Overview
Nicotine, a potent alkaloid found in plants of the nightshade family, is a crucial component of tobacco. As an addictive organic compound capable of creating dependency, repeated use of nicotine can trigger over 20 diseases, even leading to death. Quitting smoking is undoubtedly the most effective way to prolong life, but the intense addictive nature of nicotine and its withdrawal symptoms make quitting immensely challenging. Fortunately, researchers have discovered NicX in the human gut microbiota, a substance capable of degrading nicotine. Mouse experiments have shown that degrading nicotine can reduce withdrawal symptoms in these animals. This implies that using NicX to degrade nicotine could potentially serve as a viable method for smoking cessation. We aim to further enhance the nicotine degradation through 3 means: constructing a JI-NicX fusion protein, utilizing mutation techniques, and employing cell surface display system.
We prepared the relevant proteins using molecular cloning techniques and determined the enzyme activity of the protein samples using HPLC-MS. Through our analysis, we successfully reproduced the wild-type NicX protein. The point mutant NicX exhibited an increase in enzyme activity. This validates the accuracy of our prediction regarding the active site and establishes an essential foundation for future research and optimization related to the active site. Furthermore, the cell surface display system showed no difference in enzyme activity compared to the wild type, but it substantially reduced costs as it eliminated the need for purification steps. It's exactly what we were aiming for.
1st Engineering
Design
By reading extensive literature, we have learned about the primary processes and pathways involved in NicX degrading nicotine in the human body. We have also identified a range of feasible optimization methods, including point mutations, whole-cell catalysis techniques, and the addition of protein domains to enhance protein properties.
Build
In this stage, we discovered that there were no structural biology analyses available for NicX. Therefore, we used AlphaFold to predict the protein structures, including the wild-type NicX and the fusion proteins selected for surface display in the design phase.
Test
Building on AlphaFold, we analyzed the active sites, structural biology properties, and the feasibility of whole-cell catalysis technology using AutoDock-Vina. You can find specific details about this on the model page. In the end, we selected five potential active sites, four sets of surface-display anchor proteins, and one structural domain for enhancing protein stability.
Learn
In the first engineering phase, we achieved low-cost and rapid screening through literature review and modeling. We used design methods and fundamental reaction principles to screen a set of potentially successful constructs, laying the foundation for the next stage of experiments.
2st Engineering
Design
In this phase, we began designing our experiments. Based on the results of the first phase, we identified the necessary parts to construct. By reviewing the literature and leveraging previous experience, we chose to use the pET28a plasmid and DH5α or BL21 strains of E. coli as chassis cells to transform the parts we designed.
Build
Once the designs were established, we proceeded with our experiments. After six months of dedicated effort, we successfully expressed ten different proteins. However, due to specific experimental constraints, we selected three proteins that could best represent our optimization strategies for final testing. The detailed experimental procedures are as follows:
Point Mutation
The point mutation group aims to analyze the differences in activity near the predicted active sites through mutations to validate NicX's active site. Based on previous predictions, they have created five point mutations: NicX-K13D, NicX-V16G, NicX-L48Q, NicX-W52G, and NicX-Y49L. These mutants were constructed and cloned into pET-28a using PCR. The expected results are changes in enzyme activity, either enhancing or reducing it.
Constructing a J1-NicX Fusion Protein
J1 protein site group is dedicated to replicating sections of the original literature related to NicA2 and J1-NicA2. They are currently attempting to apply the protein J1 to NicX to extend the protein's half-life. In this study, the research team has constructed gene variants of NicX, including the wild-type NicX, J1-NicX, NicA2, and J1-NicA2, and expressed them in BL21 (DE3) competent cells. The expected experimental results are that the enzyme activity will diverge from the wild type after a few days.
Cell surface display system
The Surface Display Group is dedicated to using surface display technology to anchor NicX on the outer membrane of E. coli cells without the need for protein purification. This approach allows for direct degradation, reducing manufacturing costs. In this study, the group constructed four gene variants: SP-NicX-HisTag-AIDAI, LppOmpA-linker-NicX-HisTag, INPNC-NicX-HisTag, and SP-HisTag-NicX-BrkA. These constructs were cloned into the pET28a vector and expressed in BL21 (DE3) competent cells.
In our expectations, we hope that the enzyme activity parameters remain essentially unchanged, but there is a significant increase in its expression level.
Three groups with a total of 14 constructs resulted in the successful creation of 10 constructs. The main reason for failures was the gene fragmentation during PCR due to excessive length and the presence of specific secondary structures. For more specific details, please refer to the model's page.
