Difference between revisions of "Part:BBa K4722000"

Latest revision as of 15:54, 11 October 2023

NicX

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal PstI site found at 762
12
INCOMPATIBLE WITH RFC[12]
Illegal PstI site found at 762
21
COMPATIBLE WITH RFC[21]
23
INCOMPATIBLE WITH RFC[23]
Illegal PstI site found at 762
25
INCOMPATIBLE WITH RFC[25]
Illegal PstI site found at 762
Illegal NgoMIV site found at 921
1000
COMPATIBLE WITH RFC[1000]

Usage and Biology

The enzyme NicX, derived from the bacterium Bacteroides xylanisolvens^[1]^[2], exhibits a predicted core structure akin to the computational model of the established nicotine-degrading enzyme, NicA. Notably, NicX demonstrates proficiency in the degradation of nicotine. Furthermore, it has been observed that in the presence of NicX, B. xylanisolvens exhibits an enhanced capacity to degrade nicotine. Moreover, the transferability of NicX into Escherichia coli has been demonstrated, with the DNA fragment encoding the full-length NicX gene being successfully cloned into the pET28a vector through conventional molecular cloning techniques (Pro-cet-cell). This particular NicX component serves as a fundamental element in the construction of our composite part denoted as J1-NicX^[3].

Design Consideration

The genetic construct was ligated into a pET28a plasmid vector and subsequently introduced into Escherichia coli strain BL21 (DE3). Enzymatic cleavage was performed at the NcoI and XhoI restriction sites, allowing for the precise integration of NicX. The original His tag on the plasmid was retained, which is useful for subsequent protein purification steps. To enhance the stability of NicX within the human body, the J1 fusion protein was strategically linked in front of them. Specific point mutations were introduced to the NicX gene sequence with the aim of enhancing its enzymatic activity. NicX was genetically connected with other components to enable its direct translation onto the surface of BL21. This innovation eliminated the need for protein purification steps, allowing for the direct utilization of E. coli as a host for enzymes in various applications^[4]^[5]^[6].

Protein Expression

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

LC-MS

Figure 10. LC-MS experiment conditions 1

Figure 11. LC-MS experiment conditions 2

LC-MS of NicX wild type

Picture-protein name- nicotine concentration-reaction time	[S]/ng	Δ[S]/ng	Δ[S] in origin/ng	Δ[S] in μmol	V/μmol· min^-1
NicX 400μM 0.5	0.075	0.018	180	1.16	0.039
NicX 400μM 1	0.057	0.018	180	1.16	0.039

Table 1. LC-MS for NicX wild type

Figure 12. NicX 400μM 0.5

Figure 13. NicX 400μM 1

LC-MS of INPNC-NicX

Picture-protein name- nicotine concentration-reaction time	[S]/ng	Δ[S]/ng	Δ[S] in origin/ng	Δ[S] in μmol	V/μmol· min^-1
INPNC-NicX 1000x 250μM 0.5	0.02	0.001	10	0.06	0.001
INPNC-NicX 1000x 250μM 0.5	0.019	0.001	10	0.06	0.002

Table 2. LC-MS for INPNC-NicX

Figure 14. INPNC-NicX 1000x 250μM 0.5

Figure 15. INPNC-NicX 1000x 250μM 0.5

LC-MS of NicX-L48Q

Picture-protein name- nicotine concentration-reaction time	[S]/ng	Δ[S]/ng	Δ[S] in origin/ng	Δ[S] in μmol	V/μmol· min^-1
L48Q 250μM 0.5	0.02	>0.02	>200	>1.29	>0.039
L48Q 250μM 1	~0	>0.02	>200	>1.29	>0.039

Table 2. LC-MS for NicX-L48Q

In the case of the L48Q 250 μM sample, there was a minor contamination issue during the LC-MS sample preparation. We manually corrected the baseline to obtain the values. For all other groups, values were obtained automatically as described above.Due to the need for a 10,000-fold dilution of HPLC-MS samples, we conducted relevant processing during data analysis to obtain the approximate raw data for the samples.

