After building the chassis strain, we began to design experiments to verify whether it could achieve our desired results.
For the Detecting chasis:
Since our chassis strain is E. coli and does not contain tetracycline resistance, we need to verify whether the gene tet(A), tet(R), tet(O) we introduced enables E. coli to survive in water samples containing tetracycline and achieve detection purposes. Firstly, we need to test the tetracycline tolerance of the constructive detection chasis, here is the resluts in the fig.1
From the data, we can clearly see that, the constructive detection chasis obtain a tetracycline tolerance from 0-100μg/μL, and then, detecting chasis can be growth normally in the culture medium with tetracycline.
Meanwhile, we have a collaboration with the NKU-China that we sent our detecting chasis to ask for measuring minimal inhibitory concentration (MIC), and we can also get the tetracycline tolerance of this chasis which similar with our test results.
After determining that our chassis can survive tetracycline, we need to further test its performance and get the following data:
In order to further improve the accuracy of detection, we have exchanged the gene tet(A) and gene gfp. we verified those constructive chasis with the same experiment designs, and then we found that it can detect more lower concentration at 0.00005μg/ml.
The detecting chasis with gene exchanged can detect the lower concentration of tetracycline at 0.00005μg/ml in the water samples, but it need more time to growth and this section is not a stable chasis for each time to detect tetracycline.
Considering the stability of the project, we prefer to use the original tetracycline detection chasis to detect water samples.
For the Degradation chasis:
We successfully constructed four degradation chasis with lignin peroxidase, Manganese peroxidase and laccase. After the verification experiments, we got lots of data about those enzymes performances under the co-culture system in the below:
Through the data comparison above, we can get that the lignin peroxidase perform the better ability to degrade the tetracycline in the culture media and Manganese peroxidase cannot work well alone. So we retain the composite parts that including lignin peroxidase and Manganese peroxidase to degrade tetracycline together in this parts.