In the expression phase, we determined the optimal induction parameters through trial expression to ensure appropriate protein production. During the purification process, we employed a gradient-based approach to explore the conditions and successfully obtained the desired protein.
Additionally, detailed experiment procedures and information are recorded in our laboratory notebook. For a more comprehensive understanding, please refer to the notebook for specific details.
Test
During the construct validation stage, we verified our constructs using nucleic acid gel electrophoresis to confirm their success by comparing their positions on the gel.
After successful protein purification, we initially conducted protein gel verification (see images in the appendix) to ensure that the expressed protein matched our expectations and to assess protein purity. At this stage, we encountered issues with the expression of J1-NicX, but due to limitations, we had to abandon further research on J1-NicX. Additionally, due to the intrinsic properties of NicX, the results of its purification did not meet high purity standards. Therefore, we performed a grayscale analysis to estimate the protein content within the overall mixture. Unfortunately, due to sample quantity constraints, we were unable to create standard curves for precise protein quantification.
To validate the success of protein optimization, we prepared wild-type NicX enzyme activity samples to explore enzyme activity assays. Initially, we conducted UV full-spectrum scans in an attempt to identify characteristic peaks for substrates and products. Unfortunately, among the five sample sets prepared, we did not observe significant peak shifts. Subsequently, we consulted literature in hopes of finding guidance and even sought assistance from professionals to explore High-Performance Liquid Chromatography (HPLC) for analysis. However, we found that, due to the uniqueness of the product, HPLC separation was not feasible.
After extensive research, analysis, and experimentation, including probe assays and coupling methods, we discovered that HPLC-MS (High-Performance Liquid Chromatography-Mass Spectrometry) was a viable method for analyzing nicotine components. As a result, we prepared the following enzyme activity samples.
The overall system comprises a 50μL reaction volume with the following components:
1mM FMN
25 mM Tris-HCl (pH 8)
300mM NaCl
2μL of protein (approximately 10ng protein)
Nicotine gradient (μL): 0, 50, 150, 250, 400
The reaction is conducted at 37°C, and after 30 minutes and 60 minutes, it is halted by adding an equal volume of 20% acetonitrile. The reaction mix is then sealed and stored at -20°C until further analysis.
For the INPNC-NicX group, in addition to the regular samples, we also performed gradient measurements of cell density. After re-suspension, the bacterial culture had an OD600 of 1.6, which corresponds to 1.6 x 10^8 bacteria. We then prepared samples by diluting the culture 10x, 100x, and 1000x.
After sample preparation, considering cost constraints, we eventually selected the 1000x dilution of INPNC-NicX with a 250μM nicotine concentration for further testing. Both NicX-L48Q and NicX WT were included in the analysis.
Learn
We have start analysis after we got the data:
In terms of standard curve parameters and specific calculations, please refer to the model page for detailed information.
Through our analysis, we successfully reproduced the wild-type NicX protein. The point mutant NicX exhibited an increase in enzyme activity. This validates the accuracy of our prediction regarding the active site and establishes an essential foundation for future research and optimization related to the active site. Furthermore, the cell surface display system showed no difference in enzyme activity compared to the wild type, but it substantially reduced costs as it eliminated the need for purification steps. It's exactly what we were aiming for.
We also conducted a modeling analysis of experimental failures. By using 3dnafold, we predicted the conformation of DNA in solution and found that the failure in three groups was likely due to excessive length, leading to gene breakage during the PCR stage. To address this, we may need to explore solutions in the future, such as adjusting ionic concentrations in the solution.
Regarding the expression failure of J1-NicX, we believe that the main issue was related to problems in the competency transformation process, leading to reduced bacterial viability and complications during the recovery process.
Furthermore, in the protein purification process, we believe that changing vector plasmids and host cells could address issues related to overexpression of endogenous proteins and foreign expression concentrations, which is a goal for our future work.
3rd Engineering —— Our future
In the previous phase of our study, we analyzed the reasons for the failures in specific groups. In the future, we plan to optimize the expression by testing different plasmids and host cells. We also intend to further improve the successfully constructed proteins. As mentioned in our introduction, we aim to develop NicX as a drug-like molecule, exploring ways to enhance its stability and long-term preservation. Our ultimate goal is to make a positive impact and help a wider audience.
In the upcoming phase, we aim to implement the following optimizations:
• Screening plasmids and host cells to reduce endogenous protein interference and increase expression levels.
