOE8999-sg*sinR*-donor and pJOE8999-sg*tasA*-donor.

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| ![5](https://static.igem.wiki/teams/5810/results/sinr-tasa-knock-out/figure1-3-a.webp)  ![5](https://static.igem.wiki/teams/5810/results/sinr-tasa-knock-out/figure1-3-b.webp) |

Fig 1-3: pJOE8999-sgRNA-donor Sequence Alignment Output

### Targeted knockout of *B. subtilis* 168

The plasmids we successfully constructed were transformed into *Bacillus subtilis* 168 and coated on a kanamycin-resistant and 0.4 % mannose-containing plate for overnight culture.

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| ![5](https://static.igem.wiki/teams/5810/results/sinr-tasa-knock-out/figure2-1.webp) |

Fig 1-4: Kanamycin-0.4 % mannose selective plates for *Bacillus subtilis* 168 carrying the knockout plasmid.

A pair of primers upstream and downstream of the target genes was used to verify the genome of the mannose-induced knockout bacteria by PCR, and the results were positive, indicating that there were successfully knocked out colonies. The PCR products were sequenced, and the sequencing results showed that the *sinR* and *tasA* genes were missing, and the rest were completely paired, without deletion or mutation, indicating that the *sinR*-*tasA* gene of *Bacillus subtilis* was successfully knocked out.

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| ![5](https://static.igem.wiki/teams/5810/results/sinr-tasa-knock-out/figure2-2.webp) |

Fig 1-5: *Bacillus subtilis* knockout bacteria Sequence Alignment Output.

### Elimination of knockout plasmid

In order to eliminate the influence of the knockout plasmid on the subsequent experiments, we transferred the verified bacteria to the anti-free LB liquid medium for 3 consecutive rounds to eliminate the knockout plasmid. Single colonies were streaked on solid media containing and without kanamycin antibiotics, and kanamycin antibiotic-sensitive bacteria were selected. After the colony PCR verification was in line with the expectation, the colony was expanded and cultured to obtain plasmid-eliminated Δ*tasA*Δ*sinR* *Bacillus subtilis* 168, and the strain was preserved.

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| ![5](https://static.igem.wiki/teams/5810/results/sinr-tasa-knock-out/figure3.webp) |

Fig 1-6: Colony PCR verification result
M: DL5000 marker
Lane 1, 2 ,3 and 4: Using plasmid-specific primers, no plasmid-derived amplicon was detected
Lane 5, 6, 7 and 8: PCR using primers flanking the genomic target gene produced an amplicon of ~1,000 bp, consistent with the expected size upon successful knockout.

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| ![5](https://static.igem.wiki/teams/5810/results/sinr-tasa-knock-out/fig1-7new.avif) |

Fig 1-7: Experimental validation of plasmid curing
a.Kanamycin-0.4 % mannose selective plate
b.Kanamycin-selective LB plate
c.Antibiotic-free LB plate

### The formation of knockout bacteria biofilms

Congo red can be combined with TasA fibers，which was used to stain the biofilm of knockout bacteria and wild bacteria. The results showed that the biofilm formed by the knockout bacteria was lighter stained, indicating that there are no TasA fibers in the biofilm of the knockout strain, and the knockout effect was good.

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| ![5](https://static.igem.wiki/teams/5810/results/sinr-tasa-knock-out/figure5.webp) |

Fig 1-8: Result of Congo Red staining.

Collectively, we successfully constructed a *tasA*-*sinR* double-deletion mutant of *Bacillus subtilis* 168, providing a genetically defined host for subsequent functional characterization.

## 2. Adhesion Module

### 2.1 TasA Fusion Protein

By modifying the *tasA* gene in *Bacillus subtilis*, we enhanced its adhesion to polystyrene filler. In this project, we selected three potential adhesion peptides or proteins: Mfp5 from mussel foot protein and two computer-simulated polystyrene-binding peptides. These were fused with TasA to improve the bacterial affinity for polystyrene. To expand the system's applicability, we also developed a SpyTag/SpyCatcher system that resolved secretion challenges for large molecular TasA fusion proteins, enabling broader applications across various adhesion proteins.

### Construction of fusion protein

We constructed our plasmid using homologous recombination. After lac operon expression in the vector, we designed homologous arms at both ends of the target gene and used homologous recombinase to introduce the target gene into the plasmid backbone, thereby constructing pHT01-tasA plasmid. The pHT01-tasA plasmid was then used as a plasmid scaffold to construct the TasA fusion protein plasmid.

**Table 2.1-1: Fragment primer containing a homologous arm.**

| **Gene** | **Forward Primer** | **Reverse Primer** |
| --- | --- | --- |
| *tasA* | AATTAAAGGAGGAAGGATCCATGGGTATGAAAAAGAAATT | CCGCTCATTAGGCGGGCTGCTTAATTTTTATCCTCGCTATGCG |
| *mfp5* | GGTGGTGGTGGTTCTGGCGGTGGCGGTTCCATGAGCTCTGAAGAATACAA | CCGCTCATTAGGCGGGCTGCTTAGCTAGAACCGCCACCGTAGT |
| *mcherry* | GGTGGTGGTGGTTCTGGCGGTGGCGGTTCCATGGTGAGCAAGGGCGAGGA | CTCATTAGGCGGGCTGCTTACTTGTACAGCTCGTCCATGCC |
| *PS-tag1* | GGTGGATGCGCCATATGTTTGCGTGGAAGATTTTTTAAGCAGCCCGCTAATGAGCGG | AAACATATGGCGCATCCACCAGCTTCCTCCTCCTCCATTTTTATCCTCGCTATGCG |
| *PS-tag2* | TTGGTGGCGCACAATTGTTTGGCGCCATATTAGATAAGCAGCCCGCTAATGAGCGG | AAACAATTGTGCGCCACCAAAAGCTTCCTCCTCCTCCATTTTTATCCTCGCTATGCG |

We selected *Bacillus subtilis* with both endogenous *tasA* and *sinR* sequences knocked out as our engineered strain. Since the constitutive promoter plasmid carrying exogenous TasA protein exhibits toxicity to the engineered bacteria and maintains continuous expression of the foreign protein, which adversely affects growth, we designed an IPTG-inducible promoter (lac operon) preceding the exogenous *tasA* sequence.

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| ![5](https://static.igem.wiki/teams/5810/results/tasa-fusion-protein/fig-2-1n.avif) |

Fig 2.1-1: pHT01-tasA plasmid and other TasA fusion protein plasmids.

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| ![5](https://static.igem.wiki/teams/5810/results/tasa-fusion-protein/fig-2-2.avif) |

Fig 2.1-2: Sequencing results of pHT01-tasA plasmid and other TasA fusion protein plasmids.

Sequencing results of pHT01-tasA and other TasA fusion protein particles showed that the sequencing results of key regions were completely consistent with standard gene sequences, with no mutations occurring. pHT01-tasA and other TasA fusion protein particles have been successfully constructed, and the bacterial strains were preserved for functional validation.

### pHT01 plasmid functional verification

pHT01-TasA-mcherry function verification

To explore the function of the TasA fusion protein, we introduced the pHT01-TasA-mcherry plasmid into knockout bacteria, added 1mM IPTG, and induced expression at 37℃for 48 hours. The fluorescence expression results were observed under a fluorescence microscope.

