Loading
To express eukaryotic proteins using prokaryotic systems, we prioritized the use of Escherichia coli BL21 (DE3) as the chassis cell, with Escherichia coli DH5α assisting in plasmid construction to ensure efficient protein expression. After interviewing Dr. Wang Yun, a chief physician at Jiangsu Provincial People's Hospital, we recognized the importance of safety in engineered bacteria for human use. Consequently, we considered employing live bacteria therapy; Escherichia coli Nissle 1917 is a probiotic that naturally exists in the human gut, is one of the few non-endotoxic strains, and possesses antibacterial, anti-inflammatory, and gut microbiome-regulating properties. Therefore, we decided to utilize Escherichia coli Nissle 1917 as the chassis cell in subsequent experiments to enhance safety.
We aim for the multiple modules of the project to specifically act at the site of intestinal inflammation. Research indicates that nitric oxide (NO) is an ideal input signal for engineered bacteria, offering good specificity. The SoxR/SoxS oxidative stress-responsive promoter can enable targeted treatment in inflamed regions, avoiding unintended effects on the vital activities of normal cells. Therefore, we decided to use this promoter as a regulatory element to achieve targeted therapy in the context of intestinal inflammation.
To investigate the optimal induction temperature for the SoxR/SoxS oxidative stress-responsive promoter and to demonstrate its functionality at human body temperature (37°C), we constructed the plasmid pET29a-J23119-RBS-SoxR-pSoxS-RBS-eGFP-T7 and transformed it into Escherichia coli Nissle 1917. We explored the promoter's performance under different induction temperatures (16°C, 20°C, 25°C, 30°C, 37°C, and 40°C).
In the first round of DBTL, we constructed an antioxidant module using SOD-1 to help eliminate ROS and reduce oxidative damage. Our team obtained the SOD-1 sequence from BBa_K2215003, optimized its codons, and sent the related gene fragment for synthesis. We designed the plasmid pET29a-J23119-RBS-(SOD-1)-T7, as illustrated in the plasmid diagram below.
During the first round of construction, numerous random mutations occurred. Literature reviews revealed that excessive foreign SOD leads to an overproduction of H2O2 in Escherichia coli, reaching toxic levels that can cause DNA damage, resulting in a high frequency of random mutations in the sequence. Additionally, considering that BL21(DE3) has a superior protein expression capacity compared to general strains like DH5α or EcN, it is more likely to experience random mutations in the DNA sequence. To avoid this possibility, we decided to use DH5α in all subsequent SOD experiments. In the second round of DBTL, we chose to use the weaker lac promoter to induce SOD expression in Escherichia coli with IPTG. Therefore, our team redesigned the plasmid pET-29(a)-pT7-lac operator-(SOD-1+His)-T7.
In the third round of DBTL, to enhance the activity, solubility, and thermal stability of SOD, we adopted a strategy involving large primer site-directed mutagenesis and high-throughput screening to identify superior SOD variants. Mutating specific bases in proteins has proven valuable for detecting the contribution of individual amino acid side chains to protein properties. Alanine (with a side chain R = methyl) lacks unusual backbone dihedral angle preferences; for instance, glycine (R = H) also renders the side chain ineffective but introduces conformational flexibility into the protein backbone. The ability of alanine scanning mutagenesis to provide key biological insights has been demonstrated in important early examples, combining the convenience of combinatorial libraries with the insights of site-directed mutagenesis. Conventional methods have limited applicability as they require high-throughput screening or three-dimensional structures to guide the targeting of active site residues.
We conducted virtual amino acid mutations using Calculate Mutation Energy (Binding) to analyze a protein-ligand complex based on interaction forces. This approach allowed us to identify 28 key amino acids in the active site, as well as potential amino acid mutations that could enhance binding affinity, with the aim of increasing SOD enzyme activity.
We also learned to use Calculate Mutation Energy (Stability) to perform virtual amino acid mutations based on thermal stability. This allowed us to predict mutation targets that could enhance protein thermal stability and identify five optimal amino acid mutation combinations using Predict Stabilizing Mutations. These predictions will guide our rational amino acid mutations, potentially improving enzyme activity.
