Difference between revisions of "Team:BIT-China/ExperimentsOutput"

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              <ul id="left-nav">
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    <ul id="left-nav">
 
         <li>
 
         <li>
 
             <a>PROJECT</a>
 
             <a>PROJECT</a>
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                         <img src=" https://static.igem.org/mediawiki/2018/2/20/T--BIT-China--ExperimentOutputFig2.png">
 
                         <img src=" https://static.igem.org/mediawiki/2018/2/20/T--BIT-China--ExperimentOutputFig2.png">
 
                         <figcaption>Fig.2 Orp1 protein,roGFP+Linker and fusion protein roGFP-Orp1 obtained by PCR.
 
                         <figcaption>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)
+
                            1.size of Orp1 (492bp) 2. size of roGFP+Linker (825bp) 3. size of roGFP2-Orp1 (1317bp)
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                                </figcaption>
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                        </figcaption>
 
                     </figure>
 
                     </figure>
  
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             <div class="EXP-title-2">
 
             <div class="EXP-title-2">
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                 <a style="text-decoration: none;color: #131313;">Select the suitable promoter and verify the function of the gene</a>
+
                 <a style="text-decoration: none;color: #131313;">Select the suitable promoter and verify the function
 +
                    of the gene</a>
 
             </div>
 
             </div>
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             <div class="EXP-content-all">
 
             <div class="EXP-content-all">
  
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                 <div class="EXP-content">
 
                 <div class="EXP-content">
 
                     <p class="EXP-content-p">
 
                     <p class="EXP-content-p">
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                         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.  
+
                         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.
 
                     </p>
 
                     </p>
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                 </div>
 
                 </div>
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                 <div class="EXP-content">
 
                 <div class="EXP-content">
 
                     <p class="EXP-content-p">
 
                     <p class="EXP-content-p">
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                         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. <sup>[1]</sup>
+
                         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. <sup>[1]</sup>
 
                     </p>
 
                     </p>
  
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                  <p class="EXP-content-p">
+
                    <p class="EXP-content-p">
−
                         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.
+
                         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.
 
                     </p>
 
                     </p>
 
                     <figure class="EXP-Fig EXP-margin-toContentP">
 
                     <figure class="EXP-Fig EXP-margin-toContentP">
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                 </div>
 
                 </div>
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             </div>
 
             </div>
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                 <div class="EXP-content">
 
                 <div class="EXP-content">
 
                     <p class="EXP-content-p">
 
                     <p class="EXP-content-p">
−
                         According to literature<sup>[2]</sup>, 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 H<sub>2</sub>O<sub>2</sub> 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.  
+
                         According to literature<sup>[2]</sup>, 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 H<sub>2</sub>O<sub>2</sub>
 +
                        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.
 
                     </p>
 
                     </p>
 
                     <figure class="EXP-Fig EXP-margin-toContentP">
 
                     <figure class="EXP-Fig EXP-margin-toContentP">
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                     <p class="EXP-content-p">
 
                     <p class="EXP-content-p">
−
                         As Figure.7 shown, the fluorescence ratio R of roGFP2-Orp1 increases with the increase of H<sub>2</sub>O<sub>2</sub> concentration. And the fluorescence ratio is basically unchanged when the concentration of H<sub>2</sub>O<sub>2</sub> exceeds 0.8 mM.
+
                         As Figure.7 shown, the fluorescence ratio R of roGFP2-Orp1 increases with the increase of H<sub>2</sub>O<sub>2</sub>
 +
                        concentration. And the fluorescence ratio is basically unchanged when the concentration of H<sub>2</sub>O<sub>2</sub>
 +
                        exceeds 0.8 mM.
 
                     </p>
 
                     </p>
 
                     <figure class="EXP-Fig EXP-margin-toContentP">
 
                     <figure class="EXP-Fig EXP-margin-toContentP">
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                            <img src="https://static.igem.org/mediawiki/2018/1/11/T--BIT-China--ExperimentOutputFig8.png">
+
                        <img src="https://static.igem.org/mediawiki/2018/1/11/T--BIT-China--ExperimentOutputFig8.png">
−
                            <figcaption>Fig.8 Control Group</figcaption>
+
                        <figcaption>Fig.8 Control Group</figcaption>
 
                     </figure>
 
                     </figure>
−
                   
+
 
 
                     <p class="EXP-content-p">
 
                     <p class="EXP-content-p">
−
                         As Figure.8 shown, the fluorescence ratio of wide-type was not affected by the change of H<sub>2</sub>O<sub>2</sub> concentration and remained unchanged.
+
                         As Figure.8 shown, the fluorescence ratio of wide-type was not affected by the change of H<sub>2</sub>O<sub>2</sub>
 +
                        concentration and remained unchanged.
 
