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In the site of intestinal inflammation, there is a high concentration of nitric oxide (NO), which we selected as the signaling trigger for the therapeutic system. We chose the SoxR/SoxS oxidative stress-responsive promoter (BBa_K3771048) as the regulatory element. SoxR is a transcription factor containing a [2Fe-2S] cluster, while SoxS is a 53-base pair promoter sequence. In a high NO and reactive oxygen species (ROS) environment, SoxR binds to specific sequences of the SoxS promoter and undergoes a conformational change due to oxidative stress. This change causes SoxS to bend (with a bending angle of approximately 65°), bringing the -10 and -35 regions of the promoter closer together, facilitating the binding of RNA polymerase (RNAP) and other transcription factors, thereby regulating the expression of downstream genes.
In our study of the SoxR/SoxS system, we determined that the optimal final concentration of sodium nitroprusside (SNP, an NO donor) for induction is 100 μM. However, there is a lack of research on the induction temperature. Therefore, we aimed to supplement this data, hoping it would guide future studies. Additionally, we explored whether the promoter's functionality meets expectations under physiological conditions at 37°C. We designed and constructed the plasmid pET29a-J23119-RBS-SoxR-T7-pSoxS-RBS-eGFP-T7 and measured the fluorescence intensity per unit OD using a microplate reader to analyze promoter expression at different induction temperatures (20°C, 25°C, 30°C, 37°C, and 40°C).
We selected samples to be taken every 4 hours for detection, with each measurement conducted in triplicate. Using a microplate reader, we measured OD600 and fluorescence intensity (FL). After processing the data, we obtained the fluorescence intensity per unit OD600 (FL/OD600), with the results presented in the following figure.
During the design phase of SOD-1, we added basic components: SOD Pro1 (BBa_K5322014), SOD Pro2 (BBa_K5322015), SOD Pro3 (BBa_K5322016), SOD Pro4 (BBa_K5322017), SOD Pro5 (BBa_K5322018), SOD Pro6 (BBa_K5322019), SOD Pro7 (BBa_K5322020), single-point mutations based on SOD-1; SOD Ultra1 (BBa_K5322021), SOD Ultra2 (BBa_K5322022), SOD Ultra3 (BBa_K5322023), SOD Ultra4 (BBa_K5322024), double-point mutations based on SOD-1; SOD Plus (BBa_K5322025), three point mutations were made on the basis of SOD-1 to improve its enzyme activity and thermal stability.
In addition, we have added composite elements based on the contributions of previous IGEM: lac-inducible SOD-1 expression system (BBa_K5322011), which is used for the expression of SOD-1 in E. coli; NO-inducible SOD-1 expression system (BBa_K5322013), which is used to ensure that SOD-1 is expressed only in the intestinal inflammation site.
During the design phase of the adhesin, we incorporated key elements: a flexible protein linker (GGGGS) BBa_K5322001, used to connect Mfp5 and Mfp3; and the SoxS promoter BBa_K5322004, which regulates the expression of mussel foot proteins (Mfp) under high NO conditions.
In addition, building upon the contributions of previous iGEM teams, we incorporated composite parts: the constitutive Mfp3 expression system (BBa_K5322000), the constitutive Mfp5 expression system (BBa_K5322002), the NO-inducible Mfp3 expression system (BBa_K5322005), and the NO-inducible Mfp5 expression system (BBa_K5322006). Furthermore, using the flexible protein linker (BBa_K5322001) we designed, we connected Mfp3 and Mfp5 to construct the constitutive Mfp53 expression system (BBa_K5322003) and the NO-inducible Mfp53 expression system (BBa_K5322007).
