iGEM 2025 — Team baid-china

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{% block title %}Notebook{% endblock %}
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Notebook

![](https://static.igem.wiki/teams/5625/pics/team/apple.webp)
![](https://static.igem.wiki/teams/5625/pics/team/avacado.webp)
![](https://static.igem.wiki/teams/5625/pics/team/banana.webp)
![](https://static.igem.wiki/teams/5625/pics/team/corn.webp)
![](https://static.igem.wiki/teams/5625/pics/team/dragonfruit.webp)
![](https://static.igem.wiki/teams/5625/pics/team/grape.webp)
![](https://static.igem.wiki/teams/5625/pics/team/lemon.webp)
![](https://static.igem.wiki/teams/5625/pics/team/lichee.webp)
![](https://static.igem.wiki/teams/5625/pics/team/mango.webp)
![](https://static.igem.wiki/teams/5625/pics/team/mangostene.webp)
![](https://static.igem.wiki/teams/5625/pics/team/melon.webp)
![](https://static.igem.wiki/teams/5625/pics/team/orange.webp)
![](https://static.igem.wiki/teams/5625/pics/team/peach.webp)
![](https://static.igem.wiki/teams/5625/pics/team/pear.webp)
![](https://static.igem.wiki/teams/5625/pics/team/pineapple.webp)
![](https://static.igem.wiki/teams/5625/pics/team/strawberry.webp)
![](https://static.igem.wiki/teams/5625/pics/team/tomato.webp)
![](https://static.igem.wiki/teams/5625/pics/team/watermelon.webp)
![](https://static.igem.wiki/teams/5625/pics/team/brocoli.webp)
![](https://static.igem.wiki/teams/5625/pics/team/pumpkin.webp)
![](https://static.igem.wiki/teams/5625/pics/team/coconut.webp)
![](https://static.igem.wiki/teams/5625/pics/team/spinach.webp)

# Notebook

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{% extends "\_layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
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Engineering

![](https://static.igem.wiki/teams/5625/pics/team/apple.webp)
![](https://static.igem.wiki/teams/5625/pics/team/avacado.webp)
![](https://static.igem.wiki/teams/5625/pics/team/banana.webp)
![](https://static.igem.wiki/teams/5625/pics/team/corn.webp)
![](https://static.igem.wiki/teams/5625/pics/team/dragonfruit.webp)
![](https://static.igem.wiki/teams/5625/pics/team/grape.webp)
![](https://static.igem.wiki/teams/5625/pics/team/lemon.webp)
![](https://static.igem.wiki/teams/5625/pics/team/lichee.webp)
![](https://static.igem.wiki/teams/5625/pics/team/mango.webp)
![](https://static.igem.wiki/teams/5625/pics/team/mangostene.webp)
![](https://static.igem.wiki/teams/5625/pics/team/melon.webp)
![](https://static.igem.wiki/teams/5625/pics/team/orange.webp)
![](https://static.igem.wiki/teams/5625/pics/team/peach.webp)
![](https://static.igem.wiki/teams/5625/pics/team/pear.webp)
![](https://static.igem.wiki/teams/5625/pics/team/pineapple.webp)
![](https://static.igem.wiki/teams/5625/pics/team/strawberry.webp)
![](https://static.igem.wiki/teams/5625/pics/team/tomato.webp)
![](https://static.igem.wiki/teams/5625/pics/team/watermelon.webp)
![](https://static.igem.wiki/teams/5625/pics/team/brocoli.webp)
![](https://static.igem.wiki/teams/5625/pics/team/pumpkin.webp)
![](https://static.igem.wiki/teams/5625/pics/team/coconut.webp)
![](https://static.igem.wiki/teams/5625/pics/team/spinach.webp)

# Overview

Our project Pestacheck aims to develop a rapid, low-cost pesticide residue detection test strip. After careful research of the development of fast pesticide detection technologies emerging on the market, we decided to use enzyme-inhibition assay as our core principle. Combining synthetic biology, we first optimized the expression of a common indicator in enzyme inhibition assay——Acetylcholineterase (AChE) with the help of Alanine scanning. The mutant enzymes were then expressed in E.coli. However, due to the formation of inclusion bodies, we then express the enzyme in a cell-free system. Then, we integrate the AChE mutant with the highest activity and thermal stability with golden nanoparticles (AuNP) to enhance its ability to stay on abiotic surfaces. At last, we redesign a more efficient paper test strip to check for the food safety of the entire human-being.

# Cycle 1: Alanine-scanning mutagenesis

**In Silico Design**
Acetylcholinesterase (AChE) is an enzyme primarily found in muscles and nerves. According to Fuyal & Giri, it can be utilized to generate signals when pesticide residues were detected (2020). The current AChE immobilized on the strip consists a series of problems. Thus, we first decided to optimized the stability and sensitivity of the enzymes.
The method we utilized is Alanine-scanning. By measuring the ddG value (the change in free energy before and after), it is possible to determine the contribution of this residue to stability（Moreira et al., 2007). As we are trying to make the enzymes more stable, we need to identify these residues that cause low stability of the enzymes and optimize them (the sites with a relatively low ddG values).
The procedure we used is shown below:

![Figure 1. Alanine-scanning procedure](https://static.igem.wiki/teams/5625/pics/engineering/figure-1.webp)

Figure 1. Alanine-scanning procedure

**Build**
With the assistance of the Software Team, we measured the ddG values of 550 sites of AChE. The results are shown in the graph below.

![Figure 2. Alanine-scanning results](https://static.igem.wiki/teams/5625/pics/engineering/figure-2.webp)

Figure 2. Alanine-scanning results

Among all the sites, there are 5 sites that shown significant low value of ddG (below 0). They are 203, 355, 396, 403 and 466. These five residues were selected and mutated to alanine.

**Test**
After identifying the potential unstable sites during the construction phase, we initially wanted to test through computer simulations whether the mutations at these sites could indeed enhance the stability of AChE. Using molecular dynamics simulations, we measured the root mean square deviation (RMSD) values to monitor the structural fluctuations of the wild-type and mutant enzymes(Maiorov & Crippen, 1994). At the same time, we predicted the free energy changes to assess the stability and checked whether the catalytic sites remained intact. These tests helped us confirm which mutations were indeed beneficial for stability and avoid those that might reduce the enzyme activity.

![Figure 3. Modifying enzyme structure](https://static.igem.wiki/teams/5625/pics/engineering/figure-3.webp)

Figure 3. Modifying enzyme structure

The white part illustrated the original conformation as the blue part illustrated the optimized structure. The tested RMSD value is 0.833. As it’s much lower than 3, the protein backbone is staying very close to the origin conformation during the simulation. The mutation does not destabilize the enzyme.

