Engineering

The Synthesis of Resveratrol

Cycle I

Design

In the design of resveratrol synthesis in our chassis, we discovered no resveratrol synthetic pathway in Nicotiana benthamiana after analyzing preliminary metabolic data and reviewing the relevant literature. However, the concentration of p-Coumaric acid in leaves of Nicotiana benthamiana, which is the substrate of many kinds of secondary metabolites, including resveratrol, is considerable. Consequently, we were willing to express the key enzyme needed to produce resveratrol -- Stilbene synthase (STS) in our chassis. Finally, we focused on the stilbene synthase gene in grape VvSTS, which has been tested and verified valid in wild organisms[1][2]. We wanted to see whether it could enable synthesis in Nicotiana benthamiana.

Build

We retrieved the VvSTS sequence on NCBI and contacted a professional company to synthesize and optimize the fragment. We constructed the pDONR207-VvSTS and pEAQ-DEST1-VvSTS using the Gateway method and then transferred the latter into Agrobacterium GV3101. After that, we conducted transient transfection to express the VvSTS.

Test

We analyzed the synthesis of resveratrol in the leaves of transiently transfected tobacco through LC-MS/MS. As shown in Fig.1-1, there was resveratrol produced in tobacco when VvSTS was successfully imported.

Learn

Compared to blank control and blank vector, we detected the resveratrol in the wild-type tobacco after importing VvSTS, inferring that VvSTS can express efficiently in our chassis. Notably, the yield of resveratrol was poor, which may be attributed to the factor that the central part of p-Coumaric acid is used to generate Chlorogenic acid and other secondary metabolites. Therefore, we tried to make the heterologous synthesis of resveratrol in our VessaTobacco. Meanwhile, our instructors said there may be negative feedback in the upstream and suggested improving the production by releasing or alleviating the feedback loop.

Cycle II

Design

To verify the efficiency and strength of our chassis in the synthesis of secondary metabolites, we changed the wild-type tobacco into HQT-knockout tobacco and conducted transient transfection. We also found that the mutant of DAHP synthase YIARO3 in Saccharomyces can alleviate the repression of DAHP synthase from L-Phe in the upstream[3], as shown in Fig.1-2.

Build

We constructed the pDONR207-YIARO3 and pEAQ-DEST1-YIARO3 by Gateway and transferred the latter into Agrobacterium GV3101. By setting the group of single gene and double genes transfection, we hoped to observe the improvement in yield of resveratrol in VvSTS and YIARO3 simultaneously importing.

Test

We used LC-MS/MS to detect the resveratrol after injecting mutant tobacco. As we can see in Fig.1-3, ΔHQT tobacco produced more resveratrol than that of wild type. However, our anticipation that higher production of double gene groups than single gene groups did not appear.

Learn

As shown above, HQT-knockout tobacco displayed a higher yield of resveratrol than wild type, indicating that knockout HQT dispersed the flow of p-Coumaric acid to resveratrol to some extent. Nevertheless, the effect was insignificant, and YIARO3 did not increase resveratrol production. More than that, the production of resveratrol decreased by nearly 50% compared with the data generated by the last batch of tobacco. We predicted these results may be due to the following factors:

1. Because different gene combinations injected different layers of leaves. Leaves underneath have grown for a longer time and have undergone the key phase of diversion of metabolism, which contributed to no improvement after injection of YIARO3 and even decreased.
2. The sample volumes were too small to keep the homogeneity of the samples. The status of plants significantly affected the results.
3. We delayed the sampling time for 24 hours, during which the resveratrol may be partially degraded.

In conclusion, we decided to enlarge the sample volume and fix the leaf layer to inject. The sampling time was controlled in 72 - 96 h after injection.

Cycle III

Design

According to the experience in DBTL cycle Ⅱ, we placed importance on the following:

1. narrow the difference between samples with different genes injection
2. choose suitable tobacco to inject and adjust the sampling time
3. enlarge sample volume

Build

We chose suitable (four-week-age) tobaccos and injected 4 - 5 leaves for each plant. To narrow the difference between plants, we changed the method: one kind of gene group for the leaves from the same plant, and one gene group injected 2 - 3 plants. When sampling, we mixed samples with the same gene group and milled. Moreover, we extracted the metabolites using freeze-dried samples to reduce the influence of water content.

