Team:BIT-China/ExperimentsOutput

OUTPUT

To sense intracellular ROS content and express its changes quickly and intuitively, we constructed roGFP2-orp1 fusion protein and optimized it.

Increase the sensitivity of roGFP2 to hydrogen peroxide

Overview

We made codon optimization of roGFP2 gene sequences and constructed RoGFP2-Orp1 fusion protein to make roGFP2 more sensitive to the REDOX state of cells.

Specific methods:

First, we obtained the gene sequence of roGFP2 from the part:BBa_K2296006: Constitutive Promoter-RBS-roGFP2-Orp1 C82S and codon optimized it for our chassis organisms---yeast, in anticipation of better expression in yeast.

Second, we synthesized the codon-optimized roGFP2+linker sequence, obtained the sequence of Orp1 from the yeast genome and ligated them by OE-PCR. This enhances the specificity of roGFP2 for recognizing hydrogen peroxide and increases its sensitivity to H₂O₂. After that, we completed the 82nd cysteine point mutation (C82S), which made our signal output more responsive.

Fig.2 Orp1 protein,roGFP+Linker and fusion protein roGFP-Orp1 obtained by PCR. 1.size of Orp1 (492bp) 2. size of roGFP+Linker (825bp) 3. size of roGFP2-Orp1 (1317bp)

Select the suitable promoter and verify the function of the gene

Overview:

After optimizing the most important detector component roGFP2-orp1, we need to construct it into yeasts modified in regulator and feedback part. In order to make roGFP2-orp1 in a suitable redox state, we chose several promoters of different intensity and ligated them to roGFP2-orp1 through OE-PCR, then adding hydrogen peroxide to verify its function.

Specific method:

First, we obtain seven promoters of different intensity from Saccharomyces yeast genome through enzyme digestion method. They are: FBA1,TEF1,TEF2,ENO2,PCK1,PDC1 and PGI1. ^[1]

Second, we linked the promoter fragment to the previously constructed fragment roGFP2-orp1by OE-PCR and constructed it on the pESC-Trp plasmid. We screened positive results in the following of digestion，ligation and transformation of the large intestine. Finally, we constructed the fragments into our chassis organisms through yeast transformation.

Result & discussion

The response of roGFP2-Orp1 to H₂O₂

According to literature^[2], roGFP2-Orp1 green fluorescent protein shows peak value at 405nm (oxidation peak) and 488nm (reduction peak). Fluorescence ratio R (R=I408 / I488) is uesed to the redox degree of roGFP2-Orp1. Therefore, we used different H₂O₂ concentrations (independent variable) to simulate the accumulation of ROS in cells and the fluorescence ratio (dependent variable) to characterize the redox degree of roGFP2-Orp1, which means that the increase of fluorescence ratio R shows roGFP2-Orp1 is oxidized and the decrease shows reduction.

As Figure.7 shown, the fluorescence ratio R of roGFP2-Orp1 increases with the increase of H₂O₂ concentration. And the fluorescence ratio is basically unchanged when the concentration of H₂O₂ exceeds 0.8 mM.

As Figure.8 shown, the fluorescence ratio of wide-type was not affected by the change of H₂O₂ concentration and remained unchanged.

Redox reversibility of roGFP2-Orp1

Firstly, we made the cells almost be oxidation state by adding 1 mM H₂O₂ and observed the change of fluorescence ratio R (dependent variable) with time (independent variable).

As Figure.9 shown, the fluorescence ratio R decreased slightly in the range of 0 to 20 mins, because cell itself has the mechanism of scavenging ROS and H₂O₂ will decompose spontaneously. At the 23 mins, we added DTT (strong reducing agent) with the final concentration of 5 mM. As a result, the fluorescence ratio R decreased significantly.

Therefore, the redox of our roGFP2-Orp1 is reversible.

Codon optimization of roGFP2-Orp1

Determinate the DTT concentration making roGFP2-Orp1 completely reduced.

We added three different concentrations of DTT, 0.5 mM, 3 mM and 5 mM. When DTT was added, the roGFP2-Orp1 should be reduced and the fluorescence ratio R should decrease.

Fig.10 Relationship Between R and DTT Concentration (After Codon Optimization)

Fig.11 Relationship Between R and DTT Concentration (Before Odon Optimization)

As shown, when the concentration of DTT was 3 mM, the fluorescence ratio R was no longer decreased with the increase of the DTT concentration. At that time, the cells were in reduced state completely.

roGFP2-Orp1 protein expression before and after codon optimization.

To exclude the influence of individual differences, we made cell be in the same state of redox by adding 5mM DTT. In that case, roGFP2-Orp1 proteins were in the complete reduction state. Fluorescence intensity can characterize protein expression and we used fluorescence / OD₆₀₀ to approximate the expression protein of roGFP2-Orp1 in single cell.

Fig.12 Contrast After Codon Optimization with Before Odon Optimization on Protein Expression
(The Promoter is TEF1)

Fig.13 Contrast After Codon Optimization with Before Odon Optimization on Protein Expression
(The Promoter is ENO2)

Fig.14 Contrast After Codon Optimization with Before Odon Optimization on Protein Expression
(The Promoter is FBA1)

Fig.15 Contrast After Codon Optimization with Before Odon Optimization on Protein Expression
(The Promoter is TEF2)

As Figure.12~Figure.15 shown, codon optimized roGFP2-Orp1 had higher expression protein than control group without codon optimization and wide-type without fluorescent protein.

Therefore, the codon optimization improved the roGFP2-Orp1 protein expression successfully.

Reference

[1] Kewen Wang,Xue Yin,Yu Wang,Yuhua Li,The selection of promoter and its application in the metabolic engineering of saccharomyces cerevisiae[J] Biotechnology bulletin 2018, 34(6):38-47

[2] Meyer A J, Dick T P. Fluorescent protein-based redox probes[J]. Antioxidants & redox signaling, 2010, 13(5): 621-650.