![Figure 4. The mutation sites](https://static.igem.wiki/teams/5625/pics/engineering/figure-4.webp)

Figure 4. The mutation sites

**Learn**
By comparing different experimental subjects, we discovered that certain mutations could enhance stability without disrupting the catalytic site, while other mutations would introduce unnecessary flexibility. These findings enabled us to optimize the design strategy, with the focus being on stabilizing the residues that maintain activity, and guiding this in the next round of optimization in the engineering cycle.

# Cycle 2: Expression and Identification of AChE in E.coli

**Design**
In this cycle, we aim to express and purify the wild type (WT) AChE and the five point mutants of AChE in the prokaryotic system. This will help us to test the expression level of our mutated protein and its enzyme activity. We decided to use pET-32a as the expression vector. Each vector includes T7 promoter, RBS, Trx Tag, His tag, our target gene--WT (wild type), D203A, Y355A, Y396A, N404A, E466A--and T7 terminator. Among them, the Trx tag can be used to promote protein expression and improve solubility, and the His tag can be used for affinity chromatography to isolate proteins.

![Figure 5. Vector design](https://static.igem.wiki/teams/5625/pics/engineering/figure-5.webp)

Figure 5. Vector design

**Build**
To initiate our project, we first obtained the wild type AChE gene from GenScript and cloned it into our designed pET32a expression vector. This step provided us with a reliable starting plasmid carrying the target gene under the control of a T7 promoter, making it suitable for protein expression in E. coli.

![Figure 6. Gene synthesis: Lane M: KB Ladder, Lane 1:AChE plasmid, Lane 2:AChE plasmid digested by SacI and EcoRI](https://static.igem.wiki/teams/5625/pics/engineering/fig66.avif)

Figure 6. Gene synthesis: Lane M: KB Ladder, Lane 1:AChE plasmid, Lane 2:AChE plasmid digested by SacI and EcoRI

After establishing the wild type construct, we designed point mutations with reference to the results of the previous alanine scanning.

![Figure 7. Point mutation design](https://static.igem.wiki/teams/5625/pics/engineering/figure-7.webp)

Figure 7. Point mutation design

![Figure 8. Point mutation design](https://static.igem.wiki/teams/5625/pics/engineering/figure-8.webp)

Figure 8. Point mutation design

![Figure 9. Point mutation design](https://static.igem.wiki/teams/5625/pics/engineering/figure-9.webp)

Figure 9. Point mutation design

![Figure 10. Point mutation design](https://static.igem.wiki/teams/5625/pics/engineering/figure-10.webp)

Figure 10. Point mutation design

After each mutation, PCR and gel electrophoresis was used for test positive clones examination and identify by sequencing.

![Figure 11. Agarose gel electrophoresis of colony PCR product from DH5ɑ transformed](https://static.igem.wiki/teams/5625/pics/engineering/fig111.webp)

Figure 11. Agarose gel electrophoresis of colony PCR product from DH5ɑ transformed

The plasmids were then transferred into DH5-α E.coli for amplification and sequencing to confirm the accuracy of the mutations. Through this process, we successfully built a small collection of AChE constructs, including both the wild type and multiple point mutants. These plasmids formed the essential foundation for our subsequent Test phase, where expression and solubility of the different variants were examined.

![Figure 12. Transformation in E.coli](https://static.igem.wiki/teams/5625/pics/engineering/figure-12.webp)

Figure 12. Transformation in E.coli

**Test 1 Wild-Type AChE Expression in E.coli**
We first conducted an examination of the expression of wild-type AChE in E.coli. After the E. coli cells were lysed and centrifuged (usually at 4℃ with a speed of 12000 rpm for 20 minutes to separate the precipitate from the supernatant), the supernatant and the precipitate were taken for SDS-PAGE analysis. The results showed that there were no obvious target protein bands in the supernatant, while the target bands were clearly visible in the precipitate, indicating that AChE was mainly expressed in the form of Inclusion body in the E. coli cells.

![Figure 13. 4-12% reducing SDS-PAGE analysis of wild-type AChE expressed in E.coli](https://static.igem.wiki/teams/5625/pics/engineering/fig131.webp)

Figure 13. 4-12% reducing SDS-PAGE analysis of wild-type AChE expressed in E.coli

![Figure 14. 4-12% reducing SDS-PAGE analysis of wild-type AChE expressed in E.coli-2](https://static.igem.wiki/teams/5625/pics/engineering/fig141.webp)

Figure 14. 4-12% reducing SDS-PAGE analysis of wild-type AChE expressed in E.coli-2

**Test 2 Mutant AChE Expression and Identification in E.coli**
Referring to the results of wild-type protein expression, we re-expressed the wild-type protein and mutant proteins, and purified the proteins through urea denaturation and Ni-affinity chromatography. The results are shown below.

![Figure 15. 4-12% reducing SDS-PAGE analysis of purified AChE by Ni-NTA expressed in E.coli](https://static.igem.wiki/teams/5625/pics/engineering/fig151.webp)

Figure 15. 4-12% reducing SDS-PAGE analysis of purified AChE by Ni-NTA expressed in E.coli

**Learn**
During our expression tests, we observed that the majority of the recombinant protein was present in the insoluble precipitations, while only a negligible amount appeared in the supernatant. This result indicates that the protein was mainly expressed as inclusion bodies, suggesting improper folding under the reducing conditions of E. coli cytoplasm. Since correct folding of our target protein requires the formation of disulfide bonds, the insolubility implied that these structural features could not be established in the prokaryotic system.
This limitation led us to shift towards a cell-free expression system, which provides a more suitable environment for producing soluble, active protein.

# Cycle 3: Cell-Free Protein Expression

**Design**
Expressing mutant genes within the prokaryotic system, we found that this method was not efficient because of the misfolding of the protein. After conducting interview with Prof. Li, we turn to the Cell-free protein expression(CFPE) methods to express our protein. Putting the DNA template, cell-free extract(mainly includes ribosomes, tRNA, enzymes, amino acids, and polymerases), and cofactors/energies together, the cell-free system is built.

![Figure 16. Cell-Free expression system](https://static.igem.wiki/teams/5625/pics/engineering/figure-16.webp)

Figure 16. Cell-Free expression system

**Build**
In order to increase the protein expression level and the uniformity of experiments, we amplified the open reading frame of the target gene and the upstream and downstream transcriptional elements by means of PCR, and purified the PCR products. The PCR fragments were then be used as the DNA templates in the cell-free expression system.

![Figure 17. PCR fragment for cell free protein expression](https://static.igem.wiki/teams/5625/pics/engineering/figure-17.webp)

Figure 17. PCR fragment for cell free protein expression

To detect whether our protein was expressed in the in cell-free expression system, we detected the His tag by Western blot (WB). The results showed that the protein was expressed.

