Engineering

Introduction

Our iGEM project focuses on developing an eco-friendly preservative film using ε-poly-L-lysine (PLL) as the antimicrobial core material, strictly adhering to the "Design-Build-Test-Learn" (DBTL) engineering cycle. The initiative integrates R&D, production, market research, and sales across all phases. This DBTL-driven approach bridges lab innovation with real-world applications, aligning with iGEM's mission of advancing synthetic biology for societal benefit.The diagram below illustrates the key engineering cycles related to our project implementation:

Cycle-1 Heterologous Expression of pls Gene in Bacillus subtilis for ε-Polylysine Production

Design

ε-Polylysine is mainly produced naturally by Streptomyces microorganisms, but the slow growth, complex morphology, and difficult genetic manipulation of Streptomyces limit the industrial production efficiency of ε-PL. Bacillus subtilis, as a GRAS (Generally Recognized As Safe) strain, has advantages of rapid growth, clear genetic background, and ease of genetic manipulation, making it an ideal heterologous expression host. By introducing the pls gene BBa_25EMGU3Q encoding ε-polylysine synthetase into Bacillus subtilis, engineered strains can be constructed to achieve heterologous production of ε-PL[1, 2].

Fig1. Experimental design of Cycle 1.

Build

Plasmid construction requires the application of molecular cloning techniques. First, the pMA5 vector must be prepared as the backbone, which contains an ampicillin resistance gene (Amp) for selection and an appropriate origin of replication to ensure stable plasmid replication in host cells. Simultaneously, the pls gene is obtained through PCR amplification from the Streptomyces genome, with appropriate restriction enzyme sites such as BamHI and HindIII introduced at both ends of the gene during primer design to facilitate subsequent digestion and ligation operations.

The core steps of plasmid construction include double digestion, linearization of the vector, and preparation of the target gene. The pMA5 vector is double-digested with the same restriction enzymes, separated and recovered through agarose gel electrophoresis, followed by dephosphorylation treatment to prevent vector self-ligation. Meanwhile, the PCR-amplified pls gene fragment is treated with the same restriction sites to ensure proper ligation between the two components. Under the action of T4 DNA ligase, overnight ligation is performed at 16°C with the molar ratio of vector to insert controlled at approximately 1:3 to achieve optimal ligation efficiency.

The ligation products are introduced into E. coli DH5α competent cells through heat shock transformation and plated on selective medium containing ampicillin.

Fig2. (A) Part of the codon optimization of pls BBa_25EMGU3Q. (B)The structure of vector pMA5-pls. BBa_25EJCCFR

Test

Positive clones are selected and confirmed through DNA sequencing to verify the correctness of the plasmid structure and the integrity of the pls gene sequence (Figure 3A). The quality-controlled recombinant plasmid can then be used for subsequent Bacillus subtilis transformation experiments to achieve heterologous expression of the ε-polylysine synthase gene, ultimately establishing an efficient ε-polylysine-producing engineered strain. Meanwhile, we detected that the yield of ε-polylysine was approximately 0.3g/L (Figure 3B).

Fig3. (A) The sequencing results of the pls BBa_25EMGU3Q. (B) The concentration of ε-polylysine before and after introducing pls BBa_25EMGU3Q.

Learn

Our spectrum measurements of ε-polylysine production confirm the successful insertion of the pls gene BBa_25EMGU3Q into our engineered Bacillus subtilis. This genetic modification resulted in a notable increase in ε-polylysine production, rising from 0.1 g/L to 0.3 g/L. While this improvement marks progress, the concentration of ε-polylysine in Bacillus subtilis remains relatively low, especially when compared to the higher yields typically seen in Streptomyces species. This disparity indicates that while our initial modifications have been successful, further optimization is required to significantly enhance the production of ε-polylysine in our engineered bacterial strain. Moving forward, additional adjustments to the metabolic pathways and growth conditions will be crucial to achieve higher yields and bring the production levels closer to those found in Streptomyces microorganisms.

Cycle-2 Using dual gene co expression strategy to improve the production efficiency of ε - PL in Bacillus subtilis

Introduction

With these drawbacks in mind, we consulted Dr. Wei, an eminent expert in gene edition of Bacillus subtilis. After his advice and some additional research, we decided on two methods to increase its production: 1) Adding PPK2 BBa_25L5WCKD, the ATP regeneration gene from Corynebacterium glutamate for more energy. 2) Replacing the thrD gene in Bacillus subtilis with the lysC311 gene BBa_252TM36G from Corynebacterium glutamate to relieve lysine inhibition.