In general, a single round of mutation often fails to achieve the desired goals, necessitating multiple rounds of cumulative mutations. Therefore, we planned to select the best-performing mutants from the previous round as templates for subsequent iterative mutations. In the fourth round of DBTL, we chose the seven mutation sites—2, 13, 15, 24, 26, 30, and 33—that exhibited the largest improvements to form combinations, resulting in 21 pairs of mutations. Due to the first round of mutations yielding inhibition percentages greater than 70%, we diluted the samples accordingly this time. Using the Vazyme™ BCA Protein Assay Kit, we generated a protein standard curve to calculate protein concentrations, diluting samples to 30 µg/µl for subsequent analysis.
The results from the fourth round indicated that, compared to the original seven mutations, most enzyme activities did not show significant improvements. However, the combinations of 2+33, 26+33, 15+24, and 13+30 exhibited higher inhibition rates. Based on these double mutation results, we selected these four optimal combinations—13+30, 2+33, 15+24, and 26+33—as templates for further mutations, forming the following 20 pairs of combinations:
To ensure that our SOD is expressed only in the inflamed intestinal region, we leveraged the characteristic high levels of NO in this area to regulate expression using the NO-responsive promoter SoxR/SoxS. We designed the following genetic construct:
Our team employed an NO-responsive system, with the transcription factor SoxR acting as a sensor for oxidative stress and nitric oxide. The NO present in the inflamed region will activate the transcription of pSoxS, initiating the expression of downstream SOD. By expressing the SOD enzyme, we aim to eliminate excess ROS in the intestine, creating a milder environment for subsequent treatments. Based on these design principles, we used Snapgene software to design the plasmid p-SoxR-T-pSoxS-RBS-SOD-T. Due to time constraints, we only completed the plasmid design and plan to test its performance in the future.
First round of DBTL, to ensure efficient protein expression, we used Escherichia coli BL21 (DE3) and the strong constitutive promoter J23119 as the chassis cell and promoter element, respectively. We designed and constructed the plasmids pET29a-pJ23119-RBS-Mfp3/5-T7. Subsequently, we performed SDS-PAGE and Western Blot experiments for protein characterization, validating the expression of Mfp5 and Mfp3, and proceeded with further upgrades.
In the second round of DBTL, we selected the antioxidant proteins Mfp5 and Mfp3, and used a flexible linker to fuse and express Mfp53, aiming for improved therapeutic effects. Utilizing Escherichia coli BL21 (DE3) and the strong constitutive promoter J23119 as the chassis cell and promoter element, we designed and constructed the plasmid pET29a-pJ23119-RBS-Mfp53-T7. We then performed SDS-PAGE and Western Blot experiments for protein characterization, successfully validating the expression of Mfp53.
In the third round of DBTL, to enhance intestinal mucosal repair in the high NO environment of colitis, we utilized the oxidative stress-responsive SoxR/SoxS promoter for targeted treatment. Using Escherichia coli Nissle 1917 (EcN) and the SoxR/SoxS promoter as the chassis cell and promoter element, we designed and constructed the plasmid pET29a-pJ23119-SoxR-T-pSoxS-RBS-Mfp5/3-T. We then performed SDS-PAGE and Western Blot experiments for protein characterization, successfully validating the expression of Mfp5 and Mfp3, and we look forward to further improvements.
In the fourth round of DBTL, we decided to replicate the second round by using a flexible linker to express Mfp53. We selected Escherichia coli Nissle 1917 (EcN) and the SoxR/SoxS promoter as the chassis cell and promoter elements, respectively. We designed and constructed the plasmid pET29a-pJ23119-SoxR-T-pSoxS-RBS-Mfp53-T. We then performed SDS-PAGE and Western Blot experiments for protein characterization, confirming the expression of the fusion protein Mfp53.