![Figure 18. 4-12% reducing SDS-PAGE analysis of AChE expressing in Cell-free protein synthesis (CFPS) system](https://static.igem.wiki/teams/5625/pics/engineering/figure-18.webp)

Figure 18. 4-12% reducing SDS-PAGE analysis of AChE expressing in Cell-free protein synthesis (CFPS) system

![Figure 19. Western blotting analysis of AChE expressed in Cell-free protein synthesis (CFPS) system ( anti-His antibody)](https://static.igem.wiki/teams/5625/pics/engineering/fig191.webp)

Figure 19. Western blotting analysis of AChE expressed in Cell-free protein synthesis (CFPS) system ( anti-His antibody)

After confirming the successful expression of the protein in the Cell-free expression system by WB, we first purified the protein from the supernatant of the expression system using Size Exclusion Chromatography (SEC) and then verified it through SDS-PAGE analysis again.

![](https://static.igem.wiki/teams/5625/pics/engineering/fig20.avif)

Figure 20. SEC (Size Exclusion Chromatography) by AKTA system

Result shows that a single protein peak was collected by SEC, and the protein purity was further improved, with a purity greater than 95%. Confirming through SDS-PAGE again, we successfully produced high-purity active protein materials.

![](https://static.igem.wiki/teams/5625/pics/engineering/fig211.webp)

Figure 21. 4-12% reducing SDS-PAGE analysis of purified AChE expressed in Cell-free protein synthesis (CFPS) system by Ni-NTA and SEC

**Test**
We conducted AChE enzyme ability test based on the Ellman’s method to ensure that cell-free expression system can produce AChE with higher stability and higher enzyme activity.

![Figure 23. AChE activity assay](https://static.igem.wiki/teams/5625/pics/engineering/fig221.webp)

Figure 22. AChE activity assay
WT thermo: The commercial AChE enzyme purchased from Thermo Company
 CFPS:Cell-free protein synthesis

Then we apply the Differential Scanning Fluorimetry method (DSF) to characterize the thermal stability of proteins. We used a special fluorescent protein that likes to bind to the hydrophobic regions of proteins, which is wrapped inside and exposed only when the protein is unfolding. Thereby, the higher the ratio of 350nm/330nm, which represents the fluorescence intensity, the deeper the degree of protein denaturation.

| id | Capillary Name | Tm#1 |
| --- | --- | --- |
| #1 | WT | 61.8 |
| #2 | D203A | 64.8 |
| #3 | Y355A | 61.7 |
| #4 | Y396A | 61.6 |
| #5 | N404A | 61.9 |
| #6 | E466A | 64.3 |

![Figure 25. Differential Scanning Fluorimetry](https://static.igem.wiki/teams/5625/pics/engineering/fig244.avif)

Figure 23. Differential Scanning Fluorimetry

The graphs record the changes in fluorescence intensity with the change of temperature. We recorded the critical point at which the curve rises to its highest point and then flattens in the table, which represents the maximum temperature that a protein can withstand before denaturation. Comparing the maximum temperature of wild-type and mutant, we found out that the D203A mutant withstands a much higher temperature than the wild-type, while the rest are basically the same. In conclusion, the most stable mutant enzyme is D203A.

**Learn**
First of all, we learnt to build a cell- free system to express protein. This approach is more effective, convenient than the prokaryotic way. We also mastered the DSF technique, which provides us with an effective way to estimate a protein’s thermal stability. Those methods can be flexibly applied to various experiments we conduct in the future.

In addition, by testing the thermal stability, we found out that the most stable mutant Enzyme D203A was compared to the wild-type and other types of mutants. This is a crucial discovery because we will conduct our further experiment based on these enzymes.

Since the 5 mutants of AChE all expressed higher enzyme activity than the wild-type AChE, we submitted the sequence of these mutants as basic parts to the iGEM registry. Notably, few teams have ever conducted experiments with AChE. We hope our part collection can facilitate future teams to incorporate this enzyme into their design with easy access.

# Cycle 4: AuNP synthesis with AChE

**Design**
To promote the activity and stability of enzyme, we integrated our most stable mutant D203A with gold nanoparticles (AuNP). The process includes following steps:
 1) Synthesis of AuNP: 20nm gold nanoparticles were synthesized by sodium citrate reduction method (HAuCl₄ + sodium citrate, boiled at 100℃ for 15 minutes).
2) Surface modification: Sulfhydryl or amino groups were introduced onto the surface of AuNP. The carboxyl groups were activated by EDC/NHS and covalently bound to AChE (Hammami et al., 2021).

**Build**
20nm-sized were prepared through the sodium citrate reduction method. Then the carboxyl groups on the AuNP surface were activated with EDC/NHS. The following step of coupling with enzymes was carried out.

![Figure 26. Sodium citrate reduction](https://static.igem.wiki/teams/5625/pics/engineering/fig26.webp)

Figure 24. Sodium citrate reduction

**Test**
Using UV-Vis spectroscopy, we analysis the level of absorbance of each component (AuNPs, AChE and AuNPs-AChE). In the 200-220nm region, AChE shows a strong absorption at around 210nm which is originate from the deep UV absorption of proteins, while we found that AuNPs-AChE exhibits even strong absorption in this region. This indicated that the protein component absorption is enhanced in the coupling sample. In the 500-550nm region, the localized surface plasmon resonance (LSRP) peak of gold nanoparticles appears, meanwhile, the absorption peak is around 520nm which conform to the characteristics in the figure. To be more specific, peak of AuNPs-AChE during this band is slightly lower than AuNPs and the peak turn to be wider with a red shift. Those characteristics, as the common signal of protein adsorption, leading to changes in refractive index or minor aggregation. AChE alone shows no significant absorption in this region just as we expected.

![Figure 27. UV-Vis Absorption Measurement](https://static.igem.wiki/teams/5625/pics/engineering/fig27.webp)

Figure 25. UV-Vis Absorption Measurement

![Figure 28. Dynamic Light Scattering (DLS) Measurement](https://static.igem.wiki/teams/5625/pics/engineering/fig28.webp)

Figure 26. Dynamic Light Scattering (DLS) Measurement

The results of DLS measurement reveals that the hydrodynamic diameter of AuNPs-AChE particles increased significantly, indicating successful synthesis.

![Figure 29. Transmission Electron Microscopy (TEM) Characterization](https://static.igem.wiki/teams/5625/pics/engineering/fig200.avif)

Figure 27. Transmission Electron Microscopy (TEM) Characterization

**Learn**
Due to time limitations, we haven’t conduct test for the comparison between the stability of AChE-AuNP and AChE alone on test strips. This will be supplemented soon in the future. But as most literature suggested, we are confident to hypothesize that the AuNPs-AChE have got a better stationarity and activity; as a result, it may be more appropriate to place on detection strips. Furthermore, as we mastered the skill of AuNP synthesis, we documented the protocol in our Experiment page that can be adopted by future teams.

