Team:SYSU-Software/Wet Lab

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In order to verify the accuracy of the enzyme aggregation algorithm and the rationality of opto-controllable transcription factor, we designed two experiments to verify the accuracy of modelling and linker design of Phoebus.

We plan to design the linker by Phoebus and construct a GFP-CRY2 expression vector. We would express it in E.coli, and observe whether the green fluorescence can form clusters under 450nm excitation light under fluorescent microscopy. If this step succeeded, the transcription factor-CRY2 expression vector would then be constructed to verify the function of the opto-controllable transcription factor. However, due to the lack of magnification and resolution of the microscope, the pictures of our experimental results are not clear enough, so we are working with SYSU-China team to express the GFP-CRY2 fusion protein in eukaryotes (HEK-293T cells). The experiments are still ongoing and our first construction of the expression vector was not successful (Figure 1,2), so we are trying to reconstruct the expression vector with a different enzyme.

Figure 1,2 | Figure 1 is a colony PCR result, but the band size implies that the fragment size is incorrect. The construct failed, which is confirmed by the sequencing results. In figure 2, lane 1 and 2 is PCR amplification and the lane 3, 4, 5 is vector (pcDNA3.1) linearization.

In parallel, we planned to select two enzymes in a two-step reaction and construct enzyme-CRY2 fusion proteins and designed linker by Phoebus to validate opto-controllable enzyme aggregation. We have found a multi-step reflection in E.coli (Figure 3), and the rate-limiting enzymes for this reaction are aroF and pheA, which means it can be seen as a two-step reaction approximately. After using Phoebus to predict the sequence of the opto-controllable fusion protein, we plan to transform it into E.coli separately and observe if it can increase the local reaction rate through the oligomerization effect of CRY2. Our team is currently constructing the expression vectors. The following is our experimental plan.

1 Use Phoebus to predict the linker sequence of CRY2-aroF and CRY2-pheA.
2 Synthesis of these two genes through Sangon company.
3 PCR to amplify the genes and ligate them to the expression vector pcDNA3.1.
4 Using chemical transformation method to transform Plasmids into E.coli K12 competent cells.
5 Culturing bacteria on medium with Amp.
6 Lysing the bacteria, collecting and purifying the fusion protein.
7 The purified products of aroF-CRY2 and pheA-CRY2 were mixed with the substrate and reacted under 450nm light for a period of time, and then the products were measured for phenylpyruvate and phenylalanine using high performance liquid chromatography.