Data Analysis and Discussion

Firstly, for the wild-type NicX protein, we diluted the protein to a concentration of approximately 10 ng/μL before use. Considering that protein purification is not 100% due to protein properties, we roughly estimated that the protein constitutes around 50% of the total concentration. In the enzyme activity system, we added 2 μL, which is approximately 10 ng of protein. Compared to the 1 μg of protein determined in the literature, this is roughly 100 times lower, and the reaction rate is also around 100 times lower. Therefore, we can conclude that the wild-type NicX was successfully expressed and reproduced.

Furthermore, according to the Michaelis-Menten constant (Km) chart in the original paper, we found that the enzyme activity is not significantly affected by nicotine concentrations in the range of 250-400 μM. Thus, even at a nicotine concentration of 250 μM, the L48Q mutation exhibits comparable enzyme activity to NicX at 400 μM nicotine concentration. Analyzing the reaction rates, we found that even with a slightly lower nicotine concentration, L48Q has a higher enzyme reaction rate than the wild-type. Although the exact difference in reaction rates is unknown, this is sufficient to conclude that L48 is one of the active residues. As previously demonstrated, this means that our active site prediction is reasonably accurate.

For INPNC-NicX, we observed a suspicious anomalous peak in the 0.5-hour chromatogram, specifically due to column residue. The reason for this occurrence is likely improper handling of bacteria during pretreatment. However, due to the presence of residue, the final enzyme activity should be greater than 0.002 μmol/min. When we determined the concentration of INPNC-NicX at OD600 = 1.6, based on previous experiments and empirical data, we estimated the cell density to be approximately 1.6 x 10^8 cells per milliliter, which becomes 1.6 x 10^5 cells per milliliter after a 1000-fold dilution. In the system, we added 2 μL of bacterial culture, corresponding to 320 cells. Based on the previous calculation, we know that the reaction rate for 10 ng of protein is around 0.039 μmol/min. Using this information, we can infer that cells containing about 0.5 ng of protein are effectively participating in the reaction. Therefore, we can conclude that there is approximately 0.16 ng of protein per hundred cells. Additionally, we already know that 320 cells correspond to 0.5 ng of protein and a reaction rate of 0.002 μmol/min. Scaling this up, 6,400 cells correspond to 10 ng of protein and a reaction rate of around 0.04 μmol/min, consistent with the wild-type. Furthermore, in practical terms, when cultivating 1 L of medium to an OD600 of 1, there are approximately 10^8 cells, equivalent to about 16,000 ng of protein or a protein concentration of around 16 mg/mL. Additionally, surface display technology does not require cell disruption and purification, making it far more efficient than traditional intracellular expression methods.

References

↑ Chen, B., Sun, L., Zeng, G., Shen, Z., Wang, K., Yin, L., ... & Jiang, C. (2022). Gut bacteria alleviate smoking-related NASH by degrading gut nicotine. Nature, 610(7932), 562-568. https://doi.org/10.1038/s41586-022-05299-4
↑ Jiménez, J. I., Canales, Á., Jiménez-Barbero, J., Ginalski, K., Rychlewski, L., García, J. L., & Díaz, E. (2008). Deciphering the genetic determinants for aerobic nicotinic acid degradation: the nic cluster from Pseudomonas putida KT2440. Proceedings of the National Academy of Sciences, 105(32), 11329-11334.https://doi.org/10.1073/pnas.080227310
↑ Xue, S., Kallupi, M., Zhou, B., Smith, L. C., Miranda, P. O., George, O., & Janda, K. D. (2018). An enzymatic advance in nicotine cessation therapy. Chemical Communications, 54(14), 1686-1689.DOI https://doi.org/10.1039/C7CC09134F
↑ Wang, S. N., Liu, Z., Tang, H. Z., Meng, J., & Xu, P. (2007). Characterization of environmentally friendly nicotine degradation by Pseudomonas putida biotype A strain S16. Microbiology, 153(5), 1556-1565. https://doi.org/10.1099/mic.0.2006/005223-0
↑ Wang, W., Xu, P., & Tang, H. (2015). Sustainable production of valuable compound 3-succinoyl-pyridine by genetically engineering Pseudomonas putida using the tobacco waste. Scientific Reports, 5(1), 16411. https://doi.org/10.1038/srep16411
↑ Sun, F., Pang, X., Xie, T., Zhai, Y., Wang, G., & Sun, F. (2015). BrkAutoDisplay: functional display of multiple exogenous proteins on the surface of Escherichia coli by using BrkA autotransporter. Microbial Cell Factories, 14, 1-12. https://doi.org/10.1186/s12934-015-0316-3