# References

Fuyal, M., & Giri, B. (2020). A combined system of paper device and portable spectrometer for the detection of pesticide residues. *Food Analytical Methods, 13*(11), 2071–2078. https://doi.org/10.1007/s12161-020-01770-y
Moreira, I. S., Fernandes, P. A., & Ramos, M. J. (2007). Computational alanine scanning mutagenesis—An improved methodological approach. Journal of Computational Chemistry, 28(3), 644–654. https://doi.org/10.1002/jcc.20566
Maiorov, V. N., & Crippen, G. M. (1994). Significance of root-mean-square deviation in comparing three-dimensional structures of globular proteins. Journal of Molecular Biology, 235(2), 625–634. https://doi.org/10.1006/jmbi.1994.1017
Hammami, I., Alabdallah, N. M., Al Jomaa, A., & Kamoun, M. (2021). Gold nanoparticles: Synthesis, properties, and applications. Journal of King Saud University - Science, 33(7), 101560. https://doi.org/10.1016/j.jksus.2021.101560
Gregorio, N. E., Levine, M. Z., & Oza, J. P. (2019). A User's Guide to Cell-Free Protein Synthesis. Methods and protocols, 2(1), 24. https://doi.org/10.3390/mps2010024
Shetab-Boushehri S. V. (2018). Ellman's method is still an appropriate method for measurement of cholinesterases activities. EXCLI journal, 17, 798–799. https://doi.org/10.17179/excli2018-1536
Sun, C., Li, Y., Yates, E. A., & Fernig, D. G. (2020). SimpleDSFviewer: A tool to analyze and view differential scanning fluorimetry data for characterizing protein thermal stability and interactions. Protein science : a publication of the Protein Society, 29(1), 19–27. https://doi.org/10.1002/pro.3703

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{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your project. It is designed to provide sufficient information for other teams to replicate our work.{% endblock %}
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Experiments

![](https://static.igem.wiki/teams/5625/pics/team/apple.webp)
![](https://static.igem.wiki/teams/5625/pics/team/avacado.webp)
![](https://static.igem.wiki/teams/5625/pics/team/banana.webp)
![](https://static.igem.wiki/teams/5625/pics/team/corn.webp)
![](https://static.igem.wiki/teams/5625/pics/team/dragonfruit.webp)
![](https://static.igem.wiki/teams/5625/pics/team/grape.webp)
![](https://static.igem.wiki/teams/5625/pics/team/lemon.webp)
![](https://static.igem.wiki/teams/5625/pics/team/lichee.webp)
![](https://static.igem.wiki/teams/5625/pics/team/mango.webp)
![](https://static.igem.wiki/teams/5625/pics/team/mangostene.webp)
![](https://static.igem.wiki/teams/5625/pics/team/melon.webp)
![](https://static.igem.wiki/teams/5625/pics/team/orange.webp)
![](https://static.igem.wiki/teams/5625/pics/team/peach.webp)
![](https://static.igem.wiki/teams/5625/pics/team/pear.webp)
![](https://static.igem.wiki/teams/5625/pics/team/pineapple.webp)
![](https://static.igem.wiki/teams/5625/pics/team/strawberry.webp)
![](https://static.igem.wiki/teams/5625/pics/team/tomato.webp)
![](https://static.igem.wiki/teams/5625/pics/team/watermelon.webp)
![](https://static.igem.wiki/teams/5625/pics/team/brocoli.webp)
![](https://static.igem.wiki/teams/5625/pics/team/pumpkin.webp)
![](https://static.igem.wiki/teams/5625/pics/team/coconut.webp)
![](https://static.igem.wiki/teams/5625/pics/team/spinach.webp)

**The experiments are arranged in alphabetical order.**

# AChE Activity Assay Kit (Colorimetric)

1. Set up 5 assay tubes and 1 control tube:

   | Tube Number | Sample Name | Volume to Add |
   | --- | --- | --- |
   | 1 | Wild-type AChE (positive control) | 100 μL |
   | 2 | Mutant 1 | 100 μL |
   | 3 | Mutant 2 | 100 μL |
   | 4 | Mutant 3 | 100 μL |
   | 5 | Mutant 4 | 100 μL |
   | 6 | Mutant 5 | 100 μL |
   | 6 | Water (negative control) | 100 μL |
2. **Pre-incubation:**
   Add 100 μL of Reagent II to tubes 1-6 (wild-type and mutants). Tube 7 (water) receives no Reagent II at this step. Incubate all tubes at 37°C for 5 minutes.
3. **Reaction initiation:**
   Add 100 μL of Reagent IV to tubes 1-6.
   For the negative control (tube 7), add 100 μL of Reagent II and 100 μL of Reagent IV.
   Mix all tubes thoroughly.
4. **Centrifugation:**
   Centrifuge all tubes at 12,000 rpm for 5 minutes.
5. **Supernatant transfer:**
   Transfer 50 μL of the supernatant from each tube to a new corresponding tube.
6. **Color development:**
   Add 100 μL of Reagent III to each new tube.
7. **Add Reagent I as follows:**
   Tubes 2-7: 780 μL
   Wild-type positive control tube (tube 1): 850 μL
   Mix thoroughly and allow to stand at room temperature for 2 minutes. Observe color development in assay tubes. The water control should show minimal color change.
8. **Measurement:**
   Using the water tube (negative control) as the blank, measure absorbance of each tube at 412 nm using a spectrophotometer. Record the results for comparison of enzymatic activity.

# Bacillus coli amplification

1. Prepare three conical flasks, each containing 50 mL of LB liquid medium.
2. Following the table below, add different volumes of ampicillin-resistant E. coli strain and ampicillin solution to each flask.

   | Group | Medium (mL) | Ampicillin-resistant E. coli culture (μL) | Ampicillin (μL) |
   | --- | --- | --- | --- |
   | 1 | 50 | 5 | 5 |
   | 2 | 50 | 50 | 5 |
   | 3 | 50 | 50 | 50 |
3. Tighten the bottle caps or seal the openings with sterile sealing film, then shake each bottle vigorously to thoroughly mix the bacterial culture and antibiotics.
4. Place the three bottles in a 37°C constant-temperature shaking incubator, set to 260 rpm.
5. Allow the bacterial cultures to incubate continuously in the shaking incubator for approximately 19 hours (overnight).
6. Observe and record the growth of the bacterial cultures in all three bottles.

# Cell Free Protein Expression (CFPE)

CFPE is a biotechnology method that produces proteins in a solution containing the essential cellular machinery, such as ribosomes and enzymes, without using living cells. This process allows for the rapid synthesis of proteins from a DNA or mRNA template and offers significant advantages, including speed, flexibility, and the ability to express problematic proteins like those that are toxic or membrane-bound. CFPE is widely used in research for protein engineering, screening, and structural analysis, though it is generally not practical for large-scale commercial production.

