Engineering
Antibacterial Module
Design
To ensure that our project can inhibit the growth of fungi on murals, we attempted to find an antimicrobial peptide with antifungal properties. We found the antimicrobial peptide Pn-AMP1 (AP00270) in the Antimicrobial Peptide Database. The database indicates that Pn-AMP1 is extracted from Pharbitis nil and may disrupt the cell membranes of fungi, having inhibitory effects on Fusarium oxysporum and Saccharomyces cerevisiae. However, the majority of the fungi we extracted were from the genera Penicillium and Aspergillus. Therefore, it is necessary to verify the inhibitory effect of Pn-AMP1 on these two types of fungi.
In order to enable our engineered bacteria to inhibit the growth of bacteria on the murals, we introduced GRP-1 into our engineered bacteria. GRP1 is an antibacterial peptide extracted from Limosilactobacillus fermentum. It shows a good inhibitory effect on Staphylococcus aureus and Salmonella Paratyphi B. However, the inhibitory effect of this substance on the microorganisms on the murals still requires further verification.
Build
We entrusted Tsingke Biotechnology Co., Ltd. to synthesize the complete nucleotide sequence of BBa_259HIEMB and BBa_25XI83ND, and constructed pET-30a(+) - BBa_259HIEMB and pET-30a(+) - BBa_25XI83ND for the subsequent testing work.
Test
Three species of fungi and six species of bacteria were isolated from the mural samples. Antimicrobial susceptibility testing via the zone of inhibition method was conducted on each microbial strain, followed by simulated conservation tests on mural specimens pre-colonized by these microorganisms.
The inhibition zone experiment demonstrated that GRP1 has a good inhibitory effect on bacteria, and PnAMP1 has a good inhibitory effect on fungi.
a: Bacteria 4.1, b: Bacteria 6.1, c: Bacteria 7.1, d: Bacteria 8.1
The white arrow indicates the experimental group.
a: Fungal 3.1, b: Fungal 4.1, c: Fungal 5.1
In order to test the survival of our engineered bacteria on the mural, we obtained simulated mural samples from Dr. Wu Fasi's laboratory.
After inoculation with BL21-BBa_25XI83ND/BBa_259HIEMB and mural microbes, no distinct microbial plaques were observed on the simulated mural (Fig 6a). However, murals not inoculated with the antimicrobial engineered bacteria displayed large plaques and exhibited significant damage (Fig 6b), demonstrating that the antimicrobial engineered bacteria can prevent microbial growth on the murals. Murals with plaques were treated with the engineered bacteria. Calcein-AM/PI staining revealed a significant number of viable bacteria on the untreated murals (Fig 6c). However, 24 h after treatment with the engineered bacteria, widespread microbial cell death was observed on the murals (Fig 6d), demonstrating that the antimicrobial engineered bacteria can effectively treat microbial infestations in the murals. Figure 7 shows that the microbial mortality rate on the murals not treated with the antimicrobial engineered bacteria was 30.03%, while that on the murals treated with the engineered bacteria reached 68.23%.
(a) Simultaneous inoculation of antimicrobial engineered bacteria and mural microorganisms; (b) Inoculation of mural microorganisms alone. (c) Calcein-AM/PI staining results after 5 d of inoculation with mural microorganisms; (d) staining results after 5 d of incubation and 1 d of treatment with engineered bacteria. Green represents viable cells, and red represents dead cells.
Learn
The results indicate that our engineered bacteria largely fulfilled the intended objectives. In subsequent work, we will explore adjustments to the types and expression levels of antimicrobial peptides to achieve enhanced efficacy.
Pigment Degradation Module
Cycle 1
Design
In order to enable our engineered bacteria to degrade melanin and carotenoids, we introduced the laccase CueO and dye decolorizing peroxidase DyP into our engineered bacteria. CueO can degrade a variety of substrates, including melanin. DyP can utilize hydrogen peroxide to degrade various substrates including carotenoids.
Build
We entrusted Beijing Tsingke Biotech Co., Ltd. to synthesize the complete sequence and construct pET-30a(+) - BBa_256VH953 and pET-30a(+) - BBa_25OTZVL1 for subsequent experiments.
Test
As shown in Fig 8, the degradation rate of melanin by the engineered bacteria without CueO protein expression was 10.08%. However, the degradation rate of melanin by the engineered bacteria expressing CueO increased to 29.67% (Fig 8b)..
(a) Melanin standard curve; (b) Melanin degradation rate by the engineered bacteria expressing CueO.
CueO: engineered bacteria without induced expression; E-CueO: engineered bacteria with induced expression.