Test

Waiting for 72 - 96 h after injection, we harvested the samples freeze-dried for 24 h and then extracted metabolites. The same LC-MS/MS method was used to analyze resveratrol production. As shown in Fig.1-4, resveratrol production increased four times that of cycle Ⅱ. HQT mutant samples with only the VvSTS gene produced more resveratrol than the wild type. However, double genes make production lower than single VvSTS gene no matter mutants and wild types. HQT knockout plant decreased more apparent than wild type.

Learn

Changing the injection method and sampling time and taking on freeze-dry helped increase resveratrol production and decrease the difference between groups. Thus, we will keep the parameters and conditions in our later investigation. Unfortunately, the expected improvement of resveratrol yield in VvSTS and YIARO3 groups did not come out. Moreover, the difference between HQT-knockout plants and wild-type plants is not apparent. We speculated that The competitive binding enzyme YIARO3 introduced into the feedback inhibition pathway may affect other growth-essential metabolic pathways, decreasing in synthetic yield. Moreover, we needed homozygous mutants to validate our design in the following experiments.

The Synthesis of Phaselic Acid

The hydroxycinnamoyl transferase in tobacco has a low affinity for malic acid and a high affinity for quinic acid or shikimic acid. To verify the ability of the tobacco plant chassis to introduce accumulated caffeoyl-CoA into other metabolic reactions as a substrate for synthesis after the HQT gene is knocked out, we introduced the hydroxycinnamoyl transferase HCT-M from Trifolium pratense and codon-optimized it for expression in Nicotiana benthamiana to attempt the synthesis of phaselic acid, thereby verifying the chassis's ability to efficiently synthesize secondary metabolites downstream of the shikimate pathway.

Cycle I

Design

We constructed a plasmid capable of expressing the exogenous HCT-M protein in plants using the Gateway cloning method. After transforming the plasmid into Agrobacterium, we used it to transfect Nicotiana benthamiana. The transfected tobacco was then subjected to LC-MS/MS analysis to verify the chassis's ability to synthesize phaselic acid.

Build

1. We amplified the HCT-M gene fragment with an aatB tag using PCR and connected it to the pDONR207 plasmid using BP reaction to construct the entry plasmid pDONR207-HCT-M.
2. The entry plasmid was subjected to an LR reaction with pEAQ-HT-DEST1 to construct the expression plasmid pEAQ-HT-DEST1-HCT-M.
3. The constructed plasmid was transformed into Agrobacterium GV3101, and single colonies were cultured in LB medium containing Rif and kanamycin for two days before being used to infect tobacco.
4. The infected tobacco region was excised, and metabolites were extracted using methanol for LC-MS/MS analysis.

Test

The data indicates a significant increase in the concentration of phaselic acid after the infiltration of Agrobacterium, suggesting that the efficiency of tobacco transformation has been enhanced following the activation of Agrobacterium, and consequently, the ability of Nicotiana benthamiana to synthesize phaselic acid has been substantially improved. We have standardized the collection of leaf samples, resulting in more uniform data among replicate experiments. Moreover, the level of phaselic acid synthesized by the mutant tobacco is higher than that of the wild-type tobacco, indicating that the Nicotiana benthamiana chassis, after the knockout of the HQT gene, possesses a greater capacity for the synthesis of phaselic acid.

Learn

In the experiment, we cut the injected area of a single leaf as a group for LC-MS/MS detection. Due to the large differences in the status of the leaves, there was a large difference between the groups. Moreover, the specific experimental operation omitted the step of shaking activation, resulting in low Agrobacterium activity and thus an insignificant increase in phaselic acid. To improve the experimental operation details, we propose the following improvements:

1. Pick Agrobacterium from the plate, shake it for 48 hours, and then shake it overnight to inject tobacco in the best state;
2. After transforming the tobacco, only cut off the leaf area in good condition;
3. Mix the cut leaves with the same treatment before grouping to eliminate differences between groups;
4. Freeze-dry the leaves before LC-MS/MS extraction to eliminate the impact of leaf water content on the experimental results.

Cycle II

Design

After transfecting the tobacco with activated Agrobacterium, we freeze-dried the transfected tobacco and performed LC-MS/MS analysis to verify the chassis's ability to synthesize phaselic acid.