• Reconstructing J1.
• Exploring enzyme thermal stability and properties under different pH conditions.
We believe that our engineering design can be of assistance to a broader audience and provide inspiration and support to other teams within the iGEM community.
Appendix
Basic part
| Part Name |
Part Number |
Attribution |
| NicX |
BBa_K4722000 |
Ziqian Dai, Daisy |
| J1 |
BBa_K4722001 |
Yichen Wang, Frank |
| Δ50NicA2 |
BBa_K4722002 |
Anqi Chen, Angela |
| NicX-K13D |
BBa_K4722003 |
Lejun Li, James |
| NicX-V16G |
BBa_K4722004 |
Lejun Li, James |
| NicX-L48Q |
BBa_K4722005 |
Lejun Li, James |
| NicX-W52G |
BBa_K4722006 |
Yanru Huang, Max |
| NicX-Y49L |
BBa_K4722007 |
Yanru Huang, Max |
| 3-Succinoyl-pyridine |
BBa_K4722008 |
Yanbing Jie, Eileen |
| AIDA-I |
BBa_K4722009 |
Yanbing Jie, Eileen |
| LppOmpA |
BBa_K4722010 |
Jingyi Peng, Emily |
| INPNC |
BBa_K4722011 |
Junwei Xu, Wayne |
| BrkA |
BBa_K4722012 |
Meiqi Wang, Etatis |
| Histag |
BBa_K4722016 |
Junwei Xu, Wayne |
| Lpp to NicX Linker |
BBa_K4722017 |
Jingyi Peng, Emily |
Composite part
Figure 1. (a) SDS-PAGE of INPNC- NicX- histag(1989bp) & NicX(1293bp) transformed into BL21 expressing strains. Induction time: 15h M:GoldBand Plus 3-color Regular Range Protein Marker(8-180 kDa), 1:INPNC- NicX- histag(1989bp)Before induction 2, 3, 4, 5, 6:After induction; 2: 37℃ 0.3mM IPTG,3: 37℃ 0.5mM IPTG,4: 37℃ 0.7mM IPTG,5: 37℃ 1mM IPTG 6: NicX(1293bp) Before induction 7,8:After induction; 7: 37℃ 0.3mM IPTG,8: 37℃ 0.5mM IPTG (b) 1: 37℃ INPNC- NicX- histag(1989bp)Before induction 2-6:After induction; 2: 37℃ 0.3mM IPTG,3: 37℃ 0.5mM IPTG,4: 37℃ 0.7mM IPTG,5: 37℃ 1mM IPTG；6: 37℃ NicX(1293bp) Before induction 7-8:After induction; 7: 37℃ 0.3mM IPTG,8: 37℃ 0.5mM IPTG
Figure 2. (a) SDS-PAGE of INPNC- NicX-histag(1989bp) & NicX(1293bp) transformed into BL21 expressing strains. Induction time: 15h M:GoldBand Plus 3-color Regular Range Protein Marker(8-180 kDa), 3: NicX (1293bp) Before induction 1,2: After induction; 1: 37℃ 0.5mM IPTG, 2: 37℃ 0.3mM IPTG 8:INPNC- NicX- histag(1989bp) Before induction 4,5,6,7:After induction; 4: 37℃ 1mM IPTG, 5: 37℃ 0.7mM IPTG, 6: 37℃ 0.5mM IPTG,7: 37℃ 0.3mM IPTG(b)3: 37℃ NicX (1293bp) Before induction 1-2: After induction; 1: 37℃ 0.5mM IPTG, 2: 37℃ 0.3mM IPTG 8: 37℃ INPNC- NicX- histag(1989bp) Before induction 4-7:After induction; 4: 37℃ 1mM IPTG, 5: 37℃ 0.7mM IPTG, 6: 37℃ 0.5mM IPTG,7: 37℃ 0.3mM IPTG
