Design

Ochratoxin is another deadly fungal mycotoxin that has attracted widespread attention around the world. In all kinds of Ochratoxins, Ochratoxin A (OTA) is considered the most toxic, abundant, virulence, toxigenic, pollutive to agricultural products, and closest relationship with human health. It shows extremely strong hepatorenal toxicity, teratogenic and carcinogenic effects. The European Food Safety Authority emphasizes that wine ranks second exposure to OTA. Therefore, we designed this project to detect and degrade OTA.

For OTA detection, currently, methods for detecting OTA consist of instrumental analysis and immunoassay techniques. Considering the inconvenience of Ochratoxin A (OTA) detection and degradation in the food industry, we have designed a rapid detection method, detecting by DNA Aptamer. When brewing wine, manufacturers will try to remove OTA during the fermentation stage. However, there may still be instances of commercial wines exceeding OTA limits. We found carboxypeptidase A (CPA) derived from bovine pancreas which could degrade OTA. Considering the practical applicability of the degradation module, we plan to link CPA with sustainable semi-interpenetrating polymer networks (sIPN). This system was consisted of elastin-like polypeptides (ELPs), and a pair of reactive protein partners, SpyTag and SpyCatcher, which could polymerize efficiently under multiple conditions. Then, we encapsulate it in a polymeric microcapsule consisting of chitosan and sodium alginate. Living sIPN makes the multiple applications of CPA easier. At the same time, we will use a lysis circuit so that the degradation device could be released into the liquid.

To check the feasibility of sIPN, we are going to use YFP as reporter gene before.

Build

We establish short single-stranded oligonucleotides with high prominent recognition specificity and affinity to OTA (Fig. 2). G-quadruplexes will form after the binding of aptamer and OTA.

To construction the sIPN system, we linked SpyTag or SpyCatcher monomers separately with Hydrophilic elastin-like polypeptides (ELPs) as backbone (T3 or C3). We obtained the gene through complete synthesis. Then, we will construct a fusion protein of YFP and T3, so that we can verify this system.

Test

Through literature review, we found that directly using aptamers to detect OTA has low sensitivity, which might not be able to detect the minimum concentration set by food safety regulations. It does not align with our application scenario, so we decided to discard this strategy.

We checked the part by SDS-PAGE. As shown in Fig. 4, we expressed and purified T3-YFP and C3 successfully. Moreover, they are capable of forming the Spy Network.

We mixed the bacteria producing T3-YFP and the bacteria producing C3 together and then made immobilized microcapsules. At the same time, we also attempted to directly use purified T3-YFP to make microcapsule (Fig. 5 a-2.). After placing two types of immobilized microcapsules at room temperature for 12 hours, we found that the engineered bacteria and sIPN system will not leak, but small molecules of proteins will leach out of the microcapsules (Fig. 5 b).

Moreover, we have established molecular models for T3 and C3, and simulated the protein binding of T3 and C3. In addition, we have also built a mesh-like molecular model and performed kinetic simulations using GROMACS, aiming to provide theoretical guidance for subsequent experiments.

In the mathematical model for protein structure determination, we conducted a comparative analysis of several machine learning methods and ultimately opted for the random forest model to predict the secondary structure of T3 and C3. The predicted results were found to successfully align with the tertiary structure, further enhancing the feasibility of protein structure reconstruction.

Test

We require the assistance of addi­tional amplified method for ultrasensitive detection because directly using aptamers to detect OTA has low sensitivity. We found a locking hairpin recognition-triggered structure switching strategy, which could drive rolling circle amplification (RCA). The detecting of OTA could be amplified by this way, and we could achieve more accurate detection.

The protein binding experiment of T3-YFP and C3 shows that the semi-interpenetrating polymer networks could be applied in practice. We will use this system in the following experiments.

Design

For OTA detection, we came up with a strategy that a structure-switching locked hairpin (LHP) triggered rolling circle amplification (RCA) with circulate template after investigating literatures based on OTA detection results in the previous loop. If you want more details, you can navigate the site of the design page about the sensor module.

For degrading enzymes, in order to obtain a CPA with enhanced thermal stability and improved tolerance to pH variations, we truncated 110 residues from the propeptide and signal peptide to construct mature CPA (M-CPA). We linked M-CPA into SpyTag monomers, which can be polymerized with SpyCatcher monomers to form a macromolecular network structure, thereby increasing the stability and sustainable generation of M-CPA.

We need quorum sensing system to control the release of two categories of protein monomers. We used tra quorum sensing system provided by Professor Zhong and lysis E protein to construct our autolysis circuit. Bacteria will express lysis E protein abundantly at high density to achieve autolysis.

Build

We designed and purchased nucleic acid sequences, molecular beacons and magnetic beads needed for OTA detection.

At the same time, we cloned C3 and T3-M-CPA into the pQE-80L, respectively, constructed pQE-80L-C3 and pQE-80L-T3-M-CPA and we chose E. coli BL21(DE3) as the chassis to express the recombinant protein. And then we constructed a LuxI-LuxR type regulatory system with pSB1C3 and express E protein to periodic release monomers in two categories.

Test

The result under this detection strategy was still not satisfactory. The trend of florescence is very chaotic and unstable, which was not in line with our expected results.