Build

1. Pick single colonies of Agrobacterium with the target plasmid in LB medium containing Rif and kanamycin and culture for two days, then expand the culture overnight with a 1% inoculation ratio.
2. Excise the injected tobacco area, freeze-dry the leaves, extract metabolites using methanol, and perform LC-MS/MS analysis.

Test

The data showed a significant increase in the concentration of phaselic acid after injecting Agrobacterium, indicating that the exogenous HCT-M gene we injected can be expressed in tobacco and greatly improve the ability of Nicotiana benthamiana to synthesize phaselic acid. In addition, the content of phaselic acid synthesized by the mutant tobacco is higher than that of the wild-type tobacco, indicating that the Nicotiana benthamiana chassis after HQT knockout has a higher ability to synthesize phaselic acid.

Learn

The ability of mutant tobacco to synthesize phaselic acid has improved to some extent compared to the wild type, but it may not have reached the best synthesis amount of phaselic acid for the chassis. This is because the mutant tobacco has missed the best time for transfection, and the chlorogenic acid in the mutant tobacco has begun to accumulate, reducing the ability to synthesize phaselic acid, and the difference with the wild type is no longer obvious.

The Synthesis of Crocin

Cycle I

Design

ALDH is one of the critical enzymes in the crocin synthesis pathway. Its primary function is to catalyze crocetindialdehyde to crocetin. In order to transform tobacco, we plan to use Gateway cloning to construct the plasmid vector pEAQ-DEST1-ALDH. The construction of this plasmid vector involves three transformations, which need to be screened in LB medium with gentamicin, LB medium with kanamycin, and LB medium with rifampicin and kanamycin, and then use agarose gel electrophoresis and sequencing and other bacterial testing methods to determine whether the target plasmid is successfully constructed.

Build

The ALDH gene fragment was connected to pDONR207 by BP reaction, and then the plasmid was transformed into E. coli TOP10. In the biosafety cabinet, 200μl of bacterial solution was applied to LB medium containing gentamicin and cultured overnight in a 37℃ incubator. The next day, a single colony was selected for expansion and culture, and then PCR and sequencing were performed. After successful Colony PCR, pEAQ-DEST1 was connected by LR reaction. After transformation, 200μl of bacterial solution was applied to LB medium containing kanamycin and cultured overnight in a 37℃ incubator. After Colony PCR, positive plasmids were taken for Agrobacterium transformation.

Test

The overnight culture of transformed E. coli showed poor growth, with few single and small colonies. After a single colony was expanded and sent for sequencing, the results showed that ALDH was not successfully connected to the plasmid vector.

Learn

1. We analyzed the phenomenon of a few single colonies and small colonies in bacterial culture. This may be because we used a culture medium containing gentamicin to screen bacteria, which resulted in a relatively slow bacterial growth rate. Hence, we will extend the incubation time.
2. The poor sequencing results may be due to the unsuccessful BP reaction, which is related to the reaction time (overnight connection) and reaction temperature (16°C). Next time, we plan to adjust the reaction temperature to 25°C and the reaction time to 2 hours.

Cycle II

Design

Considering that the first round of experiments may have failed due to operational errors or unsatisfactory reaction conditions, we plan to repeat the Gateway cloning experiment to construct the plasmid pEAQ-DEST1-ALDH. In this round, we will optimize the experimental steps and carefully check each step to ensure the successful construction of the plasmid.

Build

In the second round of experiments, we paid more attention to the standardization of experimental operations. We first optimized the conditions of the BP reaction. The reaction temperature was adjusted to 25°C, and the reaction time was adjusted to 2 hours to improve the connection efficiency. Subsequently, we used the optimized conditions to connect the ALDH gene fragment to the pDONR207 vector and transformed it into E. coli TOP10. In the biosafety cabinet, 200μl of the bacterial solution was spread on an LB medium containing gentamicin and cultured overnight in a 37°C incubator. The next day, we selected single colony for expansion culture and performed Colony PCR and sequencing.

Test

After optimizing the BP reaction conditions, we found that there was no bacterial we wanted growth on the plate, which indicated that other problems might affect the experiment's success.