[1] Chen, B., Sun, L., Zeng, G., Shen, Z., Wang, K., Yin, L., ... & Jiang, C. (2022). Gut bacteria alleviate smoking-related NASH by degrading gut nicotine. Nature, 610(7932), 562-568. https://doi.org/10.1038/s41586-022-05299-4

[2] Jiménez, J. I., Canales, Á., Jiménez-Barbero, J., Ginalski, K., Rychlewski, L., García, J. L., & Díaz, E. (2008). Deciphering the genetic determinants for aerobic nicotinic acid degradation: the nic cluster from Pseudomonas putida KT2440. Proceedings of the National Academy of Sciences, 105(32), 11329-11334.https://doi.org/10.1073/pnas.080227310

[3] Xue, S., Kallupi, M., Zhou, B., Smith, L. C., Miranda, P. O., George, O., & Janda, K. D. (2018). An enzymatic advance in nicotine cessation therapy. Chemical Communications, 54(14), 1686-1689.DOI https://doi.org/10.1039/C7CC09134F

[4] Wang, S. N., Liu, Z., Tang, H. Z., Meng, J., & Xu, P. (2007). Characterization of environmentally friendly nicotine degradation by Pseudomonas putida biotype A strain S16. Microbiology, 153(5), 1556-1565. https://doi.org/10.1099/mic.0.2006/005223-0

[5] Wang, W., Xu, P., & Tang, H. (2015). Sustainable production of valuable compound 3-succinoyl-pyridine by genetically engineering Pseudomonas putida using the tobacco waste. Scientific Reports, 5(1), 16411. https://doi.org/10.1038/srep16411

[6] Sun, F., Pang, X., Xie, T., Zhai, Y., Wang, G., & Sun, F. (2015). BrkAutoDisplay: functional display of multiple exogenous proteins on the surface of Escherichia coli by using BrkA autotransporter. Microbial Cell Factories, 14, 1-12. https://doi.org/10.1186/s12934-015-0316-3

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[2]

[3]