For T3-M-CPA and C3, we tried various expression conditions to promote their soluble expression, but the soluble expression levels of both were still very low in Fig. 8 and Fig. 9.

We tested the parts we constructed through sequencing verification. The results showed that we had successfully constructed the plasmid. Then we tested and described the growth curve of engineering bacteria. However, the curve indicates a narrow range of oscillation for the system.

Learn

After consulting Associate Professor Junbo Chen, we learned that the circulat template could generate the higher background due to the occurrence of nonspecific RCA driven by the independent hybridization between the circulat template and the primer of the LHP. In contrast, the dumbbell template enables both specific recognition and amplification of the primer sequences, reflected by the better signal to background ratio. Thus, we changed the circulate template of the strategy into a dumbbell template for OTA detection.

For the expression results of T3-M-CPA and C3, we considered that the low protein expression might be due to the weak strength of promoter of the pQE vector, so that we cloned C3 and T3-M-CPA into a vector containing a stronger T7 promoter, respectively.

As shown in Fig. 9 b, our autolysis circuit exhibited poor activity in E. coli, and we came up with two hypotheses. The first hypothsis is that the tra system in E. coli may have a poor ability to lead periodic lysis. And the second hypothesis is that the results can not reflect on the fact due to the poor sample-taking procedures and the low precision of our instruments. If you desire to access our future plan to improve this circuit, click this site: Project Result Page

Design

In our experiments, we observed significant interference from background fluorescence. After consulting Associate Professor Junbo Chen, we learned that hybridization of circular templates with LHP primers resulted in non-specific rolling circle amplification (RCA), inevitably affecting the experimental results. Consequently, based on the information provided by the professor, we reengineered a dumbbell template for specific primer recognition and amplification.

Furthermore, we also discovered that the protein amount expressed by pQE-80L did not meet our requirements. After a thorough review of the literature, we found that the reducing environment within E. coli BL21 (DE3) adversely affects the formation of disulfide bonds in M-CPA, leading to poor protein solubility. Consequently, we reconstructed the recombinant vector using pET-29a(+) and expressed it in SHuffle T7 E. coli, which has low reductibility. This modification aimed to enhance both the efficiency and quality of protein expression.

Build and Test

Sensor module: In the experiment, we obtained desirable data using the dumbbell template (Fig .11). The fluorescence intensity rises with the increase of concentrations of OTA.

Degradation module: We constructed two recombinant vectors, pET-29a(+)-T3-M-CPA and pET29a(+)-C3. Initially, when expressed in E. coli BL21 (DE3), we achieved high expression levels. However, the solubility of T3-M-CPA did not meet the experimental requirements. Considering the impact of the reducing environment in E. coli BL21 (DE3) on disulfide bond formation, we expressed it in SHuffle T7 E. coli, but solubility remained suboptimal.

Learn

Through a review of the literature, we observed that M-CPA has been widespread utility in eukaryotic expression systems, but its performance in prokaryotic expression systems is less satisfactory. We hypothesize that this discrepancy may be linked to the formation of inclusion bodies during prokaryotic expression. The formation of inclusion bodies can be influenced by various factors, such as temperature and even the correct folding of proteins. These factors may collectively result in solubility issues encountered with M-CPA. Based on literature, we learned that the solubility issue can be addressed by adding a SUMO tag before T3-M-CPA.

Design and Build

Considering the difficulty of M-CPA expression in prokaryotic, we also found a novel and highly effective OTA degrading enzyme called ADH3, which is from Stenotrophomonas acidaminiphila, and then we designed two strategies to solve the problems mentioned above.

In the first strategy, we cloned M-CPA into pET-PC-SUMO vector, and expressed the recombinant protein in SHuffle T7 E. coli expression competent cell to reduce the formation of inclusion body. If SUMO-M-CPA can be expressed solubly, we will use TEV hydrolase to cut SUMO off.

In the second strategy, we obtained the plasmid pET46EKLIC_ADH3 from Associate Professor Longhai Dai of Hubei University. At the same time, we cloned ADH3 into pET-29a(+)-T3-M-CPA vector, constructed pET-29a(+)-T3-ADH3. Both of them were expressed in E.coli BL21(DE3).

Test

In the first strategy, the soluble expression of SUMO-M-CPA (48.5kDa) was slightly increased. Most M-CPA still existed in the form of inclusion bodies.

As for the second strategy, for ADH3, obvious target bands can be seen at 43.4 kDa as shown in Fig. 13 (lanes 4 and 5). For T3-ADH3, obvious target bands can be seen at 73.6 kDa as shown in Fig. 13 (lanes 1 and 2).

Learn

According to the result of the above experiment, it is clear that M-CPA is difficult to soluble expression in E. coli, although we have taken many strategies to help it. Fortunately, both ADH3 and T3-ADH3 can be expressed solubly, so we chose T3-ADH3 and C3 as the two monomers of the sIPN system.

In conclusion, although the experimental process was very arduous, we finally succeeded in verifying the feasibility of our idea in laboratory environment. At the same time, considering food safety, we will continue to carry out strict regulation of strains to ensure compliance with food safety requirements. In the future, we plan to implement this system in probiotics like E.coli Nissle 1917.