Learn

1. We analyzed that the failure of recombinant plasmid construction may be due to the incompatibility of the ALDH gene sequence with the Gateway vector. Gateway technology requires specific sites. If these sites are not contained in the target gene sequence or secondary structures in the sequence affect the recombination efficiency, the construction may fail.
2. We will re-evaluate the ALDH gene sequence to determine whether sequence features affect the Gateway reaction and consider using other vectors or methods for experiments.
3. We also plan to PCR amplify the ALDH gene and use other vectors for transformation to verify whether there are problems with the gene sequence itself. In the next round of experiments, we will adjust the experimental strategy based on the analysis results of this round of experiments and take corresponding measures to improve the success rate of the experiment.

Cycle III

Design

After a detailed summary of the first two rounds of experiments, we decided to continue the construction of the ALDH gene recombinant plasmid using the Gibson assembly, as it is widely used for its high efficiency and flexibility and does not rely on specific restriction enzyme sites, and has a high efficiency and success rate when assembling large fragments of DNA.

Build

In this round of experiments, we designed specific primers for PCR amplification of the vector pEAQ-DEST1 and the ALDH gene. We added a His-tag sequence to the primer design to facilitate subsequent protein purification. After amplification, we used ClonExpress II One Step Cloning kit for recombination reaction at 50°C for 12 minutes. After transformation, 200 μl of the bacterial solution was spread on LB medium containing kanamycin and cultured overnight in a 37°C incubator.

Test

After PCR amplification of the ALDH gene and the pEAQ-DEST1 plasmid backbone, agarose gel electrophoresis and gel extraction were performed to verify the PCR products. The amplification results of the pEAQ-DEST1 plasmid backbone were poor, with evident mixed bands, and the plasmid concentration was low after gel extraction.

After Gibson assembly and transformation into E. coli TOP10, we observed bacterial growth on an LB medium containing kanamycin. This indicates that the recombinant plasmid may have been successfully constructed and transformed. Further, Colony PCR also showed the expected target bands through nucleic acid electrophoresis. Unfortunately, the sequencing results showed that the plasmid was not wholly connected to the ALDH gene, and only part of the fragment was successfully recombined.

Learn

1. The PCR vector shows strong mixed bands after running on the gel. Primer design problems, resulting in nonspecific binding amplification of primers and vectors. Or the vector itself may be contaminated, affecting the specificity of PCR amplification.
2. The vector fragment is too large, resulting in low recovery efficiency during gel extraction.
3. To solve these problems, we used the Phanta enzyme system to re-amplify to improve the amplification's specificity and efficiency. However, the gel extraction efficiency is still very low.
4. In the next round, we plan to redesign primers to avoid nonspecific binding, use a more efficient gel extraction kit.

Cycle IV

Design

After a detailed summary of the previous round of Gibson assembly, we redesigned the primers and optimized the experimental conditions. To solve the problem of too large vector fragments and low gel extraction efficiency, we appropriately simplified the vector. We performed a multi-tube vector PCR system, and finally the products were extracted together. To solve the problem of vector contamination, we re-cultured the bacteria containing the vector and extracted its plasmid.

Build

In this round of experiments, we designed new specific primers for PCR amplification of the vector pEAQ-DEST1 and the ALDH gene and added a His-tag sequence to the primer design. After amplification, we used the ClonExpress II One Step Cloning Kit for the recombination reaction at 50°C for 2h. The cloned product was transformed into E. coli TOP10, the bacterial solution was then spread on an LB medium containing kanamycin and cultured overnight in a 37°C incubator. After bacterial inspection and sequencing, the plasmid was extracted for Agrobacterium transformation.

Test

Use new primers to obtain the target fragment through PCR and linearize the vector. After gel extraction, incubate the target fragment and linearized vector at 50°C for 2 hours, transform the cloned product into E. coli TOP10, and then spread the bacterial liquid on LB medium containing kanamycin and culture it in a 37°C incubator overnight.

After overnight culture, there were uniform colonies on the plate. 28 single colonies were selected for amplification and Colony PCR.

We observed bacterial growth on an LB medium containing kanamycin. This indicates that the recombinant plasmid may have been successfully constructed and transformed. Further electrophoresis also showed the expected target band. We sent the PCR product containing the target band for sequencing, and the sequencing results showed that the plasmid was successfully constructed. The plasmid was then extracted and transformed into Agrobacterium.

