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:
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.
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.
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.
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.
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
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
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
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.
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.
After establishing the wild type construct, we designed point mutations with reference to the results of the previous alanine scanning.
After each mutation, PCR and gel electrophoresis was used for test positive clones examination and identify by sequencing.
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.
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.
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.
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.
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
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
Figure 7. Point mutation design
Figure 8. Point mutation design
Figure 9. Point mutation design
Figure 10. Point mutation design
Figure 11. Agarose gel electrophoresis of colony PCR product from DH5ɑ transformed
Figure 12. Transformation 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
Figure 14. 4-12% reducing SDS-PAGE analysis of wild-type AChE expressed in E.coli-2
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
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.
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.
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.
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.
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.
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.
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.
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.
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
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
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)
Figure 20. SEC (Size Exclusion Chromatography) by AKTA system
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 22. AChE activity assay
WT thermo: The commercial AChE enzyme purchased from Thermo Company
CFPS:Cell-free protein synthesis
WT thermo: The commercial AChE enzyme purchased from Thermo Company
CFPS:Cell-free protein synthesis
Figure 23. Differential Scanning Fluorimetry
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.
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.
The results of DLS measurement reveals that the hydrodynamic diameter of AuNPs-AChE particles increased significantly, indicating successful synthesis.
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.
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 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 25. UV-Vis Absorption Measurement
Figure 26. Dynamic Light Scattering (DLS) Measurement
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
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