Cycle-2-1

Design

The synthesis process of ε-PL catalyzed by ε-PL synthase requires a large amount of ATP consumption, while PPK2 can utilize polyphosphate to produce ATP, providing sufficient energy supply for ε-PL^[3]. By coexpressing the pls and ppk2 genes, a coordinated balance between ATP supply and demand can be achieved, thereby improving the production and efficiency of engineering strains of ε-PL. In addition, the introduction of PPK2 helps to maintain the balance of intracellular phosphate metabolism and avoid cell growth inhibition caused by excessive ATP consumption.

Fig4. Experimental design of Cycle 2-1

Build

During the construction process, the ppk2 gene was first obtained from Corynebacterium glutamicum. Next, select the pMA5-pls vector constructed in cycle 1, which should have antibiotic resistance markers, replicons, and promoter regions. The ppk2 gene fragment and homologous arm were amplified by PCR, and then the vector pMA5 was digested at the BamHI site to homologous recombine the target gene onto the vector. Finally, the vector was transformed into competent Escherichia coli for cloning and screening.

Fig5. Structure of pMA5 containing pls and ppk2.

Test

Firstly, preliminary screening is conducted through antibiotic resistance, and the presence of the target gene is verified by colony PCR (Figure 6A). Subsequently, bacterial liquid sequencing was performed to ensure the integrity of the ppk2 gene (Figure 6B). Meanwhile, we detected that the yield of ε-polylysine was approximately 0.8g/L (Figure 6C).

Fig6. (A) Colony PCR results of pMA5-pls-ppk2 BBa_25404K7H (B) Alignment of sequencing results of the colony PCR products against the corresponding designs (C) The concentration of tow group of ε-polylysine.

Learn

Through the addition of the PPK2 gene, we have successfully engineered Bacillus subtilis to enhance its ability in recycling and re-utilizing cellular energy. This genetic modification empowers the strain to maintain a more efficient energy balance, which in turn accelerates the biosynthesis pathways for ε-polylysine production. As a result, the engineered Bacillus subtilis shows a noticeable increase in the overall yield of ε-polylysine from around 0.3 g/L to approximately 0.8 g/L, demonstrating the strain's capacity to leverage its energy reserves more effectively for the production of this valuable metabolite. This enhancement not only boosts the quantity of ε-polylysine produced but also provides insights into the potential for further metabolic optimization to push production levels even higher.

Cycle-2-2

Design

Aspartate kinase (AK) is an enzyme that catalyzes the formation of aspartyl phosphate from L-aspartate. It is a key enzyme in the biosynthetic pathway of aspartate family amino acids and serves as the first rate-limiting enzyme in L-lysine biosynthesis^[4]. However, in B. subtilis, there is only one aspartate kinase isozyme (AKIII) encoded by the thrD gene, whose enzymatic activity is subject to synergistic feedback inhibition and repression by L-lysine and L-valine. Studies have shown that site-directed mutagenesis of the 311th amino acid in the AKIII sequence from C. glutamicum, changing threonine to isoleucine, can relieve the feedback inhibition by L-lysine^[5]. Therefore, we replaced the AKIII in B. subtilis with the feedback-deregulated AKIII from C. glutamicum (encoded by the lysC311 gene) to regulate the carbon flux in the L-lysine biosynthetic pathway.

Fig7. Experimental Design of Cycle 2-2

Build

First, the lysC gene encoding aspartate kinase III (AKIII) is cloned from Corynebacterium glutamicum (C. glutamicum). Through site-directed mutagenesis, codon 311 of the lysC gene is modified to change the corresponding amino acid from threonine (Thr) to isoleucine (Ile), obtaining the lysC311 mutant gene BBa_252TM36G. This mutation can relieve the feedback inhibition of L-lysine on enzyme activity. Meanwhile, the original thrD gene along with its upstream and downstream regulatory sequences is obtained from the Bacillus subtilis (B. subtilis) genome as a template for homologous recombination. The lysC311 gene is inserted into the corresponding position of the thrD gene in the vector through homologous recombination technology, forming the recombinant plasmid thrD-LysC BBa_25QWQUAM. During construction, it is necessary to ensure that the lysC311 gene has the same transcription direction and regulatory pattern as the original thrD gene to maintain normal gene expression levels. Simultaneously, the CRISPR/Cas9 system is used to knock out the thrD gene in B. subtilis. First, sgRNA targeting thrD is designed using Benchling. Then, the sgRNA sequence is inserted into an expression vector containing the Cas9 gene, P43 promoter, and antibiotic resistance marker. Next, B. subtilis competent cells are prepared and co-transformed with the Cas9-sgRNA plasmid through natural transformation.