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| ![5](https://static.igem.wiki/teams/5810/results/tasa-fusion-protein/fig-2-6.avif) |

Fig 2.1-3: Fluorescence microscope examination results.
 Left picture: Observation under a fluorescence microscope in a bright field; Right picture: Observation under green excitation light of a fluorescence microscope.

According to the results of fluorescence microscopy examination, the TasA-MCherry fusion protein expressed by \(\textit{tasA}^-\textit{sinR}^-\)*/tasA-mcherry* after IPTG induction can emit red fluorescence, indicating that the TasA fusion protein we constructed can be expressed in *Bacillus subtilis*.

TasA fusion protein Congo red staining

To further verify the function of the TasA fusion protein we constructed, we performed cone red staining on the TasA protein on the surface of biofilms with wild-type *Bacillus subtilis* 168 strain(WT) introduced, *Bacillus subtilis* 168 strain with *tasA* and *sinR* genes (\(\textit{tasA}^-\textit{sinR}^-\)) knocked out, and knockout bacteria with pHT01-tasA-mfp5、pHT01-tasA-PS tag1、pHT01-tasA-PS tag2 (\(\textit{tasA}^-\textit{sinR}^-\)*/tasA-mfp5*,\(\textit{tasA}^-\textit{sinR}^-\)*/tasA-PS tag1*,\(\textit{tasA}^-\textit{sinR}^-\)/*tasA-PS tag1*) plasmid introduced. Observe the expression of the fusion protein.

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| ![5](https://static.igem.wiki/teams/5810/results/tasa-fusion-protein/fig-2-15.avif) |

Fig 2.1-4: TasA protein Congo red measurements. W = OD502/Quality conversion coefficient.
 Control group: \(\textit{tasA}^-\textit{sinR}^-\)*/tasA* strain; Positive control group: WT strain; Experimental group: \(\textit{tasA}^-\textit{sinR}^-\)*/tasA-mfp5* strain, \(\textit{tasA}^-\textit{sinR}^-\)*/tasA-PS tag1* strain, \(\textit{tasA}^-\textit{sinR}^-\)*/tasA-PS tag2* strain; Three repetitions in each group.

According to the results of Congo red staining of TasA protein, the control group with the *tasA* gene knocked out basically did not express TasA protein, while the engineered bacteria introduced with pHT01-tasA-PS-tag1 and pHT01-tasA-PS-tag2 plasmids expressed a higher content of TasA protein. It indicates that the fusion protein plasmid we constructed has a strong ability to express fusion proteins. The amount of TasA protein expressed by *Bacillus subtilis* with the pHT01-tasA-mfp5 plasmid introduced in the figure is less than that of the wild type. This might be due to the excessive molecular weight of the TasA-Mfp5 fusion protein, which has a certain impact on the secretion and assembly of the protein.

Verification of the adhesion function of TasA protein

In order to investigate the function of the pHT01-tasA plasmid, we cultured three strains: wild-type *Bacillus subtilis* 168 (WT), the tasA-sinR mutant strain (\(\textit{tasA}^-\textit{sinR}^-\)), and the \(\textit{tasA}^-\textit{sinR}^-\)*/tasA* knockout strain with the pHT01-tasA plasmid. The cultures were incubated for 24 hours in polystyrene microspheres. Following ultrasonic washing to detach adherent bacteria, the bacterial suspension was diluted with PBS and spread on agar plates for colony-forming unit (CFU) counting.

CFU counts revealed that *Bacillus subtilis* strain 168 exhibited significantly reduced adhesion capacity after *tasA* gene knockout. However, the introduction of exogenous *tasA* gene partially offset this impairment. Strains with exogenous *tasA* expression still showed lower CFU counts compared to wild-type 168 strains, likely due to insufficient levels of induced TasA protein production.

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| ![5](https://static.igem.wiki/teams/5810/results/tasa-fusion-protein/fig-2-6n.avif) |

Fig 2.1-5: Schematic diagram of dilution and coating of three types of bacteria.

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| ![5](https://static.igem.wiki/teams/5810/results/tasa-fusion-protein/fig-2-4.avif) |

Fig 2.1-6: CFU count bar chart.
 Control group: \(\textit{tasA}^-\textit{sinR}^-\) strain; Positive control group: WT strain; Experimental group: \(\textit{tasA}^-\textit{sinR}^-\)*/tasA* strain; Three repetitions in each group.

Meanwhile, we performed crystal violet staining on the *Bacillus subtilis* adhering to the polystyrene spheres. By measuring the changes in the OD570 absorbance of the bacterial liquid eluted from the small ball filter material, the influence of different strains on adhesion was further analyzed.

In the two bacterial crystal violet staining experiments, it can be seen that the OD570 measured by *Bacillus subtilis* with the exogenous *tasA* gene introduced was higher than that of the control group. The results indicate that the adhesion ability of *Bacillus subtilis* with the exogenous *tasA* gene introduced has been improved to a certain extent, and the exogenous TasA protein can enhance the adhesion ability of *Bacillus subtilis* to a certain extent.

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| ![5](https://static.igem.wiki/teams/5810/results/tasa-fusion-protein/fig-2-5.avif) |

Fig 2.1-7: Comparison of crystal violet staining results of two types of bacteria.
 Control group: \(\textit{tasA}^-\textit{sinR}^-\) strain; Experimental group: \(\textit{tasA}^-\textit{sinR}^-\)*/tasA* strain; Three repetitions in each group.

### Adhesion force test

To test the adhesion ability of the TasA fusion protein, we introduced knockout bacteria of the plasmids pHT01-tasA-Mfp5, pHT01-tasA-PS tag1, and pHT01-tasA-PS tag2 (\(\textit{tasA}^-\textit{sinR}^-\)*/tasA-Mfp5*, \(\textit{tasA}^-\textit{sinR}^-\)*/tasA-PS tag1*, \(\textit{tasA}^-\textit{sinR}^-\)*/tasA-PS tag1*) and incubated them with polystyrene microsphere filter media for 24 hours respectively. The bacteria adhering to the polystyrene beads were ultrasonically eluted and then diluted with PBS for coating. CFU counts were conducted and compared with knockout bacteria introduced with the pHT01-tasA plasmid (\(\textit{tasA}^-\textit{sinR}^-\)*/tasA*)for comparative analysis.

It can be known from the CFU count results that the adhesion force of *Bacillus subtilis* introduced with TasA fusion protein granules has been significantly improved compared with the \(\textit{tasA}^-\textit{sinR}^-\)*/tasA* strain. The adhesion of *Bacillus subtilis* introduced with the TasA-Mfp5 fusion protein plasmid was stronger than that of *Bacillus subtilis* introduced with the TasA-PS tag1/TasA-PS tag2 fusion protein plasmid.

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| ![5](https://static.igem.wiki/teams/5810/results/tasa-fusion-protein/fig-2-9n.avif) |

Fig 2.1-8: Schematic diagram of dilution and coating of granular bacteria containing different TasA fusion proteins.

