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

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                     </p>
 
                     </p>
 
                     <figure class="EXP-Fig EXP-margin-toContentP">
 
                     <figure class="EXP-Fig EXP-margin-toContentP">
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                         <img src="">
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                         <img src="https://static.igem.org/mediawiki/2018/3/3a/T--BIT-China--ExperimentOutputFig1.png">
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                         <figcaption></figcaption>
+
                         <figcaption><br></figcaption>
 
                     </figure>
 
                     </figure>
 
                     <p class="EXP-content-p">
 
                     <p class="EXP-content-p">
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                         more responsive.
 
                         more responsive.
 
                     </p>
 
                     </p>
 +
                    <figure class="EXP-Fig EXP-margin-toContentP">
 +
                        <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.
 +
                                1.size of Orp1 (492bp) 2. size of roGFP+Linker (825bp) 3. size of roGFP2-Orp1 (1317bp)
 +
                                </figcaption>
 +
                    </figure>
  
 
                 </div>
 
                 </div>
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             <div class="EXP-title-2">
 
             <div class="EXP-title-2">
−
                 <a style="text-decoration: none;color: #131313;">Select the suitable promoter and verify the function
+
                 <a style="text-decoration: none;color: #131313;">Result & discussion</a>
−
                    of the gene</a>
+
 
             </div>
 
             </div>
 
             <div class="EXP-content-all">
 
             <div class="EXP-content-all">
  
 
                 <div class="EXP-title-3 EXP-margin-Title3Up">
 
                 <div class="EXP-title-3 EXP-margin-Title3Up">
−
                     <a style="text-decoration: none;color: #131313;">Overview</a>
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                     <a style="text-decoration: none;color: #131313;">The response of roGFP2-Orp1 to H<sub>2</sub>O<sub>2</sub></a>
 
                 </div>
 
                 </div>
 
                 <div class="EXP-content">
 
                 <div class="EXP-content">
 
                     <p class="EXP-content-p">
 
                     <p class="EXP-content-p">
−
                         After optimizing the most important detector component roGFP2-orp1, we need to construct it
+
                         According to literature<sup>[1]</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.  
−
                        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>
+
−
 
+
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                </div>
+
−
 
+
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                <div class="EXP-title-3 EXP-margin-Title3Up">
+
−
                    <a style="text-decoration: none;color: #131313;">Specific methods:</a>
+
−
                </div>
+
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                <div class="EXP-content">
+
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                    <p class="EXP-content-p">
+
−
                        <b>First</b>, we obtain four promoters of different intensity from <i>Saccharomyces cerevisiae</i> genome
+
−
                        through enzyme digestion method. They are: FBA1p,TEF1p,ENO2p and TEF2p. <sup>[1]</sup>
+
 
                     </p>
 
                     </p>
 
                     <figure class="EXP-Fig EXP-margin-toContentP">
 
                     <figure class="EXP-Fig EXP-margin-toContentP">
−
                         <img src="">
+
                         <img src="https://static.igem.org/mediawiki/2018/4/4b/T--BIT-China--ExperimentOutputFig7.png">
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                         <figcaption></figcaption>
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                         <figcaption>Fig.7 Experimental Group</figcaption>
 
                     </figure>
 
                     </figure>
 +
 
                     <p class="EXP-content-p">
 
                     <p class="EXP-content-p">
−
                         <b>Second</b>, we linked the promoter fragment to the previously constructed fragment
+
                         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.
−
                        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.
+
 
                     </p>
 
                     </p>
 
                     <figure class="EXP-Fig EXP-margin-toContentP">
 
                     <figure class="EXP-Fig EXP-margin-toContentP">
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                        <img src="">
+
                            <img src="https://static.igem.org/mediawiki/2018/1/11/T--BIT-China--ExperimentOutputFig8.png">
−
                        <figcaption></figcaption>
+
                            <figcaption>Fig.8 Control Group</figcaption>
 
                     </figure>
 
                     </figure>
 +
                   
 
                     <p class="EXP-content-p">
 
                     <p class="EXP-content-p">
−
                         <b>Third</b>, we designed and performed a series of functional verification experiments.
+
                         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 class="EXP-content-p">
+
−
                        <b>——Preparation of gradient hydrogen peroxide solution</b>
+
−
                    </p>
+
−
                    <p class="EXP-content-p">
+
−
                        <b>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.
+
−
                        </b>
+
 