Test

After transformation, preliminary screening was conducted through antibiotic resistance, followed by PCR validation and sequencing to confirm the correct integration of the lysC311 gene and successful replacement of the thrD gene (Figure 8A-B). Subsequently, functional validation tests were conducted to determine if the production of L-lysine increased. Next, we will first conduct a preliminary detection of the presence of L-lysine through the color change experiment of indene ketone (Figure 8C). Subsequently, functional validation tests were conducted to determine if the production of L-lysine increased (Figure 8D). The yield of recombinant strains increases with the increase of fermentation time, while the growth of the bacterial cells of the recombinant strains is not significantly inhibited (Figure 8E).

Fig8. (A) Colony PCR results of thrD-F, plasmid backbone, thrD-R, and lysC311 (B) Alignment of sequencing results of the colony PCR products against the corresponding designs (C) The color change experiment of indene ketone (D) The concentration of two groups of ε-polylysine (E) Growth status of two groups of bacterial cells

Learn

Through the substitution of the thrD gene with the lysC311 gene from Corynebacterium glutamicum, we have successfully relieved the natural inhibition of L-lysine synthesis in Bacillus subtilis, thereby enhancing its production of L-lysine. Since L-lysine is a key precursor for the biosynthesis of polylysine, this modification has had a downstream effect of significantly boosting polylysine production in our engineered strain. Further testing revealed a marked increase in polylysine yield following the addition of the PPK2 gene and the substitution of thrD with lysC311. In addition, despite these genetic modifications, our recombinant bacteria exhibit no significant differences in growth characteristics when compared to the wild-type Bacillus subtilis.

However, we recognize that the incorporation of these changes could provide a distinct advantage for the engineered strain under natural environmental conditions. With this potential for enhanced survival and productivity, there are also associated risks that need to be carefully managed. Without a built-in mechanism to regulate its viability outside controlled environments, our strain poses a potential ecological risk if it were to inadvertently escape containment. As a result, we acknowledge the absence of a "suicide switch" in our current strain design, a crucial safeguard that would make the engineered bacteria completely dependent on artificial conditions for survival. Therefore, additional experiments are required to incorporate this safeguard, ensuring both the continued success of the project and the safe application of the engineered strain.

Cycle-3

Design

Polylysine as a natural food preservative in the food industry must strictly comply with food safety standards. Traditional engineered strains typically carry antibiotic resistance genes as selection markers, and these antibiotic residues may enter the human body through the food chain. This not only violates food safety regulations but may also promote horizontal transfer of antibiotic resistance genes, exacerbating the global antibiotic resistance crisis. Therefore, developing production strains without antibiotic resistance markers has become an urgent industry need.

We constructed a nutrient-deficient chassis bacterial system by introducing the pMA5-pls-PPK2-lac-alrA plasmid BBa_25BTTP42 into Bacillus subtilis. This plasmid combines functions for polylysine production and biosafety control: the pls gene BBa_25EMGU3Q encodes polylysine synthase, directly catalyzing polylysine biosynthesis; the PPK2 gene BBa_25L5WCKD encodes polyphosphate kinase, involved in cellular energy metabolism regulation; the lac operon system BBa_25XOTKEA enables inducible gene expression control; and the alrA gene BBa_251M08GXencodes alanine racemase, which converts L-alanine to D-alanine—a critical precursor for bacterial cell wall peptidoglycan synthesis. By deleting the endogenous alrA gene BBa_251M08GX from the host chromosome, the strain develops an absolute dependency on exogenous D-alanine, creating a natural biosafety barrier. If the strain escapes into the natural environment, the absence of D-alanine disrupts cell wall synthesis, leading to cell death and effectively preventing the environmental dissemination of genetically modified microorganisms. This design seamlessly integrates polylysine production with biosafety control, eliminating the complexity associated with multi-plasmid systems.

Fig9. Experimental design of suicide switch

Build

We constructed the pMA5-pls-PPK2-lac-alrA plasmid based on the pHT43 vector. Firstly, we obtained the alrA gene sequence of Bacillus subtilis, as well as the sequences of the polylysine synthase gene (pls) and the polyphosphate kinase gene (PPK2) from NCBI. We analyzed the multiple cloning sites of the pMA5 vector and determined suitable restriction enzyme sites for the sequential insertion of multiple genes. Leveraging the existing lacI gene and Pgrac promoter system of the vector, we designed a gene expression regulation strategy.