1. **Reagent Preparation**
   Thaw all components of the cell-free protein expression kit stored at -80°C completely on ice. After thawing, briefly centrifuge and collect the supernatant to the bottom of the tube.
2. **Reaction System Preparation (Perform on ice)**
   Prepare a 50 μL reaction system in a PCR tube according to the following proportions:

   | Component | Sample Reaction (×48) | Negative Control (×1) | Positive Control (×1) |
   | --- | --- | --- | --- |
   | Reaction Mix | 17.2 μL | 17.2 μL | 17.2 μL |
   | Template DNA\* | 10 μL | - | 10 μL |
   | E. coli Cell Extract | 7.4 μL | 7.4 μL | 7.4 μL |
   | Nuclease-free Water | to 50 μL | to 50 μL | to 50 μL |
3. **Reaction Incubation**
   Place the prepared reaction tubes in a PCR machine or constant-temperature metal bath and incubate at 30°C for 2–3 hours.
4. **Product Analysis**
   After the reaction, take 2-5 μL of the reaction mixture for SDS-PAGE electrophoresis analysis or use it for other downstream experiments.

# Cell lysis

1. Add 30 milliliters of E. coli-specific cell lysis buffer per gram of bacterial cells (adjustable ratio range: 1:10 to 1:50).
2. Thoroughly disperse and mix the bacterial cells in the lysis buffer.
3. Freeze the mixture in a freezer at -20°C or below until completely solidified.
4. When ready for lysis, remove the sample and allow it to thaw naturally.
5. Perform high-speed cryo-centrifugation on the thawed sample.
6. After centrifugation, collect the supernatant (lysis supernatant) and the pellet (cell pellet) separately.

# DNA Gel Recovery and Purification

1. Under UV light, quickly cut out the gel block containing the target DNA band.
2. Place the gel block into a centrifuge tube, weigh it, and record the weight.
3. Add 300 μL of binding buffer per 100 mg of gel.
4. Incubate the centrifuge tube in a 55°C water bath until the gel is completely dissolved.
5. Transfer the entire dissolved solution to the purification column and let it stand at room temperature for 5 minutes.
6. Centrifuge at 12,000 rpm for 1 minute, then discard the supernatant.
7. Add 700 μL of wash buffer to the purification column.
8. Centrifuge at 12,000 rpm for 1 minute and discard the supernatant.
9. Repeat steps 7 and 8 for one additional wash.
10. Centrifuge the empty column at 12,000 rpm for 2 minutes to remove residual ethanol.
11. Transfer the purification column to a new centrifuge tube and add 30-50 μL of elution buffer.
12. Incubate at room temperature for 5 minutes, then centrifuge at 12,000 rpm for 1 minute to collect the DNA solution.
13. Perform agarose gel electrophoresis analysis using 2 μL of the product.
14. Store the remaining DNA solution at -20°C.

# DNA Qualification

This experiment will use a Nano nucleic acid concentration analyzer.

1. **Initialization/Blank Calibration:** Wipe the detection platform clean with lint-free paper.
2. **Use buffer solution as blank control:** Pipette 1 μL onto the detection platform and click “Blank.”
3. After blank calibration, clean the detection platform.
4. Pipette 1 microliter of nucleic acid into the detection head for measurement.
5. After approximately several seconds, the instrument displays:
   - Nucleic acid concentration (ng/μl)
   - A260/A280: Protein contamination in nucleic acid
   - A260/A230: Ion contamination in nucleic acid

![DNA Qualification](https://static.igem.wiki/teams/5625/pics/experiment/fig2.webp)

# Dynamic Light Scattering (DLS) Measurement

The hydrodynamic diameter of AuNPs and AuNPs-AChE conjugates was measured by dynamic light scattering (DLS) using a DynaPro NanoStar instrument (Wyatt Technology, USA). Prior to measurement, samples were diluted with deionized water to an appropriate concentration to avoid multiple scattering and gently mixed. All measurements were performed at 25 °C, and the reported size represents the intensity-weighted average hydrodynamic diameter. Each sample was measured in triplicate to ensure reproducibility.

![DLS Measurement](https://static.igem.wiki/teams/5625/pics/experiment/fig3.webp)

# Enzymatic Digestion - Plasmid Vector Cloning

1. **Prepare Digestion Reaction Mixture**

   | Component | Volume |
   | --- | --- |
   | Plasmid DNA | 90 μL |
   | 10× Restriction Buffer | 10 μL |
   | Restriction Enzyme (XhoI/XhoII) | 1 μL |
   | Total Volume | 101 μL |

   Mix gently by pipetting and briefly centrifuge to collect liquid.
2. **Enzyme Digestion Reaction**
   Incubate the reaction tubes in a 37°C water bath for 1 hour.
3. **Agarose Gel Electrophoresis**
   Prepare agarose gel at an appropriate concentration (e.g., 1%).
   Add an appropriate volume of DNA loading buffer to the digested samples and mix thoroughly.
   Load samples and DNA molecular weight markers into separate lanes.
   Run electrophoresis at an appropriate voltage until bands are fully separated.
4. **Gel Imaging and Gel Cutting**
   Observe electrophoresis results under a gel imaging system.
   Identify target bands: Linearized plasmid bands migrate faster (lower position) than supercoiled plasmids.
   Precisely cut out the gel fragment containing the target band using a clean blade, removing excess gel as much as possible.
5. **Gel Recovery and Purification**
   Weigh the excised gel and add Binding Buffer proportionally. Incubate in a 55-65°C water bath, mixing intermittently until the gel is completely dissolved.
   Transfer the lysate to the collection column provided in the kit and let it stand at room temperature for 2 minutes.
   Centrifuge at 12,000 rpm for 1 minute and discard the supernatant.
   Add 700 μL Wash Buffer to the column, centrifuge at 12,000 rpm for 1 minute, and discard the supernatant.
   Repeat the washing step once. Centrifuge the empty column for 2 minutes to completely remove ethanol.
   Place the column in a clean centrifuge tube, add 30-50 μL Elution Buffer, and let stand for 2 minutes.
   Centrifuge at 12,000 rpm for 1 minute. Collect the eluate, which is purified linearized plasmid DNA.
6. **Quality Control**
   Determine the concentration and purity of the purified product using a nucleic acid concentration meter (record the A260/A280 ratio).
   Perform agarose gel electrophoresis verification with a small amount of product. Compare with uncut plasmid control to confirm band size and purity.