During the construction of the immune module programmed cell death ligand-1, we supplemented the part library with the complete mouse PD-L1 (BBa_K5322033) sequence optimized according to the Escherichia coli codon usage table, along with its protein structure prediction model. Subsequently, we performed structural modifications, retaining its functional domain to construct the PD-L1 functional domain (BBa_K5322034), aiming to reduce the molecular weight and alleviate the burden on Escherichia coli. We continued to predict the protein structure of the modified protein and utilized AlphaFold 3 for molecular docking. Based on the docking results, we demonstrated that the modified PD-L1 functional domain can bind to PD-1, confirming the effectiveness of the modification. Additionally, we uploaded the surface membrane display system Lpp-OmpA (BBa_K5322035) and the cleavable flexible protein linker GISS + TEV site (BBa_K5322036). We constructed three plasmids: pET29a-J23119-RBS-(PD-L1)-T7 (BBa_K5322030), pET29a-J23119-RBS-(PD-L1 Functional domain)-T7 (BBa_K5322031), and pET29a-J23119-Lpp-OmpA-GISS-TEVsite-PD-L1 (Functional domain)-T7 (BBa_K5322032).
| Biobricks | Short Description | Contributions from our team |
|---|---|---|
| BBa_K3771048 | SoxS Promoter | We investigated promoter performance at different induction temperatures using sodium nitroprusside (SNP) induction. |
| BBa_K2215003 | CuZn SOD | Directed evolution of the original SOD-1 to obtain SOD with higher enzyme activity. |
| BBa_K5322000 | Constitutive Mfp3 Expression System | The plasmid for constitutive expression of Mfp3 was constructed, using BL21(DE3) as the chassis cell for protein expression. Protein characterization was subsequently performed. |
| BBa_K5322001 | Protein Linker (GGGGS) | It is part of a flexible ligand group composed of small non-polar amino acids, such as glycine (Gly), and polar amino acids, such as serine (Ser). |
| BBa_K5322002 | Constitutive Mfp5 Expression System | The plasmid for constitutive expression of Mfp5 was constructed, using BL21(DE3) as the chassis cell for protein expression. Protein characterization was subsequently performed. |
| BBa_K5322003 | Constitutive Mfp53 Expression System | The plasmid for constitutive expression of Mfp53 was constructed, using BL21(DE3) as the chassis cell for protein expression. |
| BBa_K5322004 | SoxS Promoter | A promoter regulated by the SoxR transcription factor and activated by nitric oxide (NO). |
| BBa_K5322005 | NO-inducible Mfp3 Expression System | The construction of a nitric oxide-inducible Mfp3 plasmid, with Escherichia coli Nissle 1917 (EcN) as the chassis cell, involves the SoxS promoter-regulated expression of the protein under high nitric oxide (NO) conditions. Protein characterization was also performed. |
| BBa_K5322006 | NO-inducible Mfp5 Expression System | The construction of a nitric oxide-inducible Mfp5 plasmid, with Escherichia coli Nissle 1917 (EcN) as the chassis cell, involves the SoxS promoter-regulated expression of the protein under high nitric oxide (NO) conditions. Protein characterization was also performed. |
| BBa_K5322007 | NO-inducible Mfp53 Expression System | The construction of a nitric oxide-inducible Mfp53 plasmid, with Escherichia coli Nissle 1917 (EcN) as the chassis cell, involves the SoxS promoter-regulated expression of the protein under high nitric oxide (NO) conditions. |
| BBa_K5322011 | lac-inducible SOD-1 expression system | An lac-inducible SOD-1 expression plasmid was constructed, using DH5α as the chassis cell for protein expression, and enzyme activity was determined. |
| BBa_K5322013 | NO-inducible SOD-1 expression system | An NO-inducible SOD-1 expression plasmid was constructed. |
| BBa_K5322014 | SOD Pro1 | An inducible SOD-1 expression plasmid was constructed by single-point mutation of SOD-1 (The 10th base is mutated from glycine to alanine), and protein expression was performed using DH5α as the chassis cell, and enzyme activity was determined. |
| BBa_K5322015 | SOD Pro2 | An inducible SOD-1 expression plasmid was constructed by single-point mutation of SOD-1 (The 61st base is mutated from glycine to alanine), and protein expression was performed using DH5α as the chassis cell, and enzyme activity was determined. |
| BBa_K5322016 | SOD Pro3 | An inducible SOD-1 expression plasmid was constructed by single-point mutation of SOD-1 (The 64th base is mutated from phenylalanine to alanine), and protein expression was performed using DH5α as the chassis cell, and enzyme activity was determined. |