Learn

1. When designing primers, their specificity should be ensured to avoid the appearance of target bands during amplification.
2. Using multiple reaction systems to linearize the vector and then performing gel extraction together can increase the vector concentration.
3. Appropriately extending the Gibson assembly incubation time may increase the cloning efficiency.

Directed Evolution

For the production of phaselic acid, the catalytic activity of HCT-M derived from Trifolium pratense is limited due to its weak affinity for caffeoyl CoA, instead favoring the binding of feruloyl CoA, leading to the production of a significant amount of feruloyl malic acid as a byproduct. To address this issue, we have conducted directed evolution of HCT-M to screen for mutants with high substrate affinity for caffeoyl CoA.

Cycle I

Design

To construct plasmids containing various HCT-M mutants, we amplified the HCT-M gene, which includes an OmpA secretory tag and an ASA linker, using error-prone PCR to introduce mutations. Subsequently, we employed homologous recombination to ligate the mutated gene fragments into the pET-28a backbone.

Build

1. Design primers to amplify the HCT-M gene containing the OmpA secretory tag and ASA linker using error-prone PCR, and simultaneously amplify the pET-28a plasmid backbone using PCR. Recover and purify the amplified fragments from agarose gel electrophoresis.
2. Perform homologous recombination to ligate the randomly mutated HCT-M gene with the pET-28a backbone.
3. ransform the ligation products into DH5α.
4. Pick single colonies and inoculate them into LB broth containing kanamycin, culture overnight, perform colony PCR, and preserve and extract plasmids from PCR-positive cultures.

Test

Out of the 36 picked bacteria, only three were positive. Sequence some positive and negative colonies, confirming that the positive colonies have successfully ligated the targeted gene and the gene has random mutations.

Learn

This experiment revealed a low positive rate, and possible reasons include:

1. olThe low concentration of DNA after gel extraction, leading to low ligation efficiency.
2. 2. Insufficient electrophoresis time to separate linearized plasmids from the original plasmids. We will increase the concentration of fragments in the ligation reaction and extend the electrophoresis time appropriately to improve the positive rate.

Cycle II

Design

We obtained HCT-M mutant gene expression plasmids with OmpA secretory tags and ASA linkers by increasing the concentration of fragments, and transformed them into BL21 for induced expression. After purifying the bacterial proteins, we used SDS-PAGE to verify protein expression.

Build

1. We amplified the HCT-M gene containing the OmpA secretory tags and ASA linker using error-prone PCR to introduce mutations, and then ligated the mutated gene fragments into the pET-28a backbone using homologous recombination. After transforming into DH5α, we picked single colonies, confirmed positive colonies by colony PCR. We extracted plasmids and transformed them into the BL21. We also constructed a non-mutated pET-28a-HCT-M without secretory tags as a control group.
2. We picked single colonies of BL21 and cultured them overnight, then scaled up the culture with a 2% inoculation ratio. When the OD reached 0.6-0.8, we induced expression for 16 hours with a final concentration of 0.2 mM IPTG.
3. After induction, we lysed the bacteria to extract total proteins and then purified the target protein.
4. We performed SDS-PAGE electrophoresis on the purified proteins.

Test

In the control group, the purified target protein band was present, but it was not observed in the mutant bacteria. Additionally, we found that neither the control group nor the mutant bacteria had the target band in the supernatant, suggesting that the OmpA tag may not be functional.

Learn

The inability to purify the mutant protein may be due to the addition of the secretion tag causing structural changes in the protein, preventing the exposure of the His-tag, or due to mutations at critical sites leading to the formation of a large number of inclusion bodies. In the next step, we will reconstruct the pET-28a-HCT-M plasmid containing the mutated HCT-M gene without the OmpA secretion tag and ASA linker.

Cycle III

Design

We will remove the OmpA secretion tag from the plasmid through homologous recombination and transform it into BL21 bacteria for induced expression. After purifying the bacterial proteins, we will use SDS-PAGE to verify protein expression.

Build

1. We will amplify the HCT-M gene fragment from the mutated plasmid before mutation and the pET-28a plasmid backbone using PCR, and then connect them through homologous recombination to remove the OmpA secretion tag and ASA linker.
2. We will transform the pET-28a-HCT-M plasmid containing the mutated HCT-M gene into BL21, pick single colonies for cultivation, and then induce expression with IPTG.
3. We will extract and purify the proteins and perform SDS-PAGE detection.