# IPTG-induced Expression

1. Prepare a sterile 1M IPTG stock solution and aliquot it for storage at -20°C.
2. When the OD600 value of the E. coli culture reaches 0.5–0.6, prepare for induction.
3. Add the inducer at a ratio of 50 μL of 1M IPTG per 100 mL of culture.
4. Gently shake the culture flask to ensure uniform dispersion of IPTG throughout the bacterial suspension.
5. Return the flask to the shaking incubator and incubate overnight at 16–18°C (16–24 hours).
6. Following induction, harvest the bacterial cells for subsequent protein analysis or purification experiments.

# Monoclonal Colony Pick

1. Prepare several sterile shaking tubes and add 25 mL of LB liquid medium to each tube.
2. Based on the type of resistance, add 25 μL of the corresponding antibiotic to each tube (e.g., ampicillin to a final concentration of 100 μg/mL).
3. Using a sterile pipette tip, pick a morphologically regular, well-defined, and uniformly sized single colony from the transformation plate.
4. Directly transfer the tip bearing the colony into the shaking tube prepared in Step 1 containing medium and antibiotic.
5. Tightly cap the shaking tube and incubate it in a 37°C constant-temperature shaking incubator at 220 rpm for 12 to 16 hours (overnight).

# Ni-NTA Affinity Chromatography

**Buffer Recipes:**

* **Lysis Equilibration Buffer (LE Buffer):** 50 mM Na₂HPO₄, 0.3 M NaCl, pH=8.0
* **Wash Buffer:** 50 mM Na₂HPO₄, 0.3 M NaCl, 10 mM Imidazole, pH=8.0
* **Elution Buffer:** 50 mM Na₂HPO₄, 0.3 M NaCl, 250 mM Imidazole, pH=8.0

![Ni-NTA Affinity Chromatography](https://static.igem.wiki/teams/5625/pics/experiment/fig4.webp)

# PCR (Polymerase Chain Reaction)

1. **Materials**
   * Template DNA (e.g., plasmid)
   * Specific Forward & Reverse Primers
   * 2× Taq Master Mix (containing Taq DNA Polymerase, dNTPs, Mg²⁺, Reaction Buffer)
   * Sterile Nuclease-free Water
2. **Reaction Setup**

   | Component | Volume | Final Concentration/Amount |
   | --- | --- | --- |
   | 2× Taq Master Mix | 50 μL | 1× |
   | Forward Primer (10 μM) | 2 μL | 0.2 μM |
   | Reverse Primer (10 μM) | 2 μL | 0.2 μM |
   | Template DNA | 1.5 μL | Variable (e.g., 10-100 ng) |
   | Nuclease-free Water | 44.5 μL | - |
   | Total Volume | 100 μL | - |
3. **Steps**
   1. Prepare the PCR reaction mixture in an ice bath.
   2. Add the components in the following order to the sterile 0.2 mL PCR tube.
   3. Carefully move the liquid up and down to mix it evenly, avoiding the formation of bubbles.
   4. The brief centrifugation causes the liquid surface to concentrate at the bottom of the tube.
   5. Place the PCR tube in the thermal cycling instrument.
   6. Set the program for the thermal cycling instrument as follows:

   | Step | Temperature | Time | Cycles |
   | --- | --- | --- | --- |
   | Initial Denaturation | 95°C | 2 minutes | 1 |
   | Denaturation | 95°C | 30 seconds | 30 |
   | Annealing | 65°C | 40 seconds | 30 |
   | Extension | 72°C | 30 seconds | 30 |
   | Final Extension | 72°C | 1 minute | 1 |
   | Hold | 4°C | ∞ | 1 |

   7. Start the operation program of the thermal cycling instrument.
   8. After the program is completed, remove the test tube from the thermal cycling instrument.The PCR products are now ready for analysis or can be stored at -20°C.

# Plasmid DNA Isolation

1. Transfer 1.6 mL of overnight bacterial culture to a centrifuge tube. Centrifuge at 12,000 rpm for 1 minute, then aspirate the supernatant. If the bacterial volume is large, perform multiple centrifugations to collect the bacterial pellet into four centrifuge tubes.
2. Add 500 μl of Solution P1 to the centrifuge tube containing the bacterial pellet. Thoroughly resuspend the bacterial cells using a pipette or vortex mixer.
3. Add 500 μl of Solution P2 to the centrifuge tube. Gently invert the tube 6-8 times to ensure complete lysis of the bacterial cells.
4. Add 500 μl of Solution P4 to the tube. Immediately invert gently 6–8 times to mix thoroughly. A white flocculent precipitate will form. Incubate at room temperature for approximately 10 minutes, then centrifuge at 12,000 rpm for 10 minutes. A precipitate will form at the bottom of the tube.
5. Add the supernatant collected in the previous step in portions to the filtration column CS (with the column placed in the collection tube). Centrifuge at 12,000 rpm for 2 min. Collect the filtrate in a clean 2-mL centrifuge tube.
6. Add 600 μl of wash buffer PW to the CP4 adsorption column. Centrifuge at 12,000 rpm for 1 minute. Discard the waste liquid from the collection tube and place the CP4 adsorption column back into the collection tube.
7. Add 600 μl of wash buffer PW to the CP4 adsorption column. Centrifuge at 12,000 rpm for 1 minute. Discard the waste liquid from the collection tube.
8. Return the CP4 adsorption column to the collection tube and centrifuge at 12,000 rpm for 2 min.
9. Transfer the CP4 adsorption column to a clean centrifuge tube. Gently dispense 100–300 μl of elution buffer TB onto the center of the adsorption membrane. Incubate at room temperature for 2 minutes, then centrifuge at 12,000 rpm for 1 minute to collect the plasmid solution into the centrifuge tube.

![Plasmid DNA Isolation](https://static.igem.wiki/teams/5625/pics/experiment/fig5.webp)