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| ![5](https://static.igem.wiki/teams/5810/results/tasa-fusion-protein/fig-2-8.avif) |

Fig 2.1-9: Bar chart of CFU counts for strains containing different fusion protein granules.
 Control group: \(\textit{tasA}^-\textit{sinR}^-\)/tasA strain; Experimental group: \(\textit{tasA}^-\textit{sinR}^-\)*/tasA-mfp5* strain, \(\textit{tasA}^-\textit{sinR}^-\)*/tasA-PS tag1* strain, \(\textit{tasA}^-\textit{sinR}^-\)*/tasA-PS tag2* strain; Three repetitions in each group.

Meanwhile, we performed crystal violet staining on the *Bacillus subtilis* adhering to the polystyrene spheres. By measuring the changes in the \(\text{OD}\_{570}\) absorbance of the bacterial liquid eluted from the small ball filter material, the differences in adhesion forces of different fusion proteins were further analyzed. The TasA-Mfp5 fusion protein has a stronger adhesion ability compared to the TasA-PS tag1/TasA-PS tag2 fusion protein. Considering that the Mfp5 adhesion protein has a wider range of adhesion activities, we chose the TasA-Mfp5 fusion protein for verification in the subsequent experiments.

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| ![5](https://static.igem.wiki/teams/5810/results/tasa-fusion-protein/fig-2-9.avif) |

Fig 2.1-10: Comparison of crystal violet staining results of granulosa containing different TasA fusion proteins.
 Control group: \(\textit{tasA}^-\textit{sinR}^-\)/tasA strain; Experimental group: \(\textit{tasA}^-\textit{sinR}^-\)*/tasA-mfp5* strain, \(\textit{tasA}^-\textit{sinR}^-\)*/tasA-PS tag1* strain, \(\textit{tasA}^-\textit{sinR}^-\)*/tasA-PS tag2* strain; Three repetitions in each group.

During the CFU counting experiment, the number of adherents of TasA-PS tag1 was higher than that of TasA-PS tag2, but the amount of biofilm adhered by TasA-PS tag1 was greater than that of TasA-PS tag2. The difference in adhesion exhibited by the fusion protein It might be because the environment during the crystal violet staining and elution process has a significant impact on the structure of PS-tag1, resulting in a lower \(\text{OD}\_{570}\) of the TasA-PS tag1 fusion protein in Figure 2.1-11.

### Detection of adhesion force of fusion proteins in sewage environment

Due to the high concentration of heavy metal ions and low environmental pH in the heavy metal wastewater treatment environment, in order to ensure that *Bacillus subtilis* introduced with TasA-Mfp5 fusion protein still exerts adhesion in the wastewater environment, we simulated environmental conditions with different \(\text{Cd}^{2+}\) concentrations and lower pH values. To explore the influence of different environments on the adhesion of the TasA-Mfp5 fusion protein.

We designed SYG media containing different \(\text{Cd}^{2+}\) concentrations (0 mg/L, 0.25 mg/L, 0.5 mg/L, 1 mg/L, 2 mg/L), and incubated the knockout bacteria introduced with pHT01-tasA-mfp5 with polystyrene microsphere filter media for 48 hours. The bacteria adhering to the polystyrene beads were ultrasonically eluted and then diluted with PBS and coated for CFU counting.

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| ![5](https://static.igem.wiki/teams/5810/results/tasa-fusion-protein/fig-2-12n.avif) |

Fig 2.1-11: Schematic diagram of dilution and coating of TasA-Mfp5 fusion protein bacteria at different \(\text{Cd}^{2+}\) concentrations.

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| ![5](https://static.igem.wiki/teams/5810/results/tasa-fusion-protein/fig-2-13n.avif) |

Fig 2.1-12: Bar charts of CFU counts of TasA-Mfp5 fusion protein bacteria at different \(\text{Cd}^{2+}\) concentrations.

It can be known from CFU counts that within a certain range of \(\text{Cd}^{2+}\) concentrations (0-0.5mg/L), with the increase of \(\text{Cd}^{2+}\) concentration, the adhesion ability of the TasA-Mfp5 fusion protein decreases to a certain extent, but there were no significant differences.

We simultaneously designed SYG media with different pH values (pH=2, 3, 4, 5, 6, 7). The knockout bacteria introduced with pHT01-tasA-mfp5 were incubated and cultured with polystyrene microsphere filter media for 48 hours. The bacteria adhering to the polystyrene microspheres were ultrasonically eluted and then diluted with PBS and spread for CFU counting.

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| ![5](https://static.igem.wiki/teams/5810/results/tasa-fusion-protein/fig-2-14n.avif) |

Fig 2.1-13: Schematic diagram of dilution and coating of TasA-Mfp5 fusion protein bacteria under different environmental pH conditions.

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| ![5](https://static.igem.wiki/teams/5810/results/tasa-fusion-protein/fig-2-13.avif) |

Fig 2.1-14: Bar charts of CFU counts of TasA-Mfp5 fusion protein bacteria under different environmental pH conditions.

It can be known from the CFU count that when the pH value of the living environment is too low (pH=2), the growth of TasA-Mfp5 fusion protein bacteria is significantly inhibited. With the increase of pH (pH=3), the inhibitory effect of environmental pH on the growth of TasA-Mfp5 fusion protein bacteria was relieved to a certain extent; In a weakly acidic environment (pH=4-6), there was no significant difference in the CFU count of TasA-Mfp5 fusion protein bacteria, and the adhesion ability was less affected by the environmental PH. When pH= 7, the CFU count of TasA-Mfp5 fusion protein bacteria decreased significantly. This might be because the surface of the polystyrene microspheres incubated with the bacterial liquid was damaged during the experiment, and the adhesion process with the fusion protein was affected.

### 2.2 SpyTag-SpyCatcher System

### Background and Introduction

SpyTag is a short peptide that forms an isopeptide bond upon encountering its protein partner SpyCatcher. This covalent peptide interaction is a simple and powerful tool for bioconjugation and extending what protein architectures are accessible. Originated from fibronectin binding protein FbaB, SpyTag-SpyCatcher exhibit spontaneous reconstruction ability under a wide range of pH values, redox conditions and temperatures, which can perfectly fit in harsh application conditions. In the practical application of the TasAnchor system, there may be issues with the TasA fusion protein being relatively large, making it difficult to express and secrete. By inserting SpyTag-SpyCatcher into our adhesion system, we can further improve the modularity of TasAnchor, make it easier to adapt to various filter materials.

### Plasmid Construction

We use Gibson Assembly to construct pHT01-TasA-SpyTag. We transferred it into *E. coli* \(\text{DH5}\_\alpha\) to amplify, then transformed the plasmid into \(\textit{sinR}^-\textit{tasA}^-\) *B. subtilis* 168 for expression. We also use Gibson Assembly to construct pET28(a)-mCherry-SpyCatcher and pET28(a)-Mfp5-SpyCatcher, transferred into *E. coli* \(\text{DH5}\_\alpha\) to amplify, then transformed the plasmid into *E. coli* BL21(DE3) for expression.

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| ![5](https://static.igem.wiki/teams/5810/results/spytag-spycatcher-system/fig-2-1.webp) |

Fig 2.2-1: pHT01-TasA-SpyTag, pET28(a)-mCherry-SpyCatcher and pET28(a)-Mfp5-SpyCatcher plasmids.

