Overview

We created the reporter part of portable pathogen detector using auto-induced Tet-off system.

We successfully demonstrated the effectiveness of trans-activating function of tTA.

We reconstituted the ADE2 gene expression in ADE2 knock out yeast.

Figure 1. Overview of PPD reporter

Designed PPD reporter

We created a reporter as the reporter of the PPD system. So we need a trans-activating system to achieve communication between the sensor and the reporter system. Among the available systems, we chose Tet-off system.

Figure 2. Principle of Tet-off system

At first, we design a test plasmid to determine the feasibility of applying Tet-off system in our system. After AHL inducing, a tetracycline-controlled transactivator (comprising a fusion of the tetracycline repressor TetR with the C-terminal activation domain of herpes simplex virus VP16) will be expressed theoretically. So we constitutively express tTA under the control of CMV enhancer and CMV promoter in pTF6.

In another ORF, we constructed a tetracycline response element followed by CYC1 region (TATA region), which controls the expression ADE2 gene. After ADE2 gene, a CYC1 terminator is used to terminate the transcription of mRNA.

ADE2 protein is phosphoribosylaminoimidazole carboxylase, which is required for adenine biosynthesis. When yeast loses ADE2 gene, the cells are in red color, because of deprivation of adenine, while the wild type yeast is in white. If the plasmid is transformed into ADE2 knock out yeast strain, TetR domain of tTA will bind to the TRE and VP16 will activate the expression of ADE2, which can reconstitute the phenotype of ADE2 knock out strain, turning the yeast from red to white.

Experiments and Results

We constructed the reporter plasmid from cm184 plasmid. We insert ADE2 PCR product into cm184 vector, getting pTF6.

Figure 3. pCM184

Figure 4. pTF6

We tested the function of pTF6 in ADE2 knock out yeast strain. We observed an obvious color change. We used cm184 transformation group as control.

Figure 5. PPD reporter gene ADE2 expression reconstitute the white phenotype of ADE2 KO yeast

The expression of ADE2 changed the color of yeast from red to white thoroughly, which indicated that expressed tTA could activate ADE2 expression. If we extend this system in our pathogen detector machine, the trans-activating efficiency of Tet-off system can be expected.

Discussion

Using the ADE2 KO strain and ADE2 gene we successfully tested that the tet-off system worked as expected. In order to rule out the possibility that the color change was the result of cyc1 mini promoter linkage, we can use two methods. We can add the Doxycycline into the plasmid or delete the tTA region. If the red color sustains, it will be verified that the system works as expected.