Team:BIT-China/Improve

IMPROVE

Introduction

So, we set up this page specially describe the improved Part, BBa_K2765043, which can produce redox-sensitive green fluorescent protein roGFP2-Orp1 and display intracellular ROS content by its fluorescence ratio.

Source

First, we obtained the gene sequence of roGFP2 from the part: BBa_K2296006: Constitutive Promoter-RBS-roGFP2-Orp1 C82S.

Second, we synthesized the codon-optimized roGFP2+linker sequence, obtained the sequence of orp1 from the yeast genome and ligated them by OE-PCR, which enhanced the specificity and sensitivity of roGFP2 to H₂O₂.

Codon Optimization

As shown, we mutated the cysteine at position 82 of the Orp1 protein to serine, which made our signal output more responsive and increased the protein expression of roGFP2-Orp1.

Promoter Optimization

According to this measurement principle, the concentration of roGFP2-Orp1 protein can influence the final signal output strength. So, 4 promoters, FBA1p, TEF2p, TEF1p, ENO2p, with different expression intensity were screened, because we wish this BioBrick Part can output signal with suitable intensity in different situation.

Modeling Optimization

Beyond these, we also did some dry-lab for this Part. Considering that fluorescence intensity of roGFP2-Orp1 can only characterize the relative level of intracellular ROS qualitatively, but can’t give the absolute content of intracellular ROS quantitatively, we established the model based on Michaelis equation and some assumptions, which can convert fluorescence intensity of roGFP2-Orp1 into the intracellular H₂O₂ concentration. As a result, the output signal of roGFP2-Orp1 has a more intuitive meaning and higher application value. (click here for more details)

Characterization and Measurement

Measure the response of roGFP2-Orp1 to H₂O₂

According to literature^[1], roGFP2-Orp1 green fluorescent protein shows peak value at 405nm (oxidation peak) and 488nm (reduction peak). Fluorescence ratio R (R=I405 / I408) 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.2 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.3 shown, the fluorescence ratio of wide-type was not affected by the change of H₂O₂ concentration and remained unchanged.

Verify redox reversibility of roGFP2-Orp1

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

As Figure.4 shown, the fluorescence ratio R decreased slightly in the range of 0 to 20 min, because cell itself has the mechanism of scavenging ROS and H₂O₂ will decompose spontaneously. At the 23 min, we added DTT (strong reducing agent) with the final concentration of 5mM. As a result, the fluorescence ratio R decreased significantly.

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

Verify codon optimization of roGFP2-Orp1

Determinate the DTT concentration making roGFP2-Orp1 completely reduced.

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

Figure.5 Relationship Between R and DTT Concentration
(After Codon Optimization)

Figure.6 Relationship Between R and DTT Concentration
(Before Codon 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.

Characterize 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.

Figure.7 Contrast After Codon Optimization with Before Codon Optimization on Protein Expression
(The Promoter is TEF1p)

Figure.8 Contrast After Codon Optimization with Before Codon Optimization on Protein Expression
(The Promoter is ENO2p)

Figure.9 Contrast After Codon Optimization with Before Codon Optimization on Protein Expression
(The Promoter is FBA1p)

Figure.10 Contrast After Codon Optimization with Before Codon Optimization on Protein Expression
(The Promoter is TEF2p)

As Figure.7~Figure.10 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] Meyer A J, Dick T P. Fluorescent protein-based redox probes[J]. Antioxidants & redox signaling, 2010, 13(5): 621-650.

[2] Morgan B, Sobotta M C, Dick T P. Measuring EGSH and H₂O₂ with roGFP2-based redox probes[J]. Free Radical Biology & Medicine, 2011, 51(11):1943-1951.