Sequence *spytag* is inserted based on formerly constructed plasmid pHT01-TasA. Plasmid pET28(a)-Mfp5-SpyCatcher is constructed based on original pET28(a), and oligonucleotide *mfp5-spycatcher* is purchased from company. Plasmid pET28(a)-mCherry-spycatcher is constructed based on pET28(a)-Mfp5-SpyCatcher, while mCherry is derived from plasmid mCherry-Rab7a-7. Sequence of related primers are as follows:

Table 2.2-1: Related primers of SpyTag-SpyCatcher system.

| Plasmid | Forward Primer | Reverse Primer |
| --- | --- | --- |
| pHT01-TasA | TCAACCATAACGATGTGAGCAGAACCACCACCACCATTTTTATCCTCGCTATGCG | TCACATCGTTATGGTTGATGCTTACAAACCTACTAAATAAGCAGCCCGCCTAATGAG |
| pET28(a) | TGAGATCCGGCTGCTAACAA | CATGGTATATCTCCTTCTTAAAG |
| Mfp5-SpyCatcher | CTTTAAGAAGGAGATATACCATGAGCTCTGAAGAATACAA | TTGTTAGCAGCCGGATCTCATTAGTGGTGATGGTGGTGGT |
| pET28(a)-SpyCatcher | CATGGTATATCTCCTTCTTAAAG | GTAGCGGTGGTGGTGGTTCT |
| pET28(a)-SpyCatcher | CTTTAAGAAGGAGATATACCATGGTGAGCAAGGGCGAG | AGAACCACCACCACCGCTACCGCCGCCACCCTTGTACAGCTCGTCCATGC |

The sequencing results separately show that SpyTag was successfully inserted into pHT01-TasA, and pET28(a)-Mfp5-SpyCatcher and pET28(a)-mCherry-SpyCatcher are successfully constructed as well.

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| ![5](https://static.igem.wiki/teams/5810/results/spytag-spycatcher-system/fig-2-2.webp) |

Fig 2.2-2: Sequencing results of pHT01-TasA-SpyTag, pET28(a)-mCherry-SpyCatcher and pET28(a)-Mfp5-SpyCatcher.

We also performed colony PCR with corresponding primers to verify whether our plasmids are successfully transferred into *B. subtilis* or *E. coli* BL21(DE3).

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| ![5](https://static.igem.wiki/teams/5810/results/spytag-spycatcher-system/fig-2-3.webp) |

Fig 2.2-3: Colony PCR result of pHT01-TasA-SpyTag \(\textit{sinR}^-\textit{tasA}^-\) *B. subtilis* 168.

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| ![5](https://static.igem.wiki/teams/5810/results/spytag-spycatcher-system/fig-2-4.webp) |

Fig 2.2-4: Colony PCR result of pET28(a)-Mfp5-SpyCatcher *E. coli* BL21(DE3).

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| ![5](https://static.igem.wiki/teams/5810/results/spytag-spycatcher-system/fig-2-5.webp) |

Fig 2.2-5: Colony PCR result of pET28a-mCherry-SpyCatcher *E. coli* BL21(DE3).

### SpyTag-SpyCatcher Interaction Experiment

Before conducting the adhesion experiment, we decided to validate the SpyTag-SpyCatcher system in *Bacillus subtilis*. We decided to incubate the mCherry-SpyCatcher protein with *Bacillus subtilis* expressing TasA-SpyTag on the surface to observe whether there was fluorescence binding.

First, we need to purify mCherry-SpyCatcher protein. We inoculated overnight-cultured transformed *E. coli* BL21(DE3) into 50ml LB liquid medium containing 50μg/ml kanamycin at a rate of 1%. IPTG was added to 1mM when the OD600 of the culture system reached 0.6-0.8. After 14 hours of 24℃, 220rpm shaking culture, the bacteria was ultrasonic crushed, and total bacterial protein was extracted.

We use Ni-NTA prepacked column to purify our target protein since they are anchored with 6xHisTag, which can combine Ni-NTA column. Related samples underwent SDS-PAGE and Coomassie Blue staining to testify the purification procedure.

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| ![5](https://static.igem.wiki/teams/5810/results/spytag-spycatcher-system/fig-2-6.webp) |

Fig 2.2-6: pET28(a)-mCherry-SpyCatcher *E. coli* BL21(DE3) expressing red fluorescence.

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| ![5](https://static.igem.wiki/teams/5810/results/spytag-spycatcher-system/fig-2-7.webp) |

Fig 2.2-7: SDS-PAGE results of mCherry-SpyCatcher(~40.2kDa).

We incubated mCherry-SpyCatcher with the biofilm formed by pHT01-TasA-SpyTag *B. subtilis* 168 and \(\textit{sinR}^-\textit{tasA}^-\) *B. subtilis* 168, and take picture of it under fluorescence microscope. The results show that SpyCatcher can successfully bind to TasA-SpyTag on cell wall.

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| ![5](https://static.igem.wiki/teams/5810/results/spytag-spycatcher-system/fig-2-8.webp) |

Fig 2.2-8: mCherry-SpyCatcher incubated *B. subtilis* 168 under excitation laser of DsRed. (a) control (\(\textit{sinR}^-\textit{tasA}^-\) *B. subtilis* 168) (b) pHT01-TasA-SpyTag.

### Adhesion Function Examination

Since we plan to incubate Mfp5-SpyCatcher protein with transformed *B. subtilis* to test its adhesion ability, we need to extract and purify the Mfp5-SpyCatcher protein. We conducted SDS-PAGE analysis on the purified protein, demonstrating that the protein was successfully purified.

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| ![5](https://static.igem.wiki/teams/5810/results/spytag-spycatcher-system/fig-2-9.webp) |

Fig 2.2-9: SDS-PAGE results of Mfp5-SpyCatcher(~22.8kDa).

We incubated *B. subtilis* 168 with polystyrene balls and Mfp5-SpyCatcher protein, and performed gradient dilution coating to measure the colony-forming units(CFU) per volume of polystyrene balls' eluent. The result revealed that SpyTag-SpyCatcher system exhibit an even better adhesion ability than fusion protein.To further verify and quantify the adhesion ability of our system, we also use crystal violet staining to indicate the small fluctuate of bacteria concentration between eluent from different bacteria incubated-polystyrene balls.

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| ![5](https://static.igem.wiki/teams/5810/results/spytag-spycatcher-system/fig-2-10.webp) |

Fig 2.2-10: 100x dilution spread plate results of related *B. subtilis* 168.

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| ![5](https://static.igem.wiki/teams/5810/results/spytag-spycatcher-system/fig-2-11.webp) |

Fig 2.2-11: Quantified results of adhesion function examination. (a) histogram of eluent's colony-forming units; (b) histogram of crystal violet staining.

We speculate that the adhesion effect of TasA-SpyTag/Mfp5-SpyCatcher is better than that of TasA-Mfp5, which may be due to the smaller molecular weight of TasA-SpyTag, making it easier to express and secrete, and displaying more TasA-SpyTag molecules on bacterial surfaces. To this end, we will compare the amount of TasA fusion protein fibers expressed on the surface of TasA-SpyTag and TasA-Mfp5 using Congo red staining. As expected, TasA-SpyTag expression secretes more TasA fusion protein, resulting in a stronger adhesion effect than TasA-Mfp5.