Figure 3. (a) SDS-PAGE of J1-Δ50NicA2(1458bp)&J1-NicX(1446bp)&J1-NicX(1446bp) &Δ50NicA2(1305bp) transformed into BL21 expressing strains. Induction time: 15h M:GoldBand Plus 3-color Regular Range Protein Marker(8-180 kDa) 1: Δ50NicA2 (1305bp)Supernatant 3: J1-Δ50NicA2 (1458bp) Before Induction 2: After induction; 2: 37℃ 0.5mM IPTG 5: NicX(1293bp) Before induction 4: After induction; 37℃ 0.5mM IPTG 7: J1-NicX(1446bp) Before induction 6: After induction; 6: 37℃ 0.5mM IPTG 9: Δ50NicA2(1305bp) Before induction 8: After induction; 8: 37℃ 0.5mM IPTG (b) 1: Δ50NicA2 (1305bp)Supernatant 3: 37℃ J1-Δ50NicA2 (1458bp) Before Induction 2: After induction; 2: 37℃ 0.5mM IPTG 5: 37℃ NicX(1293bp) Before induction 4: After induction; 37℃ 0.5mM IPTG 7: 37℃ J1-NicX(1446bp) Before induction 6: After induction; 6: 37℃ 0.5mM IPTG 9: 37℃ Δ50NicA2(1305bp) Before induction 8: After induction; 8: 37℃ 0.5mM IPTG
Figure 4. (a) SDS-PAGE of LppOmpA-linker-NicX-histag(1770bp) transformed into BL21 expressing strains. Induction time: 15h
M:GoldBand Plus 3-color Regular Range Protein Marker(8-180 kDa) 1: NicX-W52G(1293bp)Supernatant 2: Δ50NicA2 (1305bp)Washing buffer 3:NicX-W52G(1293bp)Washing buffer 4: NicX(1293bp)Washing buffer 8: LppOmpA-linker-NicX-histag(1770bp) Before induction 5,6,7: After induction; 5: 37℃ 0.5mM IPTG,6: 37℃ 0.7mM IPTG,7: 37℃ 0.1mM IPTG 9: NicX(1293bp)Supernatant (b)1: NicX-W52G(1293bp)Supernatant 2: Δ50NicA2 (1305bp)Washing buffer 3:NicX-W52G(1293bp)Washing buffer 4: NicX(1293bp)Washing buffer 8: 37℃ LppOmpA-linker-NicX-histag(1770bp) Before induction 5-7: After induction; 5: 37℃ 0.5mM IPTG,6: 37℃ 0.7mM IPTG,7: 37℃ 0.1mM IPTG 9:NicX(1293bp)Supernatant
Figure 5. (a) SDS-PAGE of LppOmpA-linker-NicX-histag(1770bp) transformed into BL21 expressing strains. Induction time: 15h M:GoldBand Plus 3-color Regular Range Protein Marker(8-180 kDa) 1: J1-Δ50NicA2 (1458bp) 2: Δ50NicA2 (1305bp)Supernatant 3:NicX-W52G(1293bp)Supernatant 4: NicX-V16G (1293bp)Supernatant 5: J1-NicX (1446bp) 6: NicX(1293bp) 7,8,9: LppOmpA-linker-NicX-histag(1770bp) After induction; 7: 37℃ 0.1mM IPTG,8: 37℃ 0.3mM IPTG,9: 37℃ 0.5mM IPTG 10: J1-Δ50NicA2 (1458bp)Supernatant 11: NicX(1293bp)Supernatant 12: J1-NicX (1446bp)Supernatant (b) 1: J1-Δ50NicA2 (1458bp) 2: Δ50NicA2 (1305bp)Supernatant 3:NicX-W52G(1293bp)Supernatant 4: NicX-V16G (1293bp)Supernatant 5: J1-NicX (1446bp) 6: NicX(1293bp) 7-9: LppOmpA-linker-NicX-histag(1770bp) After induction; 7: 37℃ 0.1mM IPTG,8: 37℃ 0.3mM IPTG,9: 37℃ 0.5mM IPTG 10: J1-Δ50NicA2 (1458bp)Supernatant 11: NicX(1293bp)Supernatant 12: J1-NicX (1446bp)Supernatant
Figure 6. (a) SDS-PAGE of LppOmpA-linker-NicX-histag(1770bp) transformed into BL21 expressing strains. Induction time: 15h