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@@ Line 9: / Line 9: @@
 ===Usage and Biology===
-The enzyme NicX, derived from the bacterium Bacteroides xylanisolvens<ref>Chen, B., Sun, L., Zeng, G., Shen, Z., Wang, K., Yin, L., ... & Jiang, C. (2022). Gut bacteria alleviate smoking-related NASH by degrading gut nicotine. Nature, 610(7932), 562-568. https://doi.org/10.1038/s41586-022-05299-4</ref><ref>Jiménez, J. I., Canales, Á., Jiménez-Barbero, J., Ginalski, K., Rychlewski, L., García, J. L., & Díaz, E. (2008). Deciphering the genetic determinants for aerobic nicotinic acid degradation: the nic cluster from Pseudomonas putida KT2440. Proceedings of the National Academy of Sciences, 105(32), 11329-11334.https://doi.org/10.1073/pnas.080227310</ref>, exhibits a predicted core structure akin to the computational model of the established nicotine-degrading enzyme, NicA. Notably, NicX demonstrates proficiency in the degradation of nicotine. Furthermore, it has been observed that in the presence of NicX, B. xylanisolvens exhibits an enhanced capacity to degrade nicotine. Moreover, the transferability of nicX into Escherichia coli has been demonstrated, with the DNA fragment encoding the full-length NicX gene being successfully cloned into the pET28a vector through conventional molecular cloning techniques (Pro-cet-cell). This particular NicX component serves as a fundamental element in the construction of our composite part denoted as J1-NicX<ref>Xue, S., Kallupi, M., Zhou, B., Smith, L. C., Miranda, P. O., George, O., & Janda, K. D. (2018). An enzymatic advance in nicotine cessation therapy. Chemical Communications, 54(14), 1686-1689.DOI	https://doi.org/10.1039/C7CC09134F</ref>.
+The enzyme NicX, derived from the bacterium Bacteroides xylanisolvens<ref>Chen, B., Sun, L., Zeng, G., Shen, Z., Wang, K., Yin, L., ... & Jiang, C. (2022). Gut bacteria alleviate smoking-related NASH by degrading gut nicotine. Nature, 610(7932), 562-568. https://doi.org/10.1038/s41586-022-05299-4</ref><ref>Jiménez, J. I., Canales, Á., Jiménez-Barbero, J., Ginalski, K., Rychlewski, L., García, J. L., & Díaz, E. (2008). Deciphering the genetic determinants for aerobic nicotinic acid degradation: the nic cluster from Pseudomonas putida KT2440. Proceedings of the National Academy of Sciences, 105(32), 11329-11334.https://doi.org/10.1073/pnas.080227310</ref>, exhibits a predicted core structure akin to the computational model of the established nicotine-degrading enzyme, NicA. Notably, NicX demonstrates proficiency in the degradation of nicotine. Furthermore, it has been observed that in the presence of NicX, B. xylanisolvens exhibits an enhanced capacity to degrade nicotine. Moreover, the transferability of NicX into Escherichia coli has been demonstrated, with the DNA fragment encoding the full-length NicX gene being successfully cloned into the pET28a vector through conventional molecular cloning techniques (Pro-cet-cell). This particular NicX component serves as a fundamental element in the construction of our composite part denoted as J1-NicX<ref>Xue, S., Kallupi, M., Zhou, B., Smith, L. C., Miranda, P. O., George, O., & Janda, K. D. (2018). An enzymatic advance in nicotine cessation therapy. Chemical Communications, 54(14), 1686-1689.DOI	https://doi.org/10.1039/C7CC09134F</ref>.
-The origin of this partial information can be attributed to the pioneering work of Jimenez et al., as documented in their study titled 'Gut Bacteria Alleviate Smoking-Related Non-Alcoholic Steatohepatitis (NASH) by Degrading Gut Nicotine.' Additionally, the nic cluster originating from Pseudomonas putida KT2440 has relevance in this context.
 ===Design Consideration===
@@ Line 18: / Line 17: @@
 To enhance the stability of NicX within the human body, the J1 fusion protein was strategically linked in front of them.
 Specific point mutations were introduced to the NicX gene sequence with the aim of enhancing its enzymatic activity.
-NicX was genetically connected with other components to enable its direct translation onto the surface of BL21. This innovation eliminated the need for protein purification steps, allowing for the direct utilization of E. coli as a host for enzymes in various applications.