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| ![5](https://static.igem.wiki/teams/5810/results/2-2-12-n.avif) |

Fig2.2-12 Comparison of Congo red staining between Mfp5-SpyTag and SpyCatcher-Mfp5

### Conclusion

By anchoring binding proteins with TasA, we successfully constructed a platform that can easily bind to the target material. To improve the modularity of our project, we introduced SpyTag-SpyCatcehr system between TasA and binding protein, which surprisingly improved the adhesion ability of TasAnchor. But sadly, due to time limitation, we simply tested the adhesion ability of TasAnchor with Mfp5 and PS-Tag. In future experiments, we will introduce more binding proteins to TasAnchor to verify the availability as well as flexibility of this platform. We also plan to integrate this system into the chromatin of \(\textit{sinR}^-\textit{tasA}^-\) *B. subtilis* 168 to eliminate the negative effect from our plasmids.

## 3. Function Test Module

### 3.1 *epcadR-pcadR-mcherry* Sensing Functional Module

### Construction and Verification of the pHT01-*epcadR-pcadR-mcherry* Plasmid

Using *mcherry* as the reporter gene, the plasmid pHT01-epcadR-pcadR-mcherry was constructed to evaluate the response of the *epcadR-pcadR* module to environmental cadmium ion concentrations. Sequencing results confirmed the successful assembly of all functional elements.

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| ![5](https://static.igem.wiki/teams/5810/results/function-test-module/fig1.avif) |

Fig 3.1-1: Plasmid map of pHT01-*epcadR-pcadR-mcherry*.

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| ![5](https://static.igem.wiki/teams/5810/results/function-test-module/fig2.avif) |

Fig 3.1-2: Sequencing result of pHT01-*epcadR-pcadR-mcherry*.

The pHT01-epcadR-pcadR-mcherry plasmid was introduced into the *ΔtasAΔsinR* *Bacillus subtilis* 168 strain using a competent cell preparation kit. Colony PCR confirmed the successful introduction of the recombinant plasmid.

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| ![5](https://static.igem.wiki/teams/5810/results/function-test-module/fig3.avif) |

Fig 3.1-3: Colony PCR was performed to verify the successful introduction of the pHT01-*epcadR-pcadR-mcherry* recombinant plasmid.

### Growth Curves of Plasmid-Free Knockout Strains and Engineered Strains under Different Cadmium Ion Concentrations

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| ![5](https://static.igem.wiki/teams/5810/results/fig-3-1-4.avif) |

Fig 3.1-4: Growth curves of the two strains under different cadmium ion concentrations.

The results indicate that, at cadmium ion concentrations measured in mg/L, both strains exhibited rapid growth, suggesting minimal inhibitory effects at these levels. The strongest inhibition was observed at 1.5 mg/L, while 0.5 mg/L and 0.25 mg/L showed relatively weak inhibition with little difference between them. Overall, cadmium ions at the 0-1.5 mg/L scale exert only minor effects on the growth of the engineered strain, which is capable of robust growth under the experimental cadmium concentrations. Furthermore, comparison of the two strains indicates that the introduction of the exogenous plasmid has negligible impact on the cadmium tolerance of the engineered strain.

### Fluorescence Response Curves of Engineered Strains to Different Cadmium Ion Concentrations over Time

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| ![5](https://static.igem.wiki/teams/5810/results/fig-3-1-5.avif) |

Fig 3.1-5: Time-course of fluorescence intensity in engineered strains under different cadmium ion concentrations.

Under treatments with 0, 1, and 2 mmol/L cadmium ions, fluorescence intensity in the pHT01-*epcadR-pcadR-mcherry* strain showed a significant positive correlation with both cadmium concentration and incubation time. The strongest fluorescence was observed at 2 mmol/L, reaching a peak at 25 h, while the 0 mmol/L control group maintained a consistently low fluorescence level (less than 30 FU), demonstrating that cadmium ions effectively induce fluorescence expression.

### 3.2 *tasA-smtA* Adsorption Functional Module

### Construction and Verification of the pHT01-*tasA-smtA* Plasmid

We first used the pHT01-tasA plasmid as a template for reverse PCR to obtain the vector backbone. Subsequently, the *smtA* fragment was joined to this backbone via Gibson assembly, resulting in the successful construction of the pHT01-*tasA-smtA* recombinant plasmid. Sequencing results confirmed the correct assembly of all functional elements.

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| ![5](https://static.igem.wiki/teams/5810/results/function-test-module/fig7.avif) |

Fig 3.2-1: Plasmid map of pHT01-*tasA-smtA*.

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| ![5](https://static.igem.wiki/teams/5810/results/function-test-module/fig8.avif) |

Fig 3.2-2: Sequencing result of pHT01-*tasA-smtA*.

The pHT01-tasA-smtA plasmid was introduced into the *ΔtasAΔsinR Bacillus subtilis* 168 strain using a competent cell preparation kit. Colony PCR confirmed the successful introduction of the recombinant plasmid.

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| ![5](https://static.igem.wiki/teams/5810/results/function-test-module/fig9.avif) |

Fig 3.2-3: Colony PCR was performed to verify the successful introduction of the pHT01-*tasA-smtA* recombinant plasmid.

### Effects of Cadmium Ions on Biofilm Formation in Engineered Strains

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| ![5](https://static.igem.wiki/teams/5810/results/function-test-module/fig10.avif) |

Fig 3.2-4: Results of Crystal Violet Staining of Biofilms Formed by Engineered Strains under Different Cadmium Ion Concentrations.

TasA is a key structural protein involved in biofilm formation and a major component of the extracellular matrix; therefore, the metal ion adsorption capacity of the TasA-SmtA fusion protein is closely associated with the biofilm formation status of the engineered strain. To ensure that this adsorption module functions under normal biofilm conditions, we examined the effects of different cadmium ion concentrations on biofilm formation in the engineered strain.
Strains carrying pHT01-*tasA-smtA* were cultured under varying cadmium ion concentrations, and after 36 h of incubation, the formed biofilms were stained with crystal violet. Biofilm production was quantified by measuring OD₅₇₀. The results showed that within the cadmium concentration range of 0-9 mg/L, biofilm formation of the pHT01-*tasA-smtA* strain was not significantly affected. These findings indicate that the TasA-SmtA module can stably form biofilm across a wide range of environmental cadmium concentrations.

### Adsorption-desorption Curves of Cadmium Ions by Engineered Strains

SmtA protein forms reversible complexes with Cd²⁺ ions through metal-coordinating sites such as thiol and carboxyl groups. Under high pH conditions, these sites are deprotonated, facilitating Cd²⁺ adsorption, whereas under low pH conditions, protonation promotes Cd²⁺ desorption, enabling controlled release of the metal ions. Based on this mechanism, adsorption-desorption curves of the engineered strain were measured in 9 mg/L and 5 mg/L Cd²⁺ solutions, demonstrating its potential for repeated use.

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| ![5](https://static.igem.wiki/teams/5810/results/function-test-module/3-2-5.avif) |

Fig 3.2-5: Adsorption-desorption Curves of Cadmium Ions by the pHT01-tasA-smtA Engineered Strain.