                     </p>
 
                     </p>
  
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                 <div class="EXP-content">
 
                 <div class="EXP-content">
 
                     <p class="EXP-content-p">
 
                     <p class="EXP-content-p">
−
                         Firstly, we made the cells almost be oxidation state by adding 1 mM H<sub>2</sub>O<sub>2</sub> and observed the change of fluorescence ratio R (dependent variable) with time (independent variable).
+
                         Firstly, we made the cells almost be oxidation state by adding 1 mM H<sub>2</sub>O<sub>2</sub>
 +
                        and observed the change of fluorescence ratio R (dependent variable) with time (independent
 +
                        variable).
 
                     </p>
 
                     </p>
  
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                         <figcaption>Fig.9 Verify Redox Reversibility of roGFP2-Orp1</figcaption>
 
                         <figcaption>Fig.9 Verify Redox Reversibility of roGFP2-Orp1</figcaption>
 
                     </figure>
 
                     </figure>
−
                  <p class="EXP-content-p">
+
                    <p class="EXP-content-p">
−
                         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 H<sub>2</sub>O<sub>2</sub> 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.
+
                         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 H<sub>2</sub>O<sub>2</sub> 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.
 
                     </p>
 
                     </p>
−
           
+
 
 
                     <p class="EXP-content-p">
 
                     <p class="EXP-content-p">
 
                         Therefore, the redox of our roGFP2-Orp1 is reversible.
 
                         Therefore, the redox of our roGFP2-Orp1 is reversible.
 
                     </p>
 
                     </p>
−
 
 
                 </div>
 
                 </div>
−
   
 
−
 
 
             </div>
 
             </div>
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             <div class="EXP-title-2">
 
             <div class="EXP-title-2">
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                 <div class="EXP-title-3 EXP-margin-Title3Up">
 
                 <div class="EXP-title-3 EXP-margin-Title3Up">
−
                     <a style="text-decoration: none;color: #131313;">Determinate the DTT concentration making roGFP2-Orp1 completely reduced.</a>
+
                     <a style="text-decoration: none;color: #131313;">Determinate the DTT concentration making
 +
                        roGFP2-Orp1 completely reduced.</a>
 
                 </div>
 
                 </div>
 
                 <div class="EXP-content">
 
                 <div class="EXP-content">
 
                     <p class="EXP-content-p">
 
                     <p class="EXP-content-p">
−
                         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.
+
                         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.
 
                     </p>
 
                     </p>
 
                     <figure class="EXP-Fig EXP-margin-toContentP">
 
                     <figure class="EXP-Fig EXP-margin-toContentP">
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                            <img src="https://static.igem.org/mediawiki/2018/2/23/T--BIT-China--ExperimentOutputFig10.png">
+
                        <img src="https://static.igem.org/mediawiki/2018/2/23/T--BIT-China--ExperimentOutputFig10.png">
−
                            <figcaption>Fig.10 Relationship Between R and DTT Concentration (After Codon Optimization)</figcaption>
+
                        <figcaption>Fig.10 Relationship Between R and DTT Concentration (After Codon Optimization)</figcaption>
 
                     </figure>
 
                     </figure>
 
                     <figure class="EXP-Fig EXP-margin-toContentP">
 
                     <figure class="EXP-Fig EXP-margin-toContentP">
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                            <img src="https://static.igem.org/mediawiki/2018/0/0b/T--BIT-China--ExperimentOutputFig11.png">
+
                        <img src="https://static.igem.org/mediawiki/2018/0/0b/T--BIT-China--ExperimentOutputFig11.png">
−
                            <figcaption>Fig.11 Relationship Between R and DTT Concentration (Before Odon Optimization)</figcaption>
+
                        <figcaption>Fig.11 Relationship Between R and DTT Concentration (Before Odon Optimization)</figcaption>
 
                     </figure>
 
                     </figure>
 
                     <p class="EXP-content-p">
 
                     <p class="EXP-content-p">
−
                            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.
+
                        As shown, when the concentration of DTT was 3 mM, the fluorescence ratio R was no longer
−
                        </p>
+
                        decreased with the increase of the DTT concentration. At that time, the cells were in reduced
 +
                        state completely.
 +
                    </p>
  
 
                 </div>
 
                 </div>
  
 
                 <div class="EXP-title-3 EXP-margin-Title3Up">
 
                 <div class="EXP-title-3 EXP-margin-Title3Up">
−
                     <a style="text-decoration: none;color: #131313;">roGFP2-Orp1 protein expression before and after codon optimization.</a>
+
                     <a style="text-decoration: none;color: #131313;">roGFP2-Orp1 protein expression before and after
 +
                        codon optimization.</a>
 
                 </div>
 
                 </div>
 
                 <div class="EXP-content">
 
                 <div class="EXP-content">
 
                     <p class="EXP-content-p">
 
                     <p class="EXP-content-p">
−
                         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 / OD<sub>600</sub> to approximate the expression protein of roGFP2-Orp1 in single cell.
+
                         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 / OD<sub>600</sub>
 +
                        to approximate the expression protein of roGFP2-Orp1 in single cell.
 
