Part:BBa_K2765043:Experience

We sent the DNA sequence of roGFP2+linker to GENEWIZ for synthesis, and got the correct DNA fragment by sequencing analysis.

We refer to the design of PART: BBa_K2296006: Constitutive Promoter-RBS-roGFP2 - Orp1 C82S to obtain the gene sequence of orp1 from the yeast genome by PCR, and mutate the cysteine at position 82 to serine.

Finally, we ligated the roGFP2+linker with the completed mutant orp1 by OE-PCR and verified the DNA sequence of the correct roGFP2-orp1 fusion protein by sequencing.

T--BIT-China--iGEM2018-PartsOutput-Fig.1-roGFP2-orp1.png

At the same time, we introduced the gene sequences before and after optimization into yeast (the promoters are the same and the terminator are CYC1t). We selected four promoters of different intensities, adding enough DTT to the medium which made the proteins all in a reduced state. Then we measured the fluorescence intensity of roGFP2-Orp1 cited by at 488 nm (reduction peak) and the emission is at 515nm. Thus, the magnitude of the fluorescence intensity can reflect the level of protein expression to some extent. The experimental results show that the optimized fusion protein has higher expression than the original design. Among the two pictures of Fig.2(A) & Fig.2 (B) the optimized expression is at least 30 times higher than the original part.

T--BIT-China--iGEM2018-PartsOutput-proteinexperimentresult.png

In addition, we also did some other verification experiments on our optimized part. For the wetlab, we verified its response to ROS. We measured the fluorescence intensity of the roGFP2-Orp1 by adding hydrogen peroxide with different concentration gradients, and obtained the following two figures shows the response speed of roGFP2-Orp1 to hydrogen peroxide. From the Fig.3, we can see that the difference in fluorescence intensity of the roGFP2-Orp1 can distinguish different concentrations of H2O2, which proves that roGFP2-Orp1 has a high sensitivity to hydrogen peroxide response From the Fig.4 we can see that roGFP2-Orp1 responds quickly to hydrogen peroxide, and a more stable output signal is available in 5min shows the results of the sensitivity of the fusion protein to hydrogen peroxide.

T--BIT-China--iGEM2018-PartsOutput-Fig. 3-hydrogenperoxide.png

T--BIT-China--iGEM2018-PartsOutput-Fig.4-respondingspeed.png

In addition, we also conducted a fluorescence reversibility verification experiment. From the literature, we know that the fluorescence produced by roGFP2-Orp1 is reversible. On this basis, we have selected this fusion protein to characterize the intracellular ROS concentration in real-time. Therefore, we have verified the reversibility of protein fluorescence. We first added 10mM H2O2 to expose the protein to an oxidizing environment. After a while, DTT (a strong reducing agent) was added to restore it to a reduced state. It can be seen (Fig. 5) that the ratio of fluorescence intensity returned to the previous level after the addition of DTT, which proves that the response of our roGFP2-Orp1 fusion protein to ROS is reversible.

T--BIT-China--iGEM2018-PartsOutput-Fig.5-Fluorescencereversibilityverification.png

Beyond these，we also did some drylab for this part. The modeling based on Micheal equation can establish the relationship between the fluorescence intensity of the fusion protein and the intracellular hydrogen peroxide concentration, which can realize the quantitative conversion of fluorescence intensity.http://2018.igem.org/Team:BIT-China/roGFP2-Orp1MichaelisEquationModel

This part is an improvement of PART: BBa_K2296006 of 2017 Cornell team.

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