Test

We performed SDS-PAGE detection on the purified proteins and successfully purified 9 out of 13 proteins.

Semi-rational Design

Semi-rational design is based on a certain understanding of the physicochemical properties, three-dimensional structure, conformational relationship, catalytic mechanism and other information of proteins, and computer-assisted fixed-point mutation, saturation mutation, and combinatorial mutation of hotspot amino acids in the active centre or active pocket. This method is between irrational design and rational design, overcoming the shortcomings of both and reducing the technical requirements. To improve the yield of resveratrol and phaselic acid, we used a semi-rational design approach to optimize STS and HCT2 by screening favourable single-point mutation sites, and verified the effectiveness of the mutants in wet experiments. However, we encountered many problems during the semi-rational design and mutant validation process. Through multiple rounds of DBTL approach, we updated and iterated the design, mutation and purification methods, and finally completed the semi-rational design and validation of STS and HCT2, and obtained mutants possessing higher catalytic activity or better thermal stability.

Cycle I

Design

Since the existing pre-trained protein language models and shared protein interaction design platforms are more maturely developed and there are studies to support their validity, we will use the shared computer platform to perform the initial screening of single-point mutation hotspots and confirm the stability of the mutant-ligand binding by molecular docking to exclude mutation sites that are not conducive to the stability of the protein-ligand binding, in order to obtain both stability and catalytic activity of the The mutants with improved stability and catalytic activity were obtained.

Build

1. The PDB files of STS and HCT2 were uploaded to Hotspot Wizard 3.0 (Lenka Sumbalova, et al., 2018) and SaprotHub (Jin Su, et al., 2024), respectively, to export the mutation hotspot scores and related data.
2. Alternative mutants were screened based on high or low scoring and the location of the mutation site (whether it was in the active pocket or not, etc.) (see protein modelling section for details).
3. PDB files of the mutants were obtained using ChimeraX 1.8.
4. STS was docked to p-coumaroyl coenzyme A and malonyl coenzyme A, and HCT2 to caffeoyl coenzyme A and malic acid molecules using CB-Dock2(Yang Liu, et al. 2022), and a vina score was obtained as a reference for docking results.
5. The obtained docking results were opened with PyMOL and polar bonds were visualised, eliminating mutants with less than 80% of the polar bonds of the wild type.

Test

Learn

The round of design produced 18 STS mutants with 22 HCT2 mutants, considering the wet experiment time and workload, we need to continue the design scheme to screen fewer mutants into the validation session. In addition, since CB-Dock2 does not support docking of a protein to two ligands at once, the reference value of Vina Score after the second docking is weak, so we need to apply a more accurate method to further screen the mutants.

Cycle II

Design

Based on the mutants screened in the first round, we will use GROMACS to perform molecular dynamics simulations to study the movement mode of the protein-ligand-solvent system, and screen out mutant proteins with more stable system and faster convergence of the potential energy of the system to be constant through the simulations of the change of RMSD & RMSF, the change of the total potential energy of the system, the change of the radius of gyration of proteins, as well as the visualisation of the molecular movement trajectories.

Build

1. Open Babel converted the small molecule ligand format to .mol2, and the penalties were measured on CGenFF, which was too high for all four ligands and required ligand optimisation.
2. Small molecule ligands are generated on ORCA and Multifin with RESP atomic charges and optimised for chemical bonding, and finally the corresponding topology files are generated on sobtop.
3. Complexes were constructed using preprocessed protein and ligand topology files, periodic boundary conditions were set, energies were minimised, pre-equilibrated and the main simulation was completed.
4. After calibrating the system parameters, the corresponding RMSD, RMSF, change in total potential energy of the system, change curve of the radius of gyration of the protein, and molecular motion trajectory were output, and the corresponding indexes of the mutant were compared with those of the wild type to screen out the mutant proteins whose systems were more stable and whose potential energies tended to be constant more quickly.

Test

Through molecular dynamics simulations, we screened a total of 12 STS proteins and 13 HCT2 proteins into the wet experiment validation session.

