This year, NAU-CHINA has designed in a total of 57 parts, including 22 new basic parts and 13 new composite parts.

The sensor module includes the Locked Hairpin (BBa_K4613209), Dumbbell Template (BBa_K4613210) and Molecular Beacon (BBa_K4613206). Ochratoxin A (OTA) triggered the structure transformation of locked hairpin, resulting in the rolling circle amplification, leading to release of florescent signal. The signal would be detected through Real-time Quantitative PCR (qPCR) instrument.

We designed recombinant mature Carboxypeptidase A (M-CPA) (BBa_K4613001) to degrade OTA .

We designed recombinant mature Carboxypeptidase A (M-CPA) (BBa_K4613001), which has better thermal and acid-base stability compared to Carboxypeptidase A. M-CPA was constructed by deleting the 110 residues including the signal peptide and propeptide. In the presence of M-CPA, the amide bond of OTA hydrolyze and the nontoxic ochratoxin α (OTα) and L-α-phenylalanine (Phe) are produced.

Moreover, we chose an amidohydrolase derived from Stenotrophomonas acidaminiphila called ADH3 (BBa_K4613002) to degrade OTA. Soluble protein expression of ADH3 in Escherichia coli has been realized and it performed well among the OTA-detoxifying enzyme, exhibiting a 57- to 35,000-fold higher activity than other enzymes.

SpyTag, SpyCatcher, and Elastin-like Polypeptide (ELPs) (BBa_K4613010) were designed to fabricate a biohybrid semi-interpenetrating polymer networks (sIPN). These ELPs (BBa_K4613010) were fused with either multiple SpyCatcher or SpyTag sequences. Spytag and Spycatcher can polymerizate by covalent bonding to form sIPNs.

We designed an autolysis gene circuit to release sIPN in response to elevated cell density.This system is a LuxR/LuxI-type quorum sensing system from Agrobacterium tumefaciens . We used protein E from Bacteriophage Phi X 174(BBa_K2152003) to lyse cells.

If you want to see more details, you can find the usage and characterization in part registry page.

This year, NAU-CHINA designed 22 new basic parts, aiming to realize the detection and degradation of Ochratoxin A (OTA).

Locked Hairpin (BBa_K4613209), Dumbbell Template (BBa_K4613210) and Molecular Beacon (BBa_K4613206) were designed to detect OTA in a more accurate and sensitive way.

In order to degrade Ochratoxin (OTA) into nontoxic ochratoxin α (OTα) and L-α-phenylalanine (Phe), we designed M-CPA (BBa_K4613001) and ADH3 (BBa_K4613002) to express Mature Carboxypeptidase A (M-CPA) and ADH3. T3 (BBa_K4613011)stands for hydrophilic elastin-like polypeptides (ELPs) (BBa_K4613010) fused with three SpyTag sequences, while C3 (BBa_K4613012) codes for ELPs fused with triple SpyCatcher sequences. T3 and C3 were designed to fabricate a biohybrid semi-interpenetrating polymer network (sIPN).

We constructed T3-YFP (BBa_K4613015), the fusion protein of YFP (BBa_K592101) and T3 (K4613011) to verify the combination between SpyTag and SpyCatcher. We also fused M-CPA and ADH3 to the C-terminus of T3 by a 3*GS Linker (BBa_K4164025) respectively to degrade OTA efficiently and sustainably.

To release the sIPN out of the engineered bacteria, traR (BBa_K4613215) and traI (BBa_K4613216) from luxR/luxI type system (tra system) facilitate the expression of lysis E (BBa_K2152003) under Ptra (BBa_K4613006) to induce cell lysis at high cell density.

You can click on the part number for more details in the following table.

To optimize the system and better implement our expected design, we designed 13 composite components by restriction enzyme digestion and ligation and In-Fusion Cloning.

We first verified the feasibility of the combination of T3-YFP (BBa_K4613015) and C3 (BBa_K4613012) by constructing pET29a(+)-T3-YFP (BBa_K4613310) and pET29a(+)-C3 (BBa_K4613301). Considering the low intensity of pQE-80L-T3-M-CPA (BBa_K4613024) and pQE-80L-C3 (BBa_K4613025) which were expressed at low levels, we replaced it with T7 lac promoter from pET-29a(+) and purified protein to improve our system. However, the rate of solube protein expression of M-CPA and C3 were still low. To achieve better solube protein expression, we constructed the pET-PC-sumo-M-CPA (BBa_K4613311), but the circumstance was still frustrating. Luckily, we found an amidohydrolase called ADH3 which solube protein expression in Escherichia coli has been achieved. So we constructed the pET-46 Ek_LIC-ADH3 (BBa_K4613023) and pET-29a(+)-T3-ADH3(BBa_K4613303) to improve our system. We assembled Ptra、lysis E、traR and traI using In-Fusion Cloning.

This year NAU-CHINA team designed Locked Hairpin (BBa_K4613209), Dumbbell Template (BBa_K4613210), Molecular Beacon (BBa_K4613206) to achieve the detection of OTA. We combined T3-ADH3 (BBa_K4613014), C3 (BBa_K4613012) to degrade OTA through synthetic biology methods. Moreover, we assembled Ptra (BBa_K4613006), traR (BBa_K4613215) , traI (BBa_K4613216) and lysis gene E (BBa_K2152003) to release sIPN through cell autolysis.

To achieve a high level of solube protein expression of OTA-detoxifying enzyme and find the best promoter intensity, we tried different promoters and various vectors. After communicating with other teams and consulting professors, we chose T7 promoter and constructed pET-29a(+)-T3-ADH3 (BBa_K4613303), pET-29a(+)-C3 (BBa_K4613301), pSB1C3-tra-lysis (BBa_K4613888) to degrade OTA efficiently. Meanwhile, we used Locked Hairpin (BBa_K4613209), Dumbbell Template (BBa_K4613210), Molecular Beacon (BBa_K4613206) to realize the detection of OTA.