The engineered strain underwent sequential adsorption-desorption cycles, repeated three times over a total of two rounds. As shown in the figure, for the 9 mg/L Cd²⁺ solution, the pHT01-*tasA-smtA* strain maintained high and stable adsorption capacity throughout the cycles. The initial adsorption nearly achieved 100% removal of cadmium ions. Although desorption did not remove all bound Cd²⁺, the engineered strain still exhibited substantial adsorption efficiency.

For the 5 mg/L Cd²⁺ solution, the initial adsorption efficiency was relatively low; however, the strain demonstrated 100% desorption efficiency during the cycles, indicating good regenerability. Moreover, adsorption efficiency increased noticeably after the first desorption, which may be attributed to activation of SmtA proteins displayed on the cell surface under low pH conditions.

The cadmium ion adsorption efficiency of the pHT01-*tasA-smtA* engineered strain was evaluated using a 9 mg/L Cd²⁺ solution. The results showed that during the initial adsorption, the strain achieved a 100% removal rate, effectively adsorbing all Cd²⁺ in the solution. After two rounds of desorption under low pH conditions, the adsorption efficiency decreased but remained at approximately 32%. These findings indicate that the engineered strain not only efficiently removes cadmium ions from the environment but also retains substantial adsorption activity after multiple desorption cycles, demonstrating strong potential for regeneration and reuse.

### 3.3 *epcadR-pcadR-mcherry-tasA-mfp5* Sensing-adhesion Functional Module

### Construction and Verification of the Sensing-adhesion Composite Plasmid

To enable the engineered strain to adhere to biofilter materials and perform cadmium ion sensing, a dual-module “sensing-adhesion” expression plasmid was constructed. Preliminary experiments indicated that among various candidate adhesion proteins, Mfp5 exhibited the most favorable surface-binding performance. Compared with the SpyTag-SpyCatcher system, the TasA-Mfp5 system offered a simpler construction process without requiring additional protein purification steps. Based on these advantages, the TasA-Mfp5 adhesion module was integrated with the *epcadR-pcadR-mcherry* sensing module to evaluate the cadmium-responsive capability of the engineered strain in an attached state.

To enhance overall expression efficiency, the strong constitutive promoter p43 was used to replace the original promoter sequence. Subsequently, the recombinant plasmid pHT01-p43-epcadR-mcherry-p43-tasA-mfp5 was successfully obtained via homologous recombination. This plasmid endows the host strain with stable adhesion to polystyrene microspheres and enables a fluorescent response in the presence of exogenous cadmium ions.

Sequencing verification confirmed the correct construction of the target plasmid, providing an experimental foundation for subsequent quantitative detection and characterization of cadmium ion responsiveness.

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| ![5](https://static.igem.wiki/teams/5810/results/function-test-module/fig13.avif) |

Fig 3.3-1: Plasmid map of pHT01-p43-epcadR-mcherry-p43-tasA-mfp5.

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| ![5](https://static.igem.wiki/teams/5810/results/fig-3-3-2.avif) |

Fig 3.3-2: Sequencing result of pHT01-p43-epcadR-mcherry-p43-tasA-mfp5.

The pHT01-p43-epcadR-mcherry-p43-tasA-mfp5 plasmid was introduced into the *ΔtasAΔsinR Bacillus subtilis* 168 strain using a competent cell preparation kit. Colony PCR confirmed the successful introduction of the recombinant plasmid.

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| ![5](https://static.igem.wiki/teams/5810/results/function-test-module/fig15.avif) |

Fig 3.3-3: Colony PCR was performed to verify the successful introduction of the pHT01-*p43-epcadR-mcherry-p43-tasA-mfp5* plasmid into the *ΔtasAΔsinR Bacillus subtilis* 168 strain.

### Verification of Fluorescence Expression under the Adherent State

In the experiment, we incubated the polystyrene globules with the SYG film-forming medium of the engineered bacteria for 30 h, then used the incubated globules for cadmium ion adsorption experiments, and then incubated with 2mmol/L cadmium ion solution for 16h, and then observe fluorescence under a microscope.

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| ![5](https://static.igem.wiki/teams/5810/results/function-test-module/fig16.avif) |

Fig 3.3-4: Fluorescence Microscopy Observation of *ΔtasAΔsinR Bacillus subtilis* 168 Strain Carrying pHT01-*p43-epcadR-mcherry-p43-tasA-mfp5* and the *ΔtasAΔsinR Bacillus subtilis* 168 Strain.

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| ![5](https://static.igem.wiki/teams/5810/results/function-test-module/fig17.avif) |

Fig 3.3-5: Observation of Polystyrene Microspheres under Yellow Light (leftmost: *ΔtasAΔsinR Bacillus subtilis* 168 carrying pHT01-*p43-epcadR-mcherry-p43-tasA-mfp5*; second from left: *ΔtasAΔsinR Bacillus subtilis* 168).

Polystyrene microspheres with adhered pHT01-*p43-epcadR-mcherry-p43-tasA-mfp5* cells exhibited pronounced red fluorescence under a fluorescence microscope after induction with 2 mmol/L cadmium chloride, the concentration producing maximal fluorescence. The fluorescence was concentrated on the bacterial cells. From a macroscopic perspective, under yellow light, microspheres carrying the pHT01-*p43-epcadR-mcherry-p43-tasA-mfp5* strain appeared redder compared to those with the *ΔtasAΔsinR* strain.

These results demonstrate that the pHT01-*p43-epcadR-mcherry-p43-tasA-mfp5* strain can simultaneously adhere to polystyrene microspheres and respond to cadmium ions by expressing fluorescence, confirming the successful construction of the engineered strain with a dual “sensing-adhesion” functional module.

### 3.4 Verification of Adsorption-adhesion Function Modules

In order to enable engineered bacteria to finally adhere to the biological filter material to play the function of adsorbing cadmium ions, we plan to construct an expression plasmid pHT01-*p43-tasA-smtA-p43-tasA-mfp5* with a dual module of "adsorption-adhesion". However, we found that the co-expression of two TasA fusion proteins would seriously affect the growth of the strains, and the transformation efficiency of the strains was low and the growth was very slow, making it difficult to carry out experimental operations.

In the experiment, we incubated the polystyrene globules with the SYG film-forming medium of the engineered bacteria for 30 h, then used the incubated globules for cadmium ion adsorption experiments, and then incubated with 9mg/L cadmium ion solution for 15h, and then measured the cadmium ion concentration to calculate the adsorption capacity of the polystyrene globules loaded with engineering bacteria for cadmium ions.

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| ![5](https://static.igem.wiki/teams/5810/results/function-test-module/3-4-1.avif) |

Fig 3.4-1: Adsorption efficiency of cadmium ions in polystyrene spheres incubated by pHT01-*tasA-smtA* engineering bacteria.

The experimental results showed that some pHT01-*tasA-smtA* engineered bacteria could stably attach to the surface of polystyrene spheres and exert the adsorption function of Cd²⁺, and their treatment capacity for Cd²⁺ was higher than that of the control group. We speculate that if engineered bacteria expressing TasA-Mfp5 protein are used, the adsorption effect may be further increased because of its stronger adhesion effect and larger bacterial volume.