Learn

Molecular dynamics simulation is a very effective way to screen for mutants that make the system more stable and the potential to stabilise faster, and we obtained mutants with higher stability and enzyme activity at the computer prediction level through the above screening process.

Cycle III

Design

Based on the mutants screened by computer-assisted design, we constructed the mutants by reverse PCR and performed primer design and point mutation according to the requirements of the toyobo point mutation kit SMK-101.

Build

1. Primer design: The mutation site is designed at or near the 5' end of the primer and the 3' end of the primer is complementary to the template by at least 20 bases. One primer is a mutant primer and the other primer is a conventional primer.
2. Point mutation PCR: Mix each set of dispensed PCR reaction solution according to the recommendations of the instruction manual and perform PCR amplification (5-10 cycles), adjusting the extension time and the number of cycles according to the size of the plasmid and the efficiency of the primers.
3. Digest the template plasmid DNA and ligate the PCR product.
4. The plasmid was transformed into the DH5α receptor state and extracted for sequencing after recovery.

Test

In total, three rounds of point mutations were performed (two rounds for STS and one round for HCT2), and only five STS mutants were successfully constructed, no HCT2 mutants were constructed, and most of the plasmids were sequenced as wild-type.

Learn

We hypothesised that the reason for the failure of the mutant construction was that one of the primers required for the Toyo Spinning kit did not carry the mutation site, which would have introduced a large number of wild-type plasmids. In addition, the insufficient number of PCR cycles also resulted in fewer mutant plasmids. Therefore, we need to improve the point mutation method to make the products of point mutation PCR purer and produce more mutant plasmids.

Cycle IV

Design

In order to increase the efficiency and positivity of point mutation PCR, we improved the method of point mutation. This time, we used partially complementary primers with mutation sites in both primers for point mutation and increased the PCR cycle to 20 times.

Build

1. Primer design: Two primers of 25-45 base complementary lengths were designed with Tm values around 78°C. Both primers contained the expected mutation sites. In order to avoid the generation of primer dimers, the Tm values of the two primer pairs were kept below 50% as far as possible.
2. Point mutation PCR: The PCR enzyme was changed to the more efficient KOD One and 20 cycles were performed.
3. Digest template plasmid DNA.
4. The plasmid was transformed into the DH5α receptor state and extracted for sequencing after recovery.

Test

After adjusting the point mutation PCR reaction, we constructed the corresponding mutants at once.

Learn

1. The success of point mutation is higher when both primers carry the mutation site, as there is a higher probability of picking positive bacteria.
2. Extending the number of PCR cycles and using more efficient PCR enzymes to increase the concentration of point mutant fragments are critical to the success of experiments to construct point mutant proteins.

Cycle V

Design

Before the characterisation of the constructed mutant protein, the nature of the protein needs to be clarified, we learnt from the data that STS has low solubility and is easy to form inclusion bodies, so in the pre-experiment, we chose to express the wild-type STS by inducing it with 0.1 mM IPTG in LB medium at 24℃, 120 rpm for 16 h. The purpose is to avoid the formation of inclusion bodies to affect the subsequent purification and reaction. The solubility of HCT was better, so the wild-type HCT was induced in LB medium with 0.2 mM IPTG at 24℃, 150rmp for 16h to express wild-type HCT.

Build

1. Protein expression was induced under Design conditions.
2. HCT proteins were purified by non-denaturing nickel column purification, and STS proteins were purified by denaturing and non-denaturing nickel column purification, respectively, and the flow-through and each of the washes and eluents were collected.
3. The collected above liquid was run on SDS-PAGE.

Test

The protein bands in the eluate after purification from the non-denaturing nickel column were extremely weak, but the bands corresponding to the size of STS protein in the denaturing purified eluate were stronger, indicating that most of the proteins formed inclusion bodies. The HCT protein bands were more obvious in the eluate after purification from the non-denaturing nickel column, indicating that the HCT protein was successfully purified.

Learn

The LB medium was able to carry too little bacteria, resulting in insufficient normal STS proteins being purified at the end, which needed to be replaced with a medium capable of carrying a larger amount of bacteria and further lowering the temperature to reduce inclusion body production and increase the amount of normal proteins.

