Safety

Design

Thioredoxin fusion system

In our project, we used E. coli to express two heterogeneous enzymes from mycobacterium smegmatis. However, we didn’t know whether they were toxic to E. coli and whether they would become inclusion bodies because of insolubility when E. coli produced them. Therefore, we found the thioredoxin fusion proteins system. When the gene of thioredoxin in E. coli (TrxA) were co-expressed, the target proteins were inserted into the active-site loop of thioredoxin, and therefore the fusion proteins can be more soluble[1].

In addition, between the thioredoxin domain and the target gene domain, we designed some linkers to ligase two domains. The first part, which could be translated to the peptide sequence, “DDDDK”, was designed as the cleavage site of enterokinase. There were some restriction sites in the second part. The third one, which could be translated to glycine-glycine, was a flexible linker[2].

Fig. 1: BBa_K2382004 showed the gene design of the Thioredoxin fusion system construction.

Aflatoxin-degrading enzyme: F420-dependent reductase group A

F420-dependent reductases (FDR) can be divided into two classes (A and B) and be found in some species of bacteria[3]. FDR-A enzymes, has up to 100 times more activity than the other. In this class, MSMEG5998 has the best specific activity to AFB1 (10350 nmol/min/mol enzyme) and AFG1 (103210 nmol/min/mol enzyme). Therefore, we looked for the coding sequence of MSMEG5998, which was registered in NCBI in mycobacterium smegmatis and put the sequence of thioredoxin before it to form a fusion protein. For the purpose of purification through nickel-resin column, we added a 18-bp sequence which can code 6 histidines. In addition, we chose T7 promoter which contains lac operator to express this protein because it can be induced by IPTG. For the terminator, we chose BBa_B0015 because it was commonly used in E. coli. The gene design are shown in Fig. 2.

Fig. 2: BBa_K2382006 showed the gene design of the Thioredoxin-MSMEG5998 fusion protein construction.

The activator of F420: F420-dependent glucose-6-phosphate dehydrogenase

F420-dependent glucose-6-phosphate dehydrogenase (FGD) can catalyze D-glucose-6-phosphate (G6P) to become D-6-phsphogluconolactone. The chemical reaction are shown in Fig. 3[4]. This enzyme can be found in many organisms. In order to coordinate with MSMEG5998 and make the two enzymes react more naturally, we chose fgd gene also in mycobacterium smegmatis (strain MC(2) 155).To increase the solubility when E. coli produce this enzyme, we used the same design as Thioredoxin-MSMEG5998 fusion protein to form Thioredoxin-FGD fusion protein and the design is demonstrated in Fig. 4.

Fig. 3: The chemical reaction of G6P and oxidized F420 were catalyzed by FGD.

Fig. 4: BBa_K2382005 showed the gene design of the Thioredoxin-FGD fusion protein construction.

The function of our proteins in aflatoxin-induced DNA repair pathway

When mammalian cells respond to DNA damage from environmental toxins or radiations, the two key signaling components, ATM and ATR were activated and therefore phosphorylated two protein kinases, Chk1 and Chk2[5]. All of them will reduce cyclin-dependent kinase (CDK) activity through activation of p53. Finally, inhibition of CDKs, such as p21 slows down or arrests cell-cycle progression. Therefore, we can know whether aflatoxin B1 induces DNA damage in HepG2 cells indirectly through the increasing expression of some markers in p53 pathway[6]. In our hypothesis, the modified aflatoxin-degrading enzyme, MSMEG_5998 can directly alleviate the genotoxicity of aflatoxin and indirectly inhibit the activation of p53 pathway by degrading the toxin when co-treatment in HepG2 cells. The model of our hypothesis is demonstrated in Fig. 5.

Fig. 5: Our hypothesis were demonstrated in this figure.

Numerous methods for determining the level of Aflatoxin B1 in food and products have been established. As is well known, high performance liquid chromatography (HPLC) using a fluorescent detector is the most widely adopted means of monitoring Aflatoxin B1. However, HPLC has many disadvantages, including its cost, the complexity of operation of the machines involved, and the extensive preparation of samples. Since a simple and efficient technique for the routine monitoring of food such as grains, peanuts and related products is in an urgent demand, we chose immunostrip as the way to detect aflatoxin.

Traditionally, this type of test strip required a special antibody that binds to aflatoxin, called “monoclonal antibody”. However, it would take a lot of time to produce it in the processes of immunization, fusion and cloning, production of ascites, and characterization of the monoclonal antibody. What’s more, not only is the cost to keep an animal hotel high, but also hybridoma is not stable enough to maintain the quality and quantity of monoclonal antibody. Not to mention the vulnerable Van der Waals force with nanoparticle probe, if the samples are not properly treated before tested, the immunostrip will not detect anything. Therefore, to change the situations of these disadvantages, we made lots of improvement on it.

scFv- RFP fusion system

In our project, we designed a fusion protein to replace the traditional monoclonal antibody applied on the immunostrip. The fusion protein that we designed is composed of three domains,scFv, rigid linker, RFP and His-Tag (Fig. 1).

Fig. 1: Using RAPTOR X to simulate the structure and folding of fusion protein. The left side is an anti-aflatoxin scFv, and the right side is an RFP with 6X His-Tag, linked by a rigid protein linker. The simulation result demonstrates that the rigid linker can maintain the distance between two domains, and keeps them from interrupting each other or misfolding.