## 4. Safety Module

### Quorum Sensing Induced Suicide Systems

In order to ensure the biosafety of *Bacillus subtilis* applied in heavy metal wastewater treatment, we designed a density-controlled suicide system based on the quorum-sensing pathway *ComQXPA* and the toxin-antitoxin pair mazEF. This regulatory circuit allows the engineered strain to survive and function at high cell density on fillers, while automatically initiating self-elimination when cells detach and enter a low-density environment.

### 4.1 *ComQXPA* Induction System

According to the literature, the *ComQXPA* system of *B. subtilis* can regulate gene expression in response to extracellular signaling peptides ComX. When cell density is high, accumulated ComX activates the ComP-ComA cascade, inducing the \(\text{P}\_{\text{srfA}}\) promoter.

### 4.1.1 pHT01-\(\text{P}\_{\text{srfA}}\)-mCherry Plasmid construction

Using *mCherry* as a reporter gene, we constructed a plasmid pHT01-\(\text{P}\_{\text{srfA}}\)-mCherry to validate the response of the \(\text{P}\_{\text{srfA}}\) to quorum-sensing signals in *Bacillus subtilis*. This plasmid allows fluorescence reporting of promoter activity controlled by the endogenous quorum-sensing system *ComQXPA*.

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| ![5](https://static.igem.wiki/teams/5810/results/safetymodule/fig-1.webp) |

Fig 4-1: Plasmid of pHT01-\(\text{P}\_{\text{srfA}}\)-mCherry.

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| ![5](https://static.igem.wiki/teams/5810/results/safetymodule/fig-2.webp) |

Fig 4-2: Sequence results of pHT01-\(\text{P}\_{\text{srfA}}\)-mCherry.

Sequencing results confirmed the successful assembly of all functional elements.

Interestingly, a single point mutation (AAA → AAG) was detected within the coding sequence; however, as both codons encode lysine, this is a synonymous mutation that does not affect protein function. Therefore, the construction of pHT01-\(\text{P}\_{\text{srfA}}\)-mCherry was considered successful, and the plasmid was used in subsequent experiments to evaluate \(\text{P}\_{\text{srfA}}\) responsiveness and activity.

### 4.1.2 \( \text{P}\_{\text{srfA}} \) Activation Threshold Analysis

To determine the threshold at which our density-dependent system is activated, we measured the response of the \(\text{P}\_{\text{srfA}}\) to endogenous quorum-sensing signals generated by the *ComQXPA* system of *Bacillus subtilis*. Since ComX, the signaling peptide, is naturally secreted and accumulated during cell growth, no exogenous inducers were added in this experiment.

Furthermore, colony PCR verification confirmed that the recombinant plasmid was successfully transformed into *Bacillus subtilis*.

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| ![5](https://static.igem.wiki/teams/5810/results/safetymodule/fig-3.webp) |

Fig 4-3: Colony PCR result of *mCherry* validation, 1-11: fragments of *mCherry*.

Then we cultured 100 mL of *B. subtilis* harboring pHT01-\(\text{P}\_{\text{srfA}}\)-mCherry in shaking flasks at 37℃ and monitored \(\text{OD}\_{600}\) and fluorescence intensity every hour. As results shown, the fluorescence began to rise significantly when \(\text{OD}\_{600}\) reached approximately 0.8-1.0, suggesting that the \(\text{P}\_{\text{srfA}}\) promoter was activated at this population density. The fluorescence then peaked when \(\text{OD}\_{600}\) approached 1.2, corresponding to the stationary phase, and gradually decreased thereafter, consistent with the quorum-sensing activation pattern.

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| ![5](https://static.igem.wiki/teams/5810/results/safetymodule/fig-4.webp) |

Fig 4-4: Growth curve and fluorescence dynamics of *B. subtilis* harboring pHT01-\(\text{P}\_{\text{srfA}}\)-mCherry.

The results indicate that the \(\text{P}\_{\text{srfA}}\) promoter has a density activation threshold, which enables the suicide circuit to respond precisely to bacterial population changes within the reactor. This threshold aligns with our system design goal: allowing cells to survive and function under high-density biofilm conditions while triggering the self-elimination module when cell density drops.

### 4.2 MazF Toxin Proteins

MazF is a toxic protein in the mazF/mazE toxin-antitoxin system, and it is function as mRNA endonuclease. In our experimental design, we utilized the MazF protein as the toxin component in a suicide system to validate its feasibility and effectiveness in *Bacillus subtilis*. We evaluated the function of MazF protein by designing a validation gene circuit to characterize it.

### 4.2.1 pHT01-mazF Plasmid Construction

The expression of *mazF* is regulated using IPTG induction in the prophase and metaphase of characterization validation. pHT01-mazF plasmid in the genetic circuit consists of the lazO/lacI system, \(\text{P}\_{\text{Grac}}\), and *mazF*. We constructed the pHT01-mazF plasmid and demonstrated the expression of the mazF toxin protein in the chassis as well as the characterization of its bactericidal function through IPTG induction experiments.

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| ![5](https://static.igem.wiki/teams/5810/results/safetymodule/fig-5.webp) |

Fig 4-5: Plasmid of pHT01-mazF.

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| ![5](https://static.igem.wiki/teams/5810/results/safetymodule/fig-6.webp) |

Fig 4-6: Sequence of pHT01-mazF.

### 4.2.2 pHT01-mazF Plasmid Transformation

To validate the function of our constructed plasmid in the chassis organism, we transformed the pHT01-mazF plasmid into *B. subtilis* and characterized the bactericidal effect of the MazF toxin protein on the host.

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| ![5](https://static.igem.wiki/teams/5810/results/safetymodule/fig-7.webp) |

Fig 4-7: Correct result of Colony PCR. 1-3: fragments of mazF.

### 4.2.3 MazF Functional Validation

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| ![5](https://static.igem.wiki/teams/5810/results/add01.avif) |

Fig 4-8: Results of IPTG gradient plate induction experiments.

We validated the function of *mazF* through IPTG gradient plate test. The maximum induction concentrations of IPTG on the gradient plates were set at 0.6 mg/mL, 0.8 mg/mL, and 1.0 mg/mL. The experimental results demonstrated a decreasing trend in the number of colony-forming units with increasing IPTG concentrations across the gradient plates, indicating that IPTG-induced expression of *mazF* exerts a bactericidal effect.

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| ![5](https://static.igem.wiki/teams/5810/results/add02.avif) |

Fig 4-9: Results of induction experiments with different concentrations of IPTG plates.

We conducted IPTG plate induction assays at varying concentrations to further investigate the bactericidal effect of the *mazF* protein under different induction levels. As the IPTG concentration increased, the number of colony-forming units in the chassis showed a significant declining trend. We observed that the *mazF* toxin began to exhibit a noticeable bactericidal effect at an IPTG concentration of 0.4 mg/mL.

### 4.3 MazE Antitoxin Proteins

In the practical application of the topic, we need engineering bacteria to survive within a certain range of high density. At this time, MazF may also have a low amount of leakage expression, which means that bacteria need to survive in a low expression state of MazF. For this, we introduced the antitoxin MazE, which allows bacteria to survive when MazF is leaked at low levels by antagonizing MazF.