# Point Mutation Plasmid Extraction

**Note:** Our procedures do not include RNase A treatment.

1. **Adsorption Column Pretreatment**
   Add 500 μl of equilibration buffer BL to adsorption column CP4 (placed in the collection tube). Centrifuge at 12,000 rpm for 1 minute. Discard the supernatant and return the column to the collection tube. (Use columns processed on the same day.)
2. **Cell Harvesting**
   Take 5–15 ml of overnight bacterial culture, centrifuge at 12,000 rpm for 1 minute, and remove supernatant as thoroughly as possible.
   *Note: For larger volumes, centrifuge in batches to collect cells. The cell volume should be sufficient for complete lysis.*
3. **Cell Resuspension**
   Add 500 μl of Solution P1 to the cell pellet. Thoroughly resuspend the pellet using a pipette or vortex mixer.
   *Note: Complete resuspension is essential. Unmixed cell clumps will impair lysis efficiency.*
4. **Alkaline Lysis**
   Add 500 μl of Solution P2. Gently invert the tube 6-8 times to thoroughly lyse the cells.
   *Note: Mix gently; avoid vigorous shaking. The solution should become clear and viscous. Do not exceed 5 minutes for this step.*
5. **Neutralization Reaction**
   Add 500 μl of Solution P4. Immediately invert the tube gently 6-8 times to mix. Incubate at room temperature for 10 minutes, then centrifuge at 12,000 rpm for 10 minutes. Collect the supernatant.
   *Note: Mix immediately after adding P4 to prevent localized precipitation. If precipitation remains in the supernatant, centrifuge again.*
6. **Filtration Purification**
   Add the supernatant in portions to the filtration column CS. Centrifuge at 12,000 rpm for 2 minutes and collect the filtrate into a clean centrifuge tube.
7. **Isopropanol Precipitation**
   Add 0.3x volume of isopropanol to the filtrate. Mix thoroughly and transfer in aliquots to the CP4 adsorption column.
   *Note: Excess isopropanol may cause RNA contamination. Do not exceed 700 μl per column loading.*
8. **Centrifugal Adsorption**
   Centrifuge at 12,000 rpm for 1 minute. Discard the supernatant. Place the adsorption column into the recovery tube.
9. **Protein Removal**
   Add 500 μl protein removal solution PD. Centrifuge at 12,000 rpm for 1 minute. Discard the supernatant.
10. **First Wash**
    Add 600 μl wash buffer PW (containing ethanol). Centrifuge at 12,000 rpm for 1 minute. Discard the supernatant.
    *Note: Allowing PW to stand at room temperature for 2-5 minutes enhances impurity removal.*
11. **Second Wash**
    Add 600 μl wash buffer PW. Centrifuge at 12,000 rpm for 1 minute. Discard supernatant.
12. **Residual Ethanol Removal**
    Centrifuge empty column at 12,000 rpm for 2 minutes. Open cap and allow to air dry completely at room temperature for several minutes.
    *Note: Residual ethanol may interfere with downstream enzymatic reactions.*
13. **Plasmid Elution**
    Place the CP4 adsorption column into a new centrifuge tube. Add 100-300 μl of TB elution buffer by pipetting directly onto the center of the adsorption membrane. Incubate at room temperature for 2 minutes. Centrifuge at 12,000 rpm for 1 minute to collect the plasmid solution.

# Prepare LB Liquid Medium

| Component | Concentration | Amount per 100 mL |
| --- | --- | --- |
| NaCl | 1% (w/v) | 1.0 g |
| Tryptone | 1% (w/v) | 1.0 g |
| Yeast extract | 0.5% (w/v) | 0.5 g |
| Deionized water | - | To 100 mL |
| pH | 7.0 | Adjust with NaOH/HCl |

1. Weigh the quantities of NaCl, Tryptone, and Yeast Extract listed in the table above and place them in a 250 mL conical flask or beaker.
2. Add approximately 90 mL of deionized water and stir thoroughly until dissolved.
3. Monitoring with a pH meter, titrate with small amounts of 1 M NaOH or 1 M HCl solution to precisely adjust the pH to 7.0.
4. Adjust the volume of the solution to 100 mL and mix thoroughly.
5. Seal the container with aluminum foil or a lid and subject it to high-pressure steam sterilization (121°C, 20 minutes). Allow to cool to room temperature after sterilization before use.

# Prepare Tris-NaCl Solution

1. **Calculate quantities:** Common formulas: n = c × V ; n = m / M
2. **Accurately weigh components using a balance:** Fold weighing paper four times, tare the balance, transfer to beaker after weighing, and add calculated amount of Tris.
3. **Mix thoroughly** with a magnetic stirrer until solute dissolves.
4. **Adjust pH:** Insert pH electrode into solution (keep electrode submerged but not touching bottom). Read pH after value stabilizes. Each time a small amount of acid or alkali is added, the addition should be stopped until the value stabilizes before proceeding with further additions.
   • If pH is low, slowly add NaOH dropwise.
   • If pH is high, slowly add HCl dropwise.
   • Typically adjust to 7.5 as needed for specific experiment requirements.
5. **Dilution to Volume:** Transfer solution to a graduated cylinder and add water to reach the required volume.
6. **Filtration**
7. **Record** composition, concentration, and date.

# Preparation and Use of Coomassie Brilliant Blue Staining Solution and Decolorizing Solution

| Solution | Component | Amount |
| --- | --- | --- |
| Staining Solution (200 mL, Fast Staining) | Coomassie Brilliant Blue R-250 | 0.5 g |
| Methanol | 90 mL |
| Glacial Acetic Acid | 20 mL |
| ddH₂O | 90 mL |
| Destaining Solution (1 L, Fast Destaining) | Methanol | 450 mL |
| Glacial Acetic Acid | 100 mL |
| ddH₂O | 450 mL |

1. Carefully remove the gel from the glass plate after electrophoresis is complete.
2. Place the gel into a clean container.
3. Pour sufficient Coomassie Brilliant Blue staining solution into the container, ensuring the liquid completely covers the gel.
4. Place the container in the microwave and heat on high for 2 minutes.
5. Remove the container and place it on a shaker at room temperature for slow agitation during staining.
6. Drain the staining solution from the container.
7. Add sufficient volume of Coomassie Brilliant Blue destaining solution to the container, ensuring complete coverage of the gel.
8. Place the container on a shaker and destain at room temperature for 30 minutes.
9. Observe the results: protein bands should appear blue, with the gel background appearing nearly colorless.

# SDS-PAGE

SDS-PAGE, or Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis, is a widely used biochemical technique that separates proteins primarily by their molecular weight. Proteins are treated with SDS to unfold them into linear chains and coat them with a uniform negative charge, then run through a polyacrylamide gel matrix. An electric field causes these negatively charged proteins to migrate, with smaller proteins moving faster and larger proteins moving slower due to the gel's sieving effect.