M:GoldBand Plus 3-color Regular Range Protein Marker(8-180 kDa) 1: NicX-V16G(1293bp)Washing buffer 2: LppOmpA-linker-NicX-histag(1770bp) Before induction 3,4,5,6,7,8,9,: After induction; 3: 16℃ 0.3mM IPTG,4: 16℃ 0.5mM IPTG,5: 16℃ 0.7mM IPTG, 6: 37℃ 0.1mM IPTG, 7: 37℃ 0.3mM IPTG,8: 37℃ 0.5mM IPTG,9: 37℃ 0.7mM IPTG 10: NicX-W52G(1293bp)Washing buffer 11: J1-Δ50NicA2 (1458bp)Washing buffer 12: NicX(1293bp)Washing buffer 13: J1-NicX (1446bp)Washing buffer 14: J1-Δ50NicA2 (1458bp)Washing buffer
Figure 7. (a) SDS-PAGE of LppOmpA-linker-NicX-histag(1770bp) & NicX-W52G(1293bp) transformed into BL21 expressing strains. Induction time: 15h M:GoldBand Plus 3-color Regular Range Protein Marker(8-180 kDa), 1: LppOmpA-linker-NicX-histag(1770bp)Before induction 2, 3, 4:After induction; 2: 37℃ 0.7mM IPTG, 3: 37℃ 0.5mM IPTG, 4: 37℃ 0.3mM IPTG, 7,10: NicX-W52G(1293bp) Before induction 5,6,8,9:After induction; 5: 37℃ 0.7mM IPTG, 6: 37℃ 0.5mM IPTG, 8: 16℃ 0.7mM IPTG, 9: 16℃ 0.5mM IPTG (b) 1: LppOmpA-linker-NicX-histag(1770bp)Before induction 2-4:After induction; 2: 37℃ 0.7mM IPTG, 3: 37℃ 0.5mM IPTG, 4: 37℃ 0.3mM IPTG, 7: 37℃ NicX-W52G(1293bp) Before induction 5-6:After induction; 5: 37℃ 0.7mM IPTG, 6: 37℃ 0.5mM IPTG 10: 16℃ NicX-W52G(1293bp) Before induction 8-9:After induction; 5: 37℃ 0.7mM IPTG, 6: 37℃ 0.5mM IPTG, 8: 16℃ 0.7mM IPTG, 9: 16℃ 0.5mM IPTG
Figure 8. (a) SDS-PAGE of NicX-L48Q (1293bp) transformed into BL21 expressing strains. Induction time: 15h M:GoldBand Plus 3-color Regular Range Protein Marker(8-180 kDa), 6,12: Before induction 1,2,3,4,5,7,8,9,10,11:After induction; 1: 37℃ 1mM IPTG,2: 37℃ 0.7mM IPTG,3: 37℃ 0.5mM IPTG, 4: 37℃ 0.3mM IPTG, 5: 37℃ 0.1mM IPTG, 7: 16℃ 1mM IPTG, 8: 16℃ 0.7mM IPTG, 9: 16℃ 0.5mM IPTG, 10: 16℃ 0.3mM IPTG, 11: 16℃ 0.1mM IPTG (b)6: 37℃ Before induction 1-5:After induction; 1: 37℃ 1mM IPTG,2: 37℃ 0.7mM IPTG,3: 37℃ 0.5mM IPTG, 4: 37℃ 0.3mM IPTG, 5: 37℃ 0.1mM IPTG 12: 16℃ Before induction 7-11:After induction; 7: 16℃ 1mM IPTG, 8: 16℃ 0.7mM IPTG, 9: 16℃ 0.5mM IPTG, 10: 16℃ 0.3mM IPTG, 11: 16℃ 0.1mM IPTG
Figure 9. (a) SDS-PAGE of NicX-Y49L(1293bp) transformed into BL21 expressing strains. Induction time: 15h M:GoldBand Plus 3-color Regular Range Protein Marker(8-180 kDa), 1,7:Before induction 2,3,4,5,6,8,9,10,11,12: After induction; 2: 16℃ 0.1mM IPTG, 3: 16℃ 0.3mM IPTG, 4: 16℃ 0.5mM IPTG, 5: 16℃ 0.7mM IPTG, 6: 16℃ 1mM IPTG, 8: 37℃ 0.1mM IPTG, 9: 16℃ 0.3mM IPTG, 10: 16℃ 0.5mM IPTG, 11: 16℃ 0.7mM IPTG, 12: 37℃ 1mM IPTG (b) 1: 16℃Before induction 2-6:After induction; 2: 16℃ 0.1mM IPTG,3: 16℃ 0.3mM IPTG, 4: 16℃ 0.5mM IPTG, 5: 16℃ 0.7mM IPTG, 6: 16℃ 1mM IPTG, 7: 16℃Before induction 8-12:After induction;8: 37℃ 0.1mM IPTG, 9: 16℃ 0.3mM IPTG, 10: 16℃ 0.5mM IPTG, 11: 16℃ 0.7mM IPTG, 12: 37℃ 1mM IPTG