+NicX was genetically connected with other components to enable its direct translation onto the surface of BL21. This innovation eliminated the need for protein purification steps, allowing for the direct utilization of E. coli as a host for enzymes in various applications<ref>Wang, S. N., Liu, Z., Tang, H. Z., Meng, J., & Xu, P. (2007). Characterization of environmentally friendly nicotine degradation by Pseudomonas putida biotype A strain S16. Microbiology, 153(5), 1556-1565. https://doi.org/10.1099/mic.0.2006/005223-0</ref><ref>Wang, W., Xu, P., & Tang, H. (2015). Sustainable production of valuable compound 3-succinoyl-pyridine by genetically engineering Pseudomonas putida using the tobacco waste. Scientific Reports, 5(1), 16411. https://doi.org/10.1038/srep16411</ref><ref>Sun, F., Pang, X., Xie, T., Zhai, Y., Wang, G., & Sun, F. (2015). BrkAutoDisplay: functional display of multiple exogenous proteins on the surface of Escherichia coli by using BrkA autotransporter. Microbial Cell Factories, 14, 1-12. https://doi.org/10.1186/s12934-015-0316-3</ref>.
 ===Protein Expression===
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 |-
-| '''Figure 3.''' (a) SDS-PAGE of J1-▲50NicA2(1458bp)&J1-NicX(1446bp)&J1-NicX(1446bp) &▲50NicA2(1305bp) transformed into BL21 expressing strains. Induction time: 15h M:GoldBand Plus 3-color Regular Range Protein Marker(8-180 kDa) 1: ▲50NicA2 (1305bp)Supernatant 3: J1-▲50NicA2 (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: ▲50NicA2(1305bp) Before induction 8: After induction; 8: 37℃ 0.5mM IPTG (b) 1: ▲50NicA2 (1305bp)Supernatant 3: 37℃ J1-▲50NicA2 (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℃ ▲50NicA2(1305bp) Before induction 8: After induction; 8: 37℃ 0.5mM 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
 |}
@@ Line 44: / Line 42: @@
 |-
 | '''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: ▲50NicA2 (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: ▲50NicA2 (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
+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
 |}
@@ Line 51: / Line 49: @@
 |-
 | '''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-▲50NicA2 (1458bp) 2: ▲50NicA2 (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-▲50NicA2 (1458bp)Supernatant 11: NicX(1293bp)Supernatant 12: J1-NicX (1446bp)Supernatant (b) 1: J1-▲50NicA2 (1458bp) 2: ▲50NicA2 (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-▲50NicA2 (1458bp)Supernatant 11: NicX(1293bp)Supernatant 12: J1-NicX (1446bp)Supernatant
+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
 |}
@@ Line 58: / Line 56: @@
 |-
 | '''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-▲50NicA2 (1458bp)Washing buffer 12: NicX(1293bp)Washing buffer 13: J1-NicX (1446bp)Washing buffer 14: J1-▲50NicA2 (1458bp)Washing buffer
+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
 |}
@@ Line 79: / Line 77: @@
 |}
-===Enzyme Activity===
+===LC-MS===
-TBD
+{|
+| <html><img style="width:900px" src=https://static.igem.wiki/teams/4722/wiki/condition-1.png></html>
+|-
+| '''Figure 10.''' LC-MS experiment conditions 1
+|}
+{|
+| <html><img style="width:900px" src=https://static.igem.wiki/teams/4722/wiki/condition-2.png></html>
+|-
+| '''Figure 11.''' LC-MS experiment conditions 2
+|}
+<html>
+<head>
+    <title>LC-MS of NicX wild type</title>
+</head>
+<body>
+    <table border="1">
+        <tr>
+            <th>Picture-protein name-<br/>nicotine concentration-reaction time</th>
+            <th>[S]/ng</th>
+            <th>Δ[S]/ng</th>
+            <th>Δ[S] in origin/ng</th>
+            <th>Δ[S] in μmol</th>
+            <th>V/μmol·<p>min<sup>-1</sup></p></th>
+        </tr>
+        <tr>
+            <td>NicX 400μM 0.5</td>
+            <td>0.075</td>
+            <td>0.018</td>
+            <td>180</td>
+            <td>1.16</td>
+            <td>0.039</td>
+        </tr>
+        <tr>
+            <td>NicX 400μM 1</td>
+            <td>0.057</td>
+            <td>0.018</td>
+            <td>180</td>
+            <td>1.16</td>
+            <td>0.039</td>
+        </tr>
+    </table>
+</body>
+</html>
+'''Table 1.''' LC-MS for NicX wild type
+{|
+| <html><img style="width:900px" src=https://static.igem.wiki/teams/4722/wiki/lcms-nicx-400-0-5.png></html>
+|-
+| '''Figure 12.''' NicX 400μM 0.5
+|}
+{|
+| <html><img style="width:900px" src=https://static.igem.wiki/teams/4722/wiki/icms-nicx-400-1.png></html>
+|-
+| '''Figure 13.''' NicX 400μM 1
+|}
+<html>
+<head>
+    <title>LC-MS of INPNC-NicX</title>
+</head>
+<body>
+    <table border="1">
+        <tr>
+            <th>Picture-protein name-<br/>nicotine concentration-reaction time</th>
+            <th>[S]/ng</th>
+            <th>Δ[S]/ng</th>
+            <th>Δ[S] in origin/ng</th>
+            <th>Δ[S] in μmol</th>
+            <th>V/μmol·<p>min<sup>-1</sup></p></th>
+        </tr>
+        <tr>
+            <td>INPNC-NicX 1000x 250μM 0.5</td>
+            <td>0.02</td>
+            <td>0.001</td>
+            <td>10</td>
+            <td>0.06</td>
+            <td>0.001</td>
+        </tr>
+        <tr>
+            <td>INPNC-NicX 1000x 250μM 0.5</td>
+            <td>0.019</td>
+            <td>0.001</td>
+            <td>10</td>
+            <td>0.06</td>
+            <td>0.002</td>
+        </tr>