1. **Materials and Reagents:**
   * 1.5 M Tris-HCl (pH 8.8) (for separation gel)
   * 1.0 M Tris-HCl (pH 6.8) (for concentration gel)
   * 30% Acr/Bis solution (29:1)
   * TEMED (crosslinking agent)
   * 10% APS (ammonium persulfate, prepare fresh)
   * 10% SDS (10% SDS solution)
   * Double-distilled water (ddH₂O)
   * Gel casting molds, combs, pipettes, beakers, etc.
2. **Preparation of Separating Gel (10%)** (Suitable for one mini gel, approximately 5 ml)

   | Component | Volume (ml) | Final Concentration |
   | --- | --- | --- |
   | ddH₂O | 1.9 | - |
   | 30% Acr/Bis (29:1) | 1.7 | 10% Acr |
   | 1.5 M Tris (pH 8.8) | 1.3 | 375 mM Tris |
   | 10% SDS | 0.05 | 0.1% SDS |
   | Subtotal | 4.95 |  |
   | 10% APS | 0.05 | 0.1% APS |
   | TEMED | 0.002 | ~0.04% |

   **Procedure:**
   1. Add components in the order listed above (add TEMED and APS last), gently mixing to avoid creating bubbles.
   2. Swiftly pour the mixture into the gel casting mold, filling approximately two-thirds of the mold's height.
   3. Gently add a layer of isopropyl alcohol or anhydrous ethanol to the surface of the separating gel to level it. Allow it to stand for approximately 15–20 minutes until it solidifies.
   4. After solidification, pour off the isopropyl alcohol and use filter paper to absorb any residual liquid.
3. **Preparation of Concentrated Gel (5%)** (Suitable for one mini gel, approximately 2 ml)

   | Component | Volume (ml) | Final Concentration |
   | --- | --- | --- |
   | ddH₂O | 1.4 | - |
   | 30% Acr/Bis (29:1) | 0.33 | 5% Acr |
   | 1.0 M Tris (pH 6.8) | 0.25 | 125 mM Tris |
   | 10% SDS | 0.02 | 0.1% SDS |
   | Subtotal | 2.00 |  |
   | 10% APS | 0.02 | 0.1% APS |
   | TEMED | 0.002 | ~0.1% |

   **Procedure:**
   1. Add components in sequence (add TEMED and APS last), gently mixing each addition.
   2. Pour the concentrated gel onto the solidified separating gel until level.
   3. Immediately insert the comb to prevent bubble formation.
   4. Allow to stand for approximately 10-15 minutes until the concentrated gel is fully solidified.

# SEC (Size Exclusion Chromatography) by AKTA System

Size Exclusion Chromatography (SEC), also known as gel filtration chromatography, is a technique that separates proteins or other macromolecules based on their size. The AKTA system provides automated, precise control for SEC, allowing for reproducible purification and analysis of biomolecules. Larger molecules elute first because they are excluded from the pores of the stationary phase, while smaller molecules enter the pores and elute later.

![SEC by AKTA system 1](https://static.igem.wiki/teams/5625/pics/experiment/fig6.webp)
![SEC by AKTA system 2](https://static.igem.wiki/teams/5625/pics/experiment/fig7.webp)

# Transmission Electron Microscopy (TEM) Characterization

300-mesh carbon-coated copper grids (Beijing Zhongjingkeyi Technology Co., Ltd.) were first subjected to static charge removal. Aliquots of AuNPs and AuNPs–AChE solutions were then deposited onto the grids. AuNPs–AChE samples were negatively stained with 1% (w/v) uranyl acetate for 1 min. After air-drying, the samples were examined using a Talos L120C G2 transmission electron microscope (Thermo Fisher Scientific) operated at 120 kV. Representative images were acquired to observe particle morphology.

![TEM Characterization](https://static.igem.wiki/teams/5625/pics/experiment/fig8.webp)

Talos L120C G2 transmission electron microscope (Thermo Fisher Scientific)

# Transformation of E. coli with Plasmid DNA

**Purpose:** To introduce plasmid DNA containing a gene of interest into chemically competent *E. coli* cells (strain DH5α) for amplification and selection.

1. Immerse the coating rod in a 25% ethanol solution for 1 hour. Sterilize by flaming over an alcohol lamp before use. Allow to cool and set aside.
2. Take one tube of DH5α competent cells and slowly thaw at room temperature.
3. Add 1 μL of target gene plasmid DNA to the competent cells. Gently tap the tube wall to mix.
4. Let the mixture stand for 30 minutes.
5. Place the mixture in a 42°C water bath for 90 seconds of heat shock.
6. Immediately remove the tube and let it stand at room temperature for 2 minutes.
7. Add 1 mL of LB liquid medium (without antibiotics) to the tube.
8. Gently mix by pipetting up and down, then incubate the tube at 37°C for 1 hour.
9. Centrifuge the tube (4,000 rpm, 1 minute). Discard most of the supernatant, retaining approximately 100 μL of liquid to resuspend the bacterial cells.
10. Spread the entire bacterial suspension onto an antibiotic-containing LB agar plate using a spreader.
11. After the bacterial suspension is absorbed, invert the plate and incubate at 37°C for 12-16 hours.

# UV-Vis Absorption Measurement

The UV-vis absorption spectrum of samples was recorded using a UV1901PC spectrophotometer (Shanghai AOXi Scientific Instrument Co., Ltd., China). Measurements were performed at room temperature over a wavelength range of 190–1100 nm using quartz cuvettes, with deionized water as the reference.

# Western blotting analysis of AChE expressed in E.coli (anti-His antibody)

1. Sample Preparation and Gel Electrophoresis: Proteins are solubilized and loaded onto a polyacrylamide gel to separate them by size.
2. Protein Transfer: The separated proteins are transferred from the gel to PVDF membrane.
3. Blocking: The membrane is blocked with 5% BSA to prevent non-specific binding of antibodies.
4. Primary Antibody Incubation: The membrane is incubated with a primary anti-His antibody(1:1000), which binds to the His-tag on AChE protein.
5. Secondary Antibody Incubation: Goat Anti-Mouse IgG-HRP (1:5000), conjugated to horseradish peroxidase, is added to bind to the primary antibody.
6. Detection: ECL is added to the membrane(1-2mins), which reacts with the enzyme to produce a detectable signal.

# Z-PCR

1. **Reaction System Setup**
   Prepare two sets of PCR reactions (Set A using Z1 high-fidelity DNA polymerase, Set B using Z2 ultra-high-fidelity DNA polymerase). Each set includes two parallel reactions (Reaction 1 and Reaction 2):

   | Component | Reaction 1 (500 μL) | Reaction 2 (500 μL) |
   | --- | --- | --- |
   | PCR1 Reaction Mix (without polymerase) | 475 μL | - |
   | PCR2 Reaction Mix (without polymerase) | - | 475 μL |
   | Z1/Z2 DNA Polymerase | 20 μL | 20 μL |
   | DNA Template (>30 ng/μL) | 5 μL | 5 μL |
   | Nuclease-free Water | x μL (to 500 μL) | x μL (to 500 μL) |
2. **Thermal Cycling Conditions**

   | Step | Temperature | Time | Cycles |
   | --- | --- | --- | --- |
   | Initial Denaturation | 98°C | 3 minutes | 1 |
   | Denaturation | 98°C | 20 seconds | 30-33 |
   | Annealing | 52°C | 15 seconds | 30-33 |
   | Extension | 72°C | 25 sec/kb (Z2) or 60 sec/kb (Z1) | 30-33 |
   | Final Extension | 72°C | 3 minutes | 1 |
   | Hold | 4-10°C | ∞ | 1 |

![Back to Top](https://static.igem.wiki/teams/5625/pics/words/top.webp)

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