Team:BIT-China/ExperimentsOutput
To sense intracellular ROS content and express its changes quickly and intuitively, we constructed roGFP2-orp1 fusion protein and optimized it.
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.
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.
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.
First, we obtain four promoters of different intensity from Saccharomyces cerevisiae genome through enzyme digestion method. They are: FBA1p,TEF1p,ENO2p and TEF2p. [1]
Second, we linked the promoter fragment to the previously constructed fragment roGFP2-Orp1 by 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.
Third, we designed and performed a series of functional verification experiments.
——Preparation of gradient hydrogen peroxide solution
Step one: Add 10 uL 30% hydrogen peroxide solution to the PBS solution to a total volume of 10mL and mix it with oscillating solution. The hydrogen peroxide solution of 10mM/L was obtained. Step two: Dilute the 10mM/L hydrogen peroxide solution which has been prepared according to the concentration gradient below.
| Hydrogen peroxide gradient concentration | 10mM/L H2O2 | PBS |
|---|
| 0mM | 0uL | 10mL |
| 0.1mM | 100uL | 9.9 mL |
| 0.2mM | 200uL | 9.8 mL |
| 0.3mM | 300uL | 9.7 mL |
| 0.4mM | 400uL | 9.6 mL |
| 0.5mM | 500uL | 9.5 mL |
| 0.6mM | 600uL | 9.4 mL |
| 0.7 mM | 700uL | 9.3 mL |
| 0.9 mM | 900uL | 9.1 mL |
| 1.2 mM | 1200uL | 8.8 mL |
| 1.4 mM | 1400uL | 8.6 mL |
| 1.7 mM | 1700uL | 8.3 mL |
——Preparation of gradient hydrogen peroxide solution
1. CEN.PK2-1c-pESC-TRP, CEN.PK2-1c-pESC-TEF1-roGFP2-Orp1-CYC1t, CEN.PK2-1c-pESC-TEF1-roGFP2-Orp1(CORNELL)-CYC1t, were inoculated into Synthetic Dropout Tryptophan Liquid Medium (SD-TRP) and cultured overnight for 32-36 hours.
2. Add 2mL cultures into the centrifuge tube, centrifuged 1min, and the supernatant was discarded.
3. Use 1mL diluted hydrogen peroxide solution to suspend the precipitate.
4. Take 100 uL of bacterial solution into a 96-well plate for fluorescence measurement. The emission wavelength was 515 nm and the excitation wavelengths were: 405 nm and 488 nm
——Results and discussion
The fluorescence value of codon-optimized CEN.PK2-1c-pESC-TEF1-roGFP2-Orp1-CYC1t protein at 488 nm (reduction peak) was significantly higher than that of the control CEN.PK2-1c-pESC-TRP (transferred into the same open-labeled empty plasmid) and CEN.PK2-1c-pESC-TEF1-roGFP2-Orp1(CORNELL)-CYC1t (no codon-optimized gene). The results show that the roGFP2-Orp1 codon optimization is feasible and effective.
The ratio of the 488 nm (reduction peak) fluorescence value of the roGFP2-orp1 protein to the 405 nm (oxidation peak) fluorescence value varies with the hydrogen peroxide concentration. Control: As the concentration of hydrogen peroxide changes, the fluorescence ratio of the CEN.PK2-1c-pESC-TRP reduction peak to the oxidation peak fluctuates between 5-6. Experimental group: The concentration of hydrogen peroxide was significantly reduced between 0mM and 0.6mM. When the concentration of hydrogen peroxide increases, the fluorescence value at 488 nm (reduction peak) decreases, and the fluorescence value at 405 nm (oxidation peak) increases, and the ratio decreases. The results indicate that the roGFP2-Orp1 protein responds to hydrogen peroxide.
To sense intracellular ROS content and express its changes quickly and intuitively, we constructed roGFP2-orp1 fusion protein and optimized it.
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.
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.
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.
First, we obtain seven promoters of different intensity from Saccharomyces cerevisiae genome through enzyme digestion method.They are:FBA1,TEF1,TEF2,ENO2,PCK1,PDC1 and PGI1. [2]
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.
Third, we designed and performed a series of functional verification experiments.
——Preparation of gradient hydrogen peroxide solution
| Hydrogen peroxide gradient concentration | 10mM/L H2O2 | PBS |
|---|
| 0mM | 0uL | 10mL |
| 0.1mM | 100uL | 9.9 mL |
| 0.2mM | 200uL | 9.8 mL |
| 0.3mM | 300uL | 9.7 mL |
| 0.4mM | 400uL | 9.6 mL |
| 0.5mM | 500uL | 9.5 mL |
| 0.6mM | 600uL | 9.4 mL |
| 0.7 mM | 700uL | 9.3 mL |
| 0.9 mM | 900uL | 9.1 mL |
| 1.2 mM | 1200uL | 8.8 mL |
| 1.4 mM | 1400uL | 8.6 mL |
| 1.7 mM | 1700uL | 8.3 mL |
——Culture and detection of bacterial liquid
1. CEN.PK2-1c-pESC-TRP, CEN.PK2-1c-pESC-TEF1-roGFP2-Orp1-CYC1t, CEN.PK2-1c-pESC-TEF1-roGFP2-Orp1(CORNELL)-CYC1t were inoculated into Synthetic Dropout Tryptophan Liquid Medium (SD-TRP) and cultured overnight for 32-36 hours.
2. Add 2mL bacteria solution into the centrifuge tube, centrifuged 1min, and the supernatant was discarded.
3. Use 1mL diluted hydrogen peroxide solution to suspend the precipitate.
4. Take 100 uL of bacterial solution into a 96-well plate for fluorescence measurement. The emission wavelength was 510 nm and the excitation wavelengths were: 405 nm and 488 nm
——Results and discussion
The fluorescence value of codon-optimized CEN.PK2-1c-pESC-TEF1-roGFP2-Orp1-CYC1t protein at 488 nm (reduction peak) was significantly higher than that of the control CEN.PK2-1c-pESC-TRP (transferred into the same open-labeled empty plasmid) and CEN.PK2-1c-pESC-TEF1-roGFP2-Orp1(CORNELL)-CYC1t (no codon-optimized gene). The results show that the roGFP2-Orp1 codon optimization is feasible and effective.
The ratio of the 488 nm (reduction peak) fluorescence value of the roGFP2-orp1 protein to the 405 nm (oxidation peak) fluorescence value varies with the hydrogen peroxide concentration. Control: As the concentration of hydrogen peroxide changes, the fluorescence ratio of the CEN.PK2-1c-pESC-TRP reduction peak to the oxidation peak fluctuates between 5-6. Experimental group: The concentration of hydrogen peroxide was significantly reduced between 0mM and 0.6mM. When the concentration of hydrogen peroxide increases, the fluorescence value at 488 nm (reduction peak) decreases, and the fluorescence value at 405 nm (oxidation peak) increases, and the ratio decreases. The results indicate that the roGFP2-Orp1 protein responds to hydrogen peroxide.
[1][2] 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