Cycle VI

Design

Compared to LB medium, TB medium is more nutrient-rich and able to carry more amount of bacteria, so we will choose TB as the medium for this round of experiments. In addition, we reduced the temperature at which we induced protein expression to 21°C, thereby reducing the amount of inclusion body formation. In order to increase the concentration of normal STS proteins, we will ultrafiltrate the samples after Akta non-deformed purification.

Build

1. TB medium, 0.1 mM IPTG, 21°C, 120 rpm, induction for 20 hours.
2. Akta non-denaturing purified protein, ultrafiltration.
3. Bradford assay for protein concentration.

Test

Learn

1. Compared with LB medium, TB medium was able to significantly increase the bacterial volume and was suitable for increasing protein production.
2. Lowering the temperature during IPTG-induced protein expression reduces the generation of inclusion bodies.
3. Ultrafiltration can significantly increase the concentration of proteins in solution.

Cycle VII

Design

In order to characterise the enzyme activity of the purified wild-type and mutant HCT proteins, we used the L-malic acid assay kit (WST-8 method) of Biyun Tian to detect the remaining amount of malic acid in the reaction substrate. Firstly, we plotted the standard curve of malic acid concentration according to the method of the kit.

Build

1. Prepare a reaction buffer with 25 mM vitamin C and phosphate buffer to pH = 6.5.
2. Prepare the appropriate amount of colour development working solution according to the volume of 80 μl for each assay reaction. Mix 74 μl of Malate Assay Buffer, 2 μl of Enzyme Solution, 2 μl of Chromogen Solution and 2 μl of Substrate to make 80 μl of Working Solution. The working solution can be prepared into 80 μl of Working Solution.
3. Take 25 μl of Malic Acid Standard Solution (10 mM), add 475 μl of reaction buffer and mix well to formulate a concentration of 500 μM Malic Acid Standard Solution. Take 0, 1, 2, 4, 8 and 20 μl of 500 μM malic acid standard solution into the standard wells of 96-well plate respectively, and make up to 20 μl with the corresponding reaction buffer, at this time, the concentration of the standard curve is 0, 25, 50, 100, 200 and 500 μM.
4. Add 80 μl of colour development working solution to each well, mix well and react at 37ºC for 30 minutes.
5. Determine the absorbance at 450nm.

Test

Table 5-5: Results of malic acid concentration gradient determination (* represents high OD value, exceeding the upper limit of detection of the zymography)

According to the kit instructions, the measured malic acid OD values should be between 0 and 1. In this case, the OD values of all six malic acid concentrations exceeded this range and did not form a gradient.

Learn

The detection principle of the kit is that L-malic acid is oxidised to oxalacetic acid (OAA) by malate dehydrogenase (MDH), and NAD+ is reduced to NADH in this reaction; the generated NADH is reduced to orange-coloured Formazan by the electron-coupling reagent 1-mPMS (1-Methoxy-5-methylphenazinium methyl sulfate). NADH is reduced to NADH by the electron coupling reagent 1-mPMS (1-Methoxyphenazinium methyl sulfate), and the generated NADH is reduced by WST-8 to form the orange-yellow colour of Formazan, with a maximum absorption peak at around 450 nm. And vitamin C also has reducing property, it is presumed that the vitamin C in the reaction buffer interfered with the colour development reaction.

Cycle VIII

Design

In response to the previous round's speculation that vitamin C affects the colour development reaction of the malic acid assay, this speculation was verified in this round. We removed vitamin C from the reaction buffer and retained only the phosphate buffer at pH=6.5.

Build

1. Prepare a reaction buffer with phosphate buffer to pH = 6.5
2. Prepare a working solution for colour development in the same way as round 6.
3. Establish the malic acid concentration gradient using the new reaction buffer without vitamin C.
4. 80 μl of colour developing working solution was added to each well, mixed well and reacted at 37ºC for 30 minutes. Absorbance was measured at 450nm.

Test

Table 5-6: Results of malic acid concentration gradient determination

The OD values for this round of tests all fell between 0 and 1 with a clear gradient.

Learn

Vitamin C does interfere with the colour development reaction, suggesting that vitamin C has a much greater ability to reduce NAD+ than malic acid, and interferes with the colour development in the assay principle. By removing vitamin C from the reaction buffer, the malic acid concentration can be measured normally.