| BBa_K5322017 | SOD Pro4 | An inducible SOD-1 expression plasmid was constructed by single-point mutation of SOD-1 (The 118th base is mutated from valine to alanine), and protein expression was performed using DH5α as the chassis cell, and enzyme activity was determined. |
| BBa_K5322018 | SOD Pro5 | An inducible SOD-1 expression plasmid was constructed by single-point mutation of SOD-1 (The 129th base is mutated from glycine to alanine), and protein expression was performed using DH5α as the chassis cell, and enzyme activity was determined. |
| BBa_K5322019 | SOD Pro6 | An inducible SOD-1 expression plasmid was constructed by single-point mutation of SOD-1 (The 56th base is mutated from glutamine to asparagine), and protein expression was performed using DH5α as the chassis cell, and enzyme activity was determined. |
| BBa_K5322020 | SOD Pro7 | An inducible SOD-1 expression plasmid was constructed by single-point mutation of SOD-1 (The 97th base is mutated from aspartic acid to lysine), and protein expression was performed using DH5α as the chassis cell, and enzyme activity was determined. |
| BBa_K5322021 | SOD Ultra1 | An inducible SOD-1 expression plasmid was constructed by single-point mutation of SOD-1 (The 97th base is mutated from aspartic acid to lysine; The 10th base is mutated from glycine to alanine), and protein expression was performed using DH5α as the chassis cell, and enzyme activity was determined. |
| BBa_K5322022 | SOD Ultra2 | An inducible SOD-1 expression plasmid was constructed by double-point mutations of SOD-1 (The 64th base is mutated from phenylalanine to alanine, The 118th base is mutated from valine to lysine), and protein expression was performed using DH5α as the chassis cell, and enzyme activity was determined. |
| BBa_K5322023 | SOD Ultra3 | An inducible SOD-1 expression plasmid was constructed by double-point mutations of SOD-1 (The 61st base is mutated from glycine to alanine, The 56th base is mutated from glutamine to asparagine), and protein expression was performed using DH5α as the chassis cell, and enzyme activity was determined. |
| BBa_K5322024 | SOD Ultra4 | An inducible SOD-1 expression plasmid was constructed by double-point mutations of SOD-1 (The 129th base is mutated from glycine to alanine, The 97th base is mutated from aspartic acid to lysine), and protein expression was performed using DH5α as the chassis cell, and enzyme activity was determined. |
| BBa_K5322025 | SOD Plus | An inducible SOD-1 expression plasmid was constructed by three-point mutations of SOD-1 (The 64th base is mutated from phenylalanine to alanine, The 118th base is mutated from valine to alanine, The 61st base is mutated from glycine to alanine), and protein expression was performed using DH5α as the chassis cell, and enzyme activity was determined. |
| BBa_K5322030 | Programmed Cell Death 1 Ligand 1 [Mus musculus] Expression System | This plasmid is used to express the full-length mouse PD-L1 protein. |
| BBa_K5322031 | Programmed Cell Death 1 Ligand 1 Functional Domain [Mus musculus] Expression System | This plasmid is used to express the truncated functional domain of mouse PD-L1. |
| BBa_K5322032 | Lpp-OmpA-Programmed Cell Death 1 Ligand 1 Functional Domain [Mus musculus] Expression System | This plasmid is used for the surface display of the truncated functional domain of mouse PD-L1 in Escherichia coli, incorporating a GISS protein linker and a TEV site to demonstrate the success of the surface display. |
| BBa_K5322033 | Programmed Cell Death 1 Ligand 1 [Mus musculus] | The mouse PD-L1 sequence, optimized according to the Escherichia coli codon usage table. |
| BBa_K5322034 | Programmed Cell Death 1 Ligand 1 Functional Domain [Mus musculus] | The sequence of the mouse PD-L1 functional domain, optimized according to the Escherichia coli codon usage table. |
| BBa_K5322035 | Lpp-OmpA Surface Display System | The Escherichia coli surface display system can present passenger proteins on the outer membrane of E. coli. |
| BBa_K5322036 | Cleavable Flexible Protein Linker (GISS + TEV site) | Composed of the flexible protein linker GISS and a TEV site, it is used for validation of surface display. |
| BBa_K5322037 | pET29a-J23119-RBS-SoxR-T7-pSoxS-RBS-eGFP-T7 | The eGFP expression system regulated by the SoxR/SoxS oxidative stress promoter is used to investigate protein expression under the pSoxS promoter at different induction temperatures. |