Subsequently, a multi-step cloning strategy was employed, and primers containing vector homology arms were designed. The primers should also include appropriate GC content (40-60%) and annealing temperature (55-65°C) to avoid the formation of secondary structures and primer dimers. First, the pls gene was inserted into the appropriate site of the pMA5 vector to construct an intermediate vector; then, the PPK2 and alrA genes were inserted sequentially, ultimately obtaining the pMA5-pls-PPK2-lac-alrA plasmid containing the complete gene cluster. During the construction process, the original chloramphenicol resistance gene (Cm) needed to be removed to ensure that the final plasmid did not contain any antibiotic resistance markers.

Meanwhile, the endogenous alrA gene on the chromosome of Bacillus subtilis was knocked out using the CRISPR/Cas9 system. First, sgRNA targeting the alrA gene was designed by Benchling to ensure high specificity and avoid off-target effects. Then, the sgRNA sequence was inserted into an expression vector containing the Cas9 gene, P43 promoter, and a temporary antibiotic resistance marker. B. subtilis competent cells were prepared, and the Cas9-sgRNA plasmid was first introduced to complete the knockout of the alrA gene. After successful verification, the modified pMA5-pls-PPK2-lac-alrA plasmid was transferred. Finally, an engineered strain completely resistant to antibiotics was obtained through screening.

Fig10. Structure of pMA5-pls-PPK2-lac-alrA BBa_25BTTP42.

Test

After transformation, initial screening was conducted through antibiotic resistance, and resistant colonies were selected for PCR verification (Figure 11A). For successful strains, their growth defect phenotype was verified on a medium without D-alanine (Figure 11B). Subsequently, pMA5-pls-PPK2-lac-alrA BBa_25BTTP42 was transferred, and IPTG was added to detect its growth recovery in a medium lacking D-alanine (Figure 11C), confirming the establishment of an auxotrophic phenotype.

Fig11. (A) Colony PCR results of pMA5-pls-PPK2-lac-alrA BBa_25BTTP42(B) Growth status of two groups (C) Growth of Bacillus subtilis without and with the addition of 0.5 mM IPTG

Film Production cycle

Design

We tried to find a film capable for loading ε-PLL and can undergo biodegradation. After trying various biosynthetic films^[6][7][8], we chose sodium alginate-chitosan bilayer and try to enhance its mechanical performance by adding calcium chloride as another plasticizers to increase cross-linking degree. We then introduced ε-PLL in the film and tested the mechanical properties and antibacterial properties^[9][10]. We finally determined the optimal concentration of each component in the antibacterial film.

Build

We made three biosynthetic films: poly-L-lysine–chitosan–PLA films, sodium alginate-chitosan bilayer films, and chitosan–pullulan composite films. After Kirby-Bauer test we chose sodium alginate-chitosan bilayer film which has good combination with ε-PLL and good tensile strength.

Fig12. Sodium alginate-chitosan bilayer

First we excluded ε-PLL to test the influence of sodium alginate, chitosan, and drying time on film performance including both anti-bacterial effect and mechanical performance. To improve the cross-linking of our film, we tried to spray different concentration of CaCl₂ between two layers as another plasticizers.

Then we introduced ε-PLL to the chitosan solution to find its optimal concentration. This step is significant because the concentration of ε-PLL is negatively correlated with mechanical performance but positively correlated with anti-bacterial performance. The area of our film is fixed at 64 square centimeters.

Last we add chitosan in the ε-PLL solution produced by purification step and make our final product.

Fig13. film produced

Test

We implemented Kirby-Bauer test to measure the antibacterial property of the film. By measuring the circle size we can determine the best concentration of each component in the film.

Fig14. Kirby-Bauer test

We conducted tensile strength test using tensile testing machine to measure the mechanical performance of our film^[11].

Fig15. Tensile strength test of different heating time

We conduct food anti-bacterial test of three kind of foods( beef, salmon and blueberry) and used the extract from its surface to conduct bateria quantification test. Our film has much better antibacterial properties than plastic wrap of the same category.

Fig16. Food antibacterial test :beef(left) salmon(middle) blueberry(right)

Fig17. BaCteria quantification test:beef(left) salmon(middle) blueberry(right)

Learn

We have developed a film with high mechanical performance and antibacterial properties with 3% calcium chloride , 0.1g/10mL sodium alginate, 0.04g/10mL ε-PLL and 4 hour drying time. We would seek further improvement in the stretchability of the film in spite of its excellent tensile strength.