Team:CIEI-BJ/Design
In our project, we aimed to establish a yeast system which simultaneously detects and degrades AFT-B1. This system will have high practical value in dealing with aflatoxin in fermentation industry, such as for Pu’er and other agricultural products. Moreover, our system also provides a robust screening platform for identification of novel aflatoxin-detoxing enzymes in further research and development. It includes three modules: activation, detection and degradation.
We realized an inducible system for detection and degradation is inevitable. We thought of yeast two-hybrid system, that would be a suitable choice. In the yeast two-hybrid system, the transcription-activation domain (AD) and DNA binding domain (BD) come close through the interaction of the two fusion proteins with AD and BD respectively, activating the expression of genes downstream of the Gal1 promoter. For detection of AFT-B1, we will use two variable fragments, Sc-Fv1 and Sc-Fv2, of the antibody against AFT-B1, fusing with AD and BD, respectively. Once aflatoxin B1 (AFT-B1) presents, it is going to interact with both peptides and draws AD and BD close together. Then, the downstream genes will be activated.
So, the activation module includes two DNA parts for constitutive expression of the two fusion peptides (antibodies, Sc-Fv1 and Sc-Fv2.). With the presence of AFT-B1, it interacts with both peptides and draws AD and BD together, hence triggers the activities of other two modules.
Figure 1. Parts of the activation module
Then comes our detection module (Figure 2). Normally, the yeast two-hybrid system uses the HIS3 gene in genetically engineered chromosome as a selection marker to indicate whether activation module works. However, our preliminary assay showed a much slow growth of yeast cells when histidine selection medium with presence of AFT-B1 was used. This leads to the inconveniency for our further work. To avoid a pressure from growth, we came with an idea to use fluorescent protein as a reporter, therefore, yeast cells could grow in a medium with histidine, and only lacking tryptophan and leucine.
The detection module contains the inducible promoter and a gene for EYFP (enhanced yellow fluorescent protein). The induction of EYFP expression is activated by AFT-B1, and detected either by microscopic observation or by Western blot analysis of the expressed fluorescent protein (Figure 2).
Figure 2. Our detection system
The degradation module (Figure 3) has the same inducible promoter as for the expression of EYFP in th detection module. But instead, we used it to drive the expression of candidate genes for AFT-B1-degrading enzymes. We cloned at least four genes which were previously reported to degrade AFT-B1, including aflatoxin-detoxifizyme (ADTZ), thioredoxin-MSMEG 5998 fusion protein (F420), and Manganese peroxidase(MNP) after the inducble Gal1 promoter. So the detection and degradation modules are to be simultaneously acitvated by AFT-B1.
Figure 3. Parts of degradation module
In summary, by using our yeast system, when AFT-B1 presents, the activation module starts to function and activates the expression of both enzyme gen and EYFP in the other two modules. The degradation of AFT-B1 by the induced enzyme is simultaneously monitored by the EYFP signal without an inference of yeast growth. In addition, our system can be used for screening novel detoxing enzymes by cloning a cDNA pool from any organism to the degradation module, and evaluated by detecting the EYFP signal during growth with AFT-B1.
Figure 4. The yeast system for detection and degradation of AFT-B1