+    </table>
+</body>
+</html>
+'''Table 2.''' LC-MS for INPNC-NicX
+{|
+| <html><img style="width:900px" src=https://static.igem.wiki/teams/4722/wiki/inpnc-nicx-250-1.png></html>
+|-
+| '''Figure 14.''' INPNC-NicX 1000x 250μM 0.5
+|}
+{|
+| <html><img style="width:900px" src=https://static.igem.wiki/teams/4722/wiki/inpnc-nicx-1000x-250-0-5.png></html>
+|-
+| '''Figure 15.''' INPNC-NicX 1000x 250μM 0.5
+|}
+<html>
+<head>
+    <title>LC-MS of NicX-L48Q</title>
+</head>
+<body>
+    <table border="1">
+        <tr>
+            <th>Picture-protein name-<br/>nicotine concentration-reaction time</th>
+            <th>[S]/ng</th>
+            <th>Δ[S]/ng</th>
+            <th>Δ[S] in origin/ng</th>
+            <th>Δ[S] in μmol</th>
+            <th>V/μmol·<p>min<sup>-1</sup></p></th>
+        </tr>
+        <tr>
+            <td>L48Q 250μM 0.5</td>
+            <td>0.02</td>
+            <td>>0.02</td>
+            <td>>200</td>
+            <td>>1.29</td>
+            <td>>0.039</td>
+        </tr>
+        <tr>
+            <td>L48Q 250μM 1</td>
+            <td>~0</td>
+            <td>>0.02</td>
+            <td>>200</td>
+            <td>>1.29</td>
+            <td>>0.039</td>
+        </tr>
+    </table>
+</body>
+</html>
+'''Table 2.''' LC-MS for NicX-L48Q
+In the case of the L48Q 250 μM sample, there was a minor contamination issue during the LC-MS sample preparation. We manually corrected the baseline to obtain the values. For all other groups, values were obtained automatically as described above.Due to the need for a 10,000-fold dilution of HPLC-MS samples, we conducted relevant processing during data analysis to obtain the approximate raw data for the samples.
+===Data Analysis and Discussion===
+Firstly, for the wild-type NicX protein, we diluted the protein to a concentration of approximately 10 ng/μL before use. Considering that protein purification is not 100% due to protein properties, we roughly estimated that the protein constitutes around 50% of the total concentration. In the enzyme activity system, we added 2 μL, which is approximately 10 ng of protein. Compared to the 1 μg of protein determined in the literature, this is roughly 100 times lower, and the reaction rate is also around 100 times lower. Therefore, we can conclude that the wild-type NicX was successfully expressed and reproduced.
+Furthermore, according to the Michaelis-Menten constant (Km) chart in the original paper, we found that the enzyme activity is not significantly affected by nicotine concentrations in the range of 250-400 μM. Thus, even at a nicotine concentration of 250 μM, the L48Q mutation exhibits comparable enzyme activity to NicX at 400 μM nicotine concentration. Analyzing the reaction rates, we found that even with a slightly lower nicotine concentration, L48Q has a higher enzyme reaction rate than the wild-type. Although the exact difference in reaction rates is unknown, this is sufficient to conclude that L48 is one of the active residues. As previously demonstrated, this means that our active site prediction is reasonably accurate.
+For INPNC-NicX, we observed a suspicious anomalous peak in the 0.5-hour chromatogram, specifically due to column residue. The reason for this occurrence is likely improper handling of bacteria during pretreatment. However, due to the presence of residue, the final enzyme activity should be greater than 0.002 μmol/min. When we determined the concentration of INPNC-NicX at OD600 = 1.6, based on previous experiments and empirical data, we estimated the cell density to be approximately 1.6 x 10^8 cells per milliliter, which becomes 1.6 x 10^5 cells per milliliter after a 1000-fold dilution. In the system, we added 2 μL of bacterial culture, corresponding to 320 cells. Based on the previous calculation, we know that the reaction rate for 10 ng of protein is around 0.039 μmol/min. Using this information, we can infer that cells containing about 0.5 ng of protein are effectively participating in the reaction. Therefore, we can conclude that there is approximately 0.16 ng of protein per hundred cells. Additionally, we already know that 320 cells correspond to 0.5 ng of protein and a reaction rate of 0.002 μmol/min. Scaling this up, 6,400 cells correspond to 10 ng of protein and a reaction rate of around 0.04 μmol/min, consistent with the wild-type. Furthermore, in practical terms, when cultivating 1 L of medium to an OD600 of 1, there are approximately 10^8 cells, equivalent to about 16,000 ng of protein or a protein concentration of around 16 mg/mL. Additionally, surface display technology does not require cell disruption and purification, making it far more efficient than traditional intracellular expression methods.
 ===References===