As shown in Fig 9, the degradation rate of β-carotene by the engineered bacteria without DyP protein was 11.99%, while that by the engineered bacteria expressing DyP increased to 35.32% (Fig 9b).
(a) β-carotene standard curve; (b) β-carotene degradation rate by the engineered bacteria expressing DyP.
DyP: engineered bacteria without induced expression; E-DyP: engineered bacteria with induced expression.
Learn
Our constructed pET-30a-CueO and pET-30a-Dyp enzymes were effective at degrading melanin and β-carotene, respectively, but the degradation efficiency was low, potentially limiting their practical application. Based on literature analysis, this may be due to the low efficiency of the functional enzymes in capturing the pigments, necessitating the addition of a pigment-capturing protein.
Cycle 2
Design
Because CueO and DyP alone have limited effectiveness in pigment degradation, we introduced a modified scaffoldin (BBa_25TLZ7K5) into the engineered bacteria. This scaffoldin binds to CueO and DyP via Dockerin, enhancing the degradation of melanin and β-carotene.
Test
As shown in Fig 11, after incubation with melanin, the absorbance of melanin decreased (Fig 11a), and the color of melanin changed from dark blue to lighter (Fig 11c). The melanin degradation efficiency increased to 59.07%, significantly higher than that of E-CueO (Fig 11b). The CueO-scaffoldin engineered bacteria further enhanced the melanin degradation efficiency of CueO.
(a) UV-visible spectrum of melanin after degradation by CueO-scaffoldin-expressing bacteria; (b) Melanin degradation efficiency by the bacteria; (c) Visual representation of melanin decolorization by CueO-scaffoldin-expressing bacteria.
As shown in Fig 12, after incubation with β-carotene, the absorbance of β-carotene decreased (Fig 12a), and the color of β-carotene changed from dark yellow to light (Fig 12c). The degradation efficiency of β-carotene increased to 49.57%, significantly higher than that of E-DyP (Fig 12b). The DyP-scaffoldin-expressing bacteria further enhanced the degradation efficiency of DyP for β-carotene.
(a) UV-visible spectrum of β-carotene after degradation by DyP-scaffoldin-expressing bacteria; (b) β-carotene degradation efficiency by the bacteria; (c) Visualization of β-carotene decolorization by DyP-scaffoldin-expressing bacteria.
pH-Responsive Switch
Design
To enable our engineered bacteria to regulate the synthesis of products according to pH changes, we need to design a regulatory element. We used the Pasr promoter, which can significantly enhance expression strength in acidic environments. We combined the Pasr promoter (BBa_K4716000), TetR (BBa_K5237005), and PtetO3 promoter (BBa_K3606007) to enable the transcription of some genes to be turned off while others are turned on at different pH levels.
Build
We constructed BBa_25WCSXND and introduced fluorescent proteins downstream of the two promoters as reporter proteins. We tested the function of the regulatory element by detecting changes in fluorescence intensity.
Test
We cultivated our engineered bacteria at different pH levels and used an enzyme detector to measure the trend of fluorescence protein fluorescence intensity over time. The increase and decrease in fluorescence intensity reflect the activation and deactivation of fluorescent protein synthesis.
Eight gradients were set up within the pH range of 4 to 7 for the experiment. The fluorescence intensity of EGFP showed a downward trend over time at pH = 4.25 and pH = 5, and an upward trend at pH = 5.5. The trends were not obvious at other pH values.
Eight gradients were set up within the pH range of 4 to 7 for the experiment. The fluorescence intensity of mCherry showed an upward trend over time at pH values ranging from 4 to 4.75, and a downward trend at pH 5. The trend was not obvious at other pH values.
Learn
Our part met the expected results under certain conditions when the pH was less than 5.5. However, the performance of Part under other conditions is confusing. The fluorescence intensities of the two fluorescent proteins did not show any significant increase or decrease trend when the pH value was greater than 5.5. The fluorescence intensity of mCHerry decreased perplexingly within the pH range of 4.5 to 5. We believe that it is possible that the transcriptional activation strength of PtetO3 at pH > 5.5 is not optimal, and the decrease of mCherry might have other causes. These require further experimentation for verification and improvement.
Suicide Module
Cycle 1
Design
To prevent genetic leakage beyond the repair materials, we designed a suicide module. When the engineered E. coli carrying this plasmid detach from the repair materials, the suicide system can be activated to prevent genetic leakage.