Due to the hyperactive immune response of CD8+ T cells at the site of intestinal inflammation, we plan to use the PD-1/PD-L1 immune checkpoint to inhibit the activity of CD8+ cytotoxic T cells and downregulate the immune response at the inflammation site. For the convenience of further experiments, we selected mouse PD-L1 (BBa_K5322033) and optimized its codons, designing the plasmid pET29a-J23119-RBS-PD-L1 (Mus)-T7, as shown in the figure below.
Considering that mouse PD-L1 is an animal protein with a complex structure and a large molecular weight, expressing it in prokaryotic Escherichia coli poses significant challenges, indicating issues with this design. Therefore, we need to explore better methods for expressing the PD-L1 protein.
According to the descriptions of mouse PD-L1 in the NCBI database (ADK70951.1, ADK70950.1, Q9NZQ7.1, AAH66841.1), the functional domain of PD-L1—the immunoglobulin variable (IgV) domain (CD20947)—is located between amino acids 20 and 130 out of a total of 290 amino acids. To ensure that the functionality of the truncated protein is not affected, we retained the first 19 amino acids at the N-terminus and the last 20 amino acids (131-150) at the C-terminus, aiming to minimize any impact on the IgV domain. Subsequently, we used AlphaFold2, a tool developed by Google DeepMind, to predict the structures of both the full-length and truncated PD-L1 proteins, yielding the following results.
 |
|
 |
|
 |
|
 |
|
 |
|
Based on the predictions from AlphaFold 2, it is evident that the main functional domain of the truncated PD-L1 remains unaffected. Accordingly, we designed an expression system for the PD-L1 functional domain: pET29a-J23119-RBS-PD-L1 (functional domain)-T7, as illustrated in the following plasmid diagram.
Subsequently, Google released AlphaFold3, which we used to predict the protein structure again and performed molecular docking to demonstrate that our truncated PD-L1 can successfully bind to PD-1, thereby exerting its function. The results are shown in the following figure.
|
|
|
|
|
|
|
|
|
|
 |
|
 |
|
 |
|
 |
|
 |
|
During the project, we considered that PD-L1 might be encapsulated by mussel foot protein Mfp during expression and release, which could reduce therapeutic efficacy. To address this, we proposed using the surface display system Lpp-OmpA to present our passenger protein PD-L1 on the outer membrane of Escherichia coli. This approach would facilitate its binding to PD-1. Additionally, to demonstrate the successful surface display, we inserted a flexible protein linker and a TEV protease recognition site between Lpp-OmpA and the PD-L1 functional domain. By incubating the cells with TEV protease, PD-L1 could be cleaved from OmpA. If the PD-L1 functional domain is detected in the supernatant, it would indirectly confirm the success of the surface display. The plasmid design is shown in the figure.
To achieve optimal results in bacterial therapy, we designed a pLuxI-regulated PhiX174E lysis system, pLuxⅠ-RBS-PhiX174E-rrnB T1. When the engineered bacteria detect NO, they synthesize mussel foot protein Mfp, anchoring the probiotic EcN at the site of intestinal inflammation, thereby establishing a dominant microbial population. However, the therapeutic protein SOD cannot cross membranes, so to ensure its efficacy, we utilize the principle of quorum sensing among bacteria. As the population density of EcN increases at the inflammation site, the quorum sensing system is activated, leading to the expression of PhiX174E protein, which subsequently induces bacterial lysis and releases the therapeutic protein, achieving optimal therapeutic effects.
Biosafety has always been a key concern for biologists. To protect biosafety and avoid potential issues like gene contamination, we designed the pDawn-MzaF safety module. pDawn is a blue light-induced promoter that can activate downstream gene expression under natural light or a single blue light source. MazF is an RNAse that efficiently degrades bacterial mRNA, thereby inhibiting bacterial growth and reproduction.
To achieve lysis and self-destruction, we designed the plasmid pET29a-LuxI-PhiX174E-pDawn-MazF. We successfully constructed this plasmid and conducted growth curve measurements of the quorum sensing system to further validate the feasibility of the project.