### 4.3.1 pHT01-mazF-\(\text{P}\_{43}\)-mazE Plasmid Construction

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| ![5](https://static.igem.wiki/teams/5810/results/safetymodule/fig-10.webp) |

Fig 4-10: Plasmid of pHT01-mazF-\(\text{P}\_{43}\)-mazE.

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| ![5](https://static.igem.wiki/teams/5810/results/safetymodule/fig-11.webp) |

Fig 4-11: Figure 4-7 Correct result of Colony PCR.
1-12: fragments of mazF-mazE;
13: No-Template Control (NTC).

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| ![5](https://static.igem.wiki/teams/5810/results/safetymodule/fig-12.webp) |

Fig 4-12: Sequence of pHT01-mazF-\(\text{P}\_{43}\)-mazE.

As the third circuit of our biosafety design, we successfully constructed the plasmid pHT01-\(\text{P}\_{\text{Grac}}\)-mazF-\(\text{P}\_{43}\)-mazE. The construction process involved PCR amplification of the p43 promoter and the mazE gene fragment, followed by enzymatic linearization of the plasmid pHT01 -mazF. These fragments were then assembled via multi-fragment homologous recombination, ultimately forming an IPTG-regulated suicide system.

### 4.3.2 MazE Functional Validation

To validate the toxic effect of *mazF* and the inhibitory role of *mazE* against the toxin in pHT01-mazF-\(\text{P}\_{43}\)-mazE, we prepared IPTG concentration gradient plates ranging from 0 mg/mL to 1.2 mg/mL across eight gradients. The bacterial culture of Plasmid of pHT01-mazF-\(\text{P}\_{43}\)-mazE was diluted to an appropriate concentration, plated, and incubated, after which colony numbers were counted.

The results showed that as IPTG concentration increased, the induction of the \(\text{P}\_{\text{Grac}}\) promoter was enhanced, leading to elevated expression of the mazF gene. In contrast, the constitutive expression of mazE mediated by the \(\text{P}\_{43}\) promoter provided limited counteracting effects, resulting in an overall increase in colony numbers of the plasmid of pHT01-mazF-\(\text{P}\_{43}\)-mazE engineered bacteria.

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| ![5](https://static.igem.wiki/teams/5810/results/safetymodule/fig-13.webp) |

Fig 4-13: Reduction in colony counts by IPTG induction.

When the IPTG concentration range was 0-0.4, we found that compared with the MazF control group, we found that the number of colonies in the experimental group expressing MazE was only slightly reduced, indicating that MazE could provide better antagonistic effect when MazF was expressed in the low concentration range. This circuit enables regulated expression of the toxin protein MazF, while the constitutive expression of the antagonist protein MazE ensures bacterial stability under normal working conditions, preventing leaky expression of the toxin gene. Furthermore, it provides a foundational vehicle for the subsequent introduction of the quorum-sensing module in overall biosafety part.

### 4.4 Overall Biosafety Part

### 4.4.1 pHT01-\(\text{P}\_{\text{srfA}}\)-mazF-\(\text{P}\_{43}\)-mazE Plasmid Construction

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| ![5](https://static.igem.wiki/teams/5810/results/safetymodule/fig-4-14n.avif) |

Fig 4-14: Plamid of pHT01-\(\text{P}\_{\text{srfA}}\)-mazF-\(\text{P}\_{43}\)-mazE.

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| ![5](https://static.igem.wiki/teams/5810/results/safetymodule/fig-15.webp) |

Fig 4-15: Correct result of Colony PCR. 1-5: fragments of \(\text{P}\_{\text{srfA}}\)-mazF-\(\text{P}\_{43}\)-mazE.

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| ![5](https://static.igem.wiki/teams/5810/results/safetymodule/fig-4-16n.avif) |

Fig 4-16: Sequence results of pHT01-\(\text{P}\_{\text{srfA}}\)-mazF-\(\text{P}\_{43}\)-mazE.

As the final and complete part of our biosafety part, we constructed a plasmid with Quorum Sensing Induced promoter \(\text{P}\_{\text{srfA}}\), which will be activated by Endogenous phosphorylation; with toxin protein gene *mazF* controlled by *lacI*, and its antagonist protein gene *mazE*, we finally completed the effect that when the engineered bacteria escape into the environment, the suicide switch is activated without affecting their normal growth in a colony state in the working environment.

To ensure this effect is noticeable, we conducted a 12-hour-trace to form its growth curve between wild type and the engineered bacteria.

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| ![5](https://static.igem.wiki/teams/5810/results/4-17-n.avif) |

Fig 4-17: Growth curve for wild type strain and pHT01-\(\text{P}\_{\text{srfA}}\)-mazF-\(\text{P}\_{43}\)-mazE strain

We can notice that, compared with WT, pHT01-\(\text{P}\_{\text{srfA}}\)-mazF-\(\text{P}\_{43}\)-mazE strain indeed show lower trend in growth curve at first 4 hours, which indicates the suicide system performs well when bacteria concentration is low.

## 5. Outlook

In the TasAnchor experiment, we improved and expanded the surface display system of *Bacillus subtilis* TasA by fusing it with Mfp5, which has broad binding affinity, and PS tags, which have specific binding affinity to polystyrene. This enhanced the colonization and biofilm formation effects of Bacillus subtilis on polystyrene biofilter filter media, and also verified the feasibility of using the SpyTag-SpyCatcher system for adhesion. Subsequently, we introduced cadmium ion induction and adsorption circuits into the engineering bacteria separately, verifying their adsorption, induction, and elution of cadmium ions. We attempted to combine the adhesion module and cadmium ion treatment module to demonstrate that the engineering bacteria can perform cadmium ion treatment under the condition of being fixed on a polystyrene solid substrate. At the same time, we also designed a biosafety module and validated a density controlled suicide switch to ensure that when bacteria detach from the solid matrix, the suicide switch can be activated to prevent leakage. The components of TasAnchor are modular design, which can address the adhesion of engineered bacteria in different environments by replacing different binding proteins.

In the iGEM competition cycle, the number of repetitions and technical iterations of our experiments is limited. Now we only try to adhere to one material——polystyrene foam filter material, and only use three binding proteins for verification. If we want to further confirm the TasAnchor system, we need to use more materials and binding proteins for verification, and enhance the credibility of the system by simulating actual environments, which is also the direction of our future experiments.

In terms of specific details of the experiment, we found that overexpression of TasA related proteins may affect the normal growth of the strain. Therefore, we used an inducible promoter *pGrac*, which indicates that in practical use, the system needs to adhere to a solid substrate through pre-treatment under IPTG conditions, and then transfer to a specific environment to exert its function. This may affect the sustainability of the system and limit the application scenarios of engineered bacteria. Through discussions with experts, we will ensure the constitutive expression and stability of protein expression by fusing the binding protein behind the TasA protein on endogenous chromosomes in the future.

Finally, it is currently unclear whether our TasAnchor can grow and function properly in more complex wastewater environments. Therefore, we need to further optimize the stability of each of our solution strategies and design hardware and delivery strategies in conjunction with the actual environment of the sewer system to improve the efficacy of our engineered bacteria.