We employed the extracellular nuclease gene nuc from Serratia as the suicide gene. The signal peptide sequence was removed to enable the nuclease to degrade DNA within the chassis E. coli, thereby inducing bacterial suicide. The suicide gene is driven by a moderately weak E. coli constitutive promoter J23105 (BBa_ J23105), coupled with the RBS sequence J61100 (BBa_ J61100). Downstream of the promoter, we incorporated the operator sites O1, O2, and O3 from the E. coli λ phage. These operator sites can bind to the λ phage CI protein（BBa_C0051）to repress gene expression. The CI protein is expressed under the control of the arabinose-inducible promoter pBAD（BBa_I13453）. In the absence of arabinose, this promoter forms a specific structure that inhibits transcription. When arabinose is present, the araC protein binds to arabinose and promotes transcription of the downstream gene.
In the presence of arabinose, the PBAD promoter successfully binds RNA polymerase and initiates transcription, expressing CI protein to suppress the expression of the nuclease nuc. In the absence of arabinose, the expression of nuc is no longer repressed by CI protein, ultimately leading to genomic DNA degradation and bacterial suicide. This design allows us to add arabinose to the repair materials, enabling normal bacterial function. Upon removal of the repair materials from the mural, the engineered bacteria activate the suicide mechanism, effectively preventing genetic leakage.
Build
We obtained the suicide module flanked by biobrick prefix and suffix through full-sequence synthesis and transformed it into E. coli strains DH5α and BL21. The functionality of the suicide plasmid DNA was then assessed.
Test
The pSB1A3 plasmid containing the suicide module was transformed into E. coli strains DH5α and BL21, respectively. The bacteria were plated on LB solid medium with varying concentrations. However, no suicide phenomenon was observed in the E. coli.
Five different arabinose concentration gradients were set (0, 0.01%, 0.1%, 0.5%, and 1%). After transforming 1 μL of the suicide plasmid, 100 μL of bacterial suspension was plated per dish. All plates contained ampicillin. The number of E. coli colonies grown in media with different concentrations showed no significant concentration-dependent differences.
To investigate the specific reasons for the failure, we designed a forward primer with a PstI restriction site at the terminus and a reverse primer with an EcoRI restriction site. The nuclease gene carrying the J61100 RBS was cloned individually and ligated into the pUC19 vector using restriction enzyme digestion and ligation. Upon inducing protein expression with IPTG, no significant bactericidal activity was observed.
E. coli transformed with the pUC19 vector plasmid carrying the nuclease gene was induced using 50 mM IPTG. No significant difference was observed between the two groups.
Learn
Our suicide plasmid failed to meet the requirements, as it did not induce bacterial suicide under growth conditions lacking arabinose. Additionally, the isolated expression of the nuclease gene alone did not result in engineered bacterial suicide. This necessitates further refinement of the suicide plasmid.
Cycle 2
Design
Since the initial nuclease did not achieve the desired bactericidal effect, we sought an alternative suicide gene and identified the protein MazF. MazF is a toxin protein in the E. coli toxin-antitoxin system, functioning as a sequence-specific endoribonuclease that cleaves single-stranded RNA at ACA sequences to induce bacterial suicide. We plan to use the method of restriction and ligation to link our regulatory elements with the MazF suicide gene, incorporating biobrick interfaces at both ends for convenient vector integration.
Build
We performed full gene synthesis of the MazF gene carrying an RBS and ligated it into the pET-30a vector. The functionality was tested via IPTG induction. Subsequently, fusion PCR was employed to connect it with the regulatory elements, constructing a suicide module carrying the MazF toxin gene.
Test
Induction with 50 mM IPTG to express the MazF protein demonstrated effective bactericidal activity under induction conditions.
E. coli transformed with the pET-30a vector plasmid carrying the MazF gene was induced using 50 mM IPTG. No E. coli growth was observed on the IPTG-containing plates.
To integrate MazF into the suicide module, we employed restriction enzyme digestion and ligation for modification. We separately cloned the upstream regulatory sequence of the suicide module and the MazF gene sequence. The upstream regulatory sequence was first ligated into the pSB1A3 vector, followed by restriction enzyme digestion and ligation to incorporate the MazF toxin protein gene at the end of the suicide regulatory sequence. This process successfully yielded the modified vector.In the presence of 1% arabinose, the engineered bacteria exhibited normal growth. However, in the absence of arabinose, the cell death program was successfully executed, and no bacterial colonies grew on the culture medium.
Subsequently, to test the survival of the engineered bacteria on the mural surface, we obtained mural samples from Dr. Wu Fasi's laboratory. E. coli carrying EGFP was sprayed onto the mural surface, and the number of surviving cells was observed under a microscope. The experiment demonstrated that the majority of the engineered bacteria failed to survive on the mural surface (Fig 20), aligning with our expectations.
Learn
The MazF gene effectively achieves the intended suicide effect and, after being incorporated into the suicide module, fully meets our requirements by activating the death program in the absence of arabinose. Furthermore, it cannot survive independently on the mural.