                     </p>
 
                     </p>
 
                     <figure class="EXP-Fig EXP-margin-toContentP">
 
                     <figure class="EXP-Fig EXP-margin-toContentP">
 
                         <img src="https://static.igem.org/mediawiki/2018/4/40/T--BIT-China--ExperimentOutputFig12.png">
 
                         <img src="https://static.igem.org/mediawiki/2018/4/40/T--BIT-China--ExperimentOutputFig12.png">
−
                         <figcaption>Fig.12 Contrast After Codon Optimization with Before Odon Optimization on Protein Expression
+
                         <figcaption>Fig.12 Contrast After Codon Optimization with Before Odon Optimization on Protein
−
                                <br>(The Promoter is TEF1)
+
                            Expression
−
                                </figcaption>
+
                            <br>(The Promoter is TEF1)
 +
                        </figcaption>
 
                     </figure>
 
                     </figure>
 
                     <figure class="EXP-Fig EXP-margin-toContentP">
 
                     <figure class="EXP-Fig EXP-margin-toContentP">
−
                            <img src="https://static.igem.org/mediawiki/2018/5/5b/T--BIT-China--ExperimentOutputFig13.png">
+
                        <img src="https://static.igem.org/mediawiki/2018/5/5b/T--BIT-China--ExperimentOutputFig13.png">
−
                            <figcaption> Fig.13 Contrast After Codon Optimization with Before Odon Optimization on Protein Expression
+
                        <figcaption> Fig.13 Contrast After Codon Optimization with Before Odon Optimization on Protein
−
                                    <br>(The Promoter is ENO2)
+
                             Expression
−
                                   
+
                             <br>(The Promoter is ENO2)
−
                                    </figcaption>
+
−
                        </figure>
+
−
                        <figure class="EXP-Fig EXP-margin-toContentP">
+
−
                             <img src="https://static.igem.org/mediawiki/2018/8/89/T--BIT-China--ExperimentOutputFig14.png">
+
−
                             <figcaption>Fig.14 Contrast After Codon Optimization with Before Odon Optimization on Protein Expression
+
−
                                    <br>(The Promoter is FBA1)                                  
+
−
                                    </figcaption>
+
−
                        </figure>
+
  
−
                         <figure class="EXP-Fig EXP-margin-toContentP">
+
                         </figcaption>
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                             <img src="https://static.igem.org/mediawiki/2018/8/8d/T--BIT-China--ExperimentOutputFig15.png">
+
                    </figure>
−
                            <figcaption>Fig.15 Contrast After Codon Optimization with Before Odon Optimization on Protein Expression
+
                    <figure class="EXP-Fig EXP-margin-toContentP">
−
                                    <br>(The Promoter is TEF2)                                                                    
+
                        <img src="https://static.igem.org/mediawiki/2018/8/89/T--BIT-China--ExperimentOutputFig14.png">
−
                                    </figcaption>
+
                        <figcaption>Fig.14 Contrast After Codon Optimization with Before Odon Optimization on Protein
−
                        </figure>
+
                             Expression
 +
                            <br>(The Promoter is FBA1)
 +
                        </figcaption>
 +
                    </figure>
 +
 
 +
                    <figure class="EXP-Fig EXP-margin-toContentP">
 +
                        <img src="https://static.igem.org/mediawiki/2018/8/8d/T--BIT-China--ExperimentOutputFig15.png">
 +
                        <figcaption>Fig.15 Contrast After Codon Optimization with Before Odon Optimization on Protein
 +
                            Expression
 +
                            <br>(The Promoter is TEF2)
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                    <a style="text-decoration: none;color: #131313;">Reference</a>
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                <a style="text-decoration: none;color: #131313;">Reference</a>
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                         [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
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                         [1] Kewen Wang,Xue Yin,Yu Wang,Yuhua Li,The selection of promoter and its application in the
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                        metabolic engineering of saccharomyces cerevisiae[J] Biotechnology bulletin 2018, 34(6):38-47
 
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                         [2] Meyer A J, Dick T P. Fluorescent protein-based redox probes[J]. Antioxidants & redox signaling, 2010, 13(5): 621-650.
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                         [2] Meyer A J, Dick T P. Fluorescent protein-based redox probes[J]. Antioxidants & redox
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                        signaling, 2010, 13(5): 621-650.
 
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Revision as of 23:45, 17 October 2018

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.

Fig.3-6 Four promoters obtained by PCR
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.

Fig.7 Experimental Group

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.

Fig.8 Control Group

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).

Fig.9 Verify Redox Reversibility of roGFP2-Orp1

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.

Contact

Institute of Biotransformation and synthetic biosystem School of Chemistry and Chemical Engineering.

Beijing Institute of Technology

100081, Beijing

Email:lichun@bit.edu.cn

Copyright © 2018 BIT-China