Team:Tianjin/Result/CFPS

Results of CFPS

Overview

We utilized the cell-free system to express the enzymes which had been modified in 22 different sites. Then we used the proteins successfully expressed to degrade PET. Our expected goal was to screen mutations with higher enzyme activities than the wild type PETase. .

Detailed results

Finally, we used PET as the substrate. Excessive PET film was been put into 20 times diluted Enzyme solution (unpurified CFPS system after expression).
After static reaction at 39℃ for 5 days, we detected the characteristic adsorption peak of the product ,MHET, which has no other characteristic adsorption peak except in 260nm.

.

                  Fig.1. Spectral scan for the degradation product MHET

We screened the samples with no other characteristic adsorption peak except in 260nm.

.

Fig.2.Spectral scan for the degradation product MHET(Samples with no other absorption peak except in 260nm)

We had been detected the fluorescence signals at 479 nm (emission wavelength) in wells of 96 well plate at the end of the expressions in the CFPS system.

.

                  Fig.3. Screened plasmids expressions in the CFPS system

According to the OD values and the RFU values of each mutation seperatelly, we got the relative activities of modified enzymes.

.

                                    Fig.4. Relative activities of enzymes.

                                    Fig.5. Relative activities of enzymes(n=3).

Summary

We successfully screened two mutants (R90A &I208V) with higher enzyme activity by site-directed mutation.