                     </p>
 
                     </p>
  
  
−
                    <div class="table-head">
 
−
                        <table>
 
−
                            <colgroup>
 
−
                                <col style="width: 30%;" />
 
−
                                <col style="width: 30%;" />
 
−
                                <col style="width: 40%;" />
 
−
                            </colgroup>
 
−
                            <thead>
 
−
                                <tr style="font-size: 20px;">
 
−
                                    <th>Hydrogen peroxide gradient concentration</th>
 
−
                                    <th>10mM/L H<sub>2</sub>O<sub>2</sub></th>
 
−
                                    <th>PBS</th>
 
−
                                </tr>
 
−
                            </thead>
 
−
                        </table>
 
−
                    </div>
 
−
                    <div class="table-body">
 
−
                        <table>
 
−
                            <colgroup>
 
−
                                <col style="width: 30%;" />
 
−
                                <col style="width: 30%;" />
 
−
                                <col style="width: 40%;" />
 
−
                            </colgroup>
 
−
                            <tbody style="font-size: 16px;">
 
−
                                <tr>
 
−
                                    <td>0mM</td>
 
−
                                    <td>0uL</td>
 
−
                                    <td>10mL</td>
 
−
                                </tr>
 
  
 +
                </div>
  
−
                                <tr>
+
                <div class="EXP-title-3 EXP-margin-Title3Up">
−
                                    <td>0.1mM</td>
+
                    <a style="text-decoration: none;color: #131313;">Redox reversibility of roGFP2-Orp1</a>
−
                                    <td>100uL</td>
+
                </div>
−
                                    <td>9.9 mL</td>
+
                <div class="EXP-content">
−
                                </tr>
+
−
 
+
−
                                <tr>
+
−
                                    <td>0.2mM</td>
+
−
                                    <td>200uL</td>
+
−
                                    <td>9.8 mL</td>
+
−
                                </tr>
+
−
 
+
−
                                <tr>
+
−
                                    <td>0.3mM</td>
+
−
                                    <td>300uL</td>
+
−
                                    <td>9.7 mL</td>
+
−
                                </tr>
+
−
 
+
−
                                <tr>
+
−
                                    <td>0.4mM</td>
+
−
                                    <td>400uL</td>
+
−
                                    <td>9.6 mL</td>
+
−
                                </tr>
+
−
 
+
−
                                <tr>
+
−
                                    <td>0.5mM</td>
+
−
                                    <td>500uL</td>
+
−
                                    <td>9.5 mL</td>
+
−
                                </tr>
+
−
 
+
−
                                <tr>
+
−
                                    <td>0.6mM</td>
+
−
                                    <td>600uL</td>
+
−
                                    <td>9.4 mL</td>
+
−
                                </tr>
+
−
 
+
−
                                <tr>
+
−
                                    <td>0.7 mM</td>
+
−
                                    <td>700uL</td>
+
−
                                    <td>9.3 mL</td>
+
−
                                </tr>
+
−
 
+
−
                                <tr>
+
−
                                    <td>0.9 mM</td>
+
−
                                    <td>900uL</td>
+
−
                                    <td>9.1 mL</td>
+
−
                                </tr>
+
−
 
+
−
                                <tr>
+
−
                                    <td>1.2 mM</td>
+
−
                                    <td>1200uL</td>
+
−
                                    <td>8.8 mL</td>
+
−
                                </tr>
+
−
 
+
−
                                <tr>
+
−
                                    <td>1.4 mM</td>
+
−
                                    <td>1400uL</td>
+
−
                                    <td>8.6 mL</td>
+
−
                                </tr>
+
−
 
+
−
                                <tr>
+
−
                                    <td>1.7 mM</td>
+
−
                                    <td>1700uL</td>
+
−
                                    <td>8.3 mL</td>
+
−
                                </tr>
+
−
 
+
−
                            </tbody>
+
−
                        </table>
+
−
                    </div>
+
−
 
+
 
                     <p class="EXP-content-p">
 
                     <p class="EXP-content-p">
−
                         <b>——Preparation of gradient hydrogen peroxide solution</b>
+
                         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 class="EXP-content-p">
+
−
                        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.
+
−
                    </p>
+
−
                    <p class="EXP-content-p">
+
−
                        2. Add 2mL cultures into the centrifuge tube, centrifuged 1min, and the supernatant
+
−
                        was discarded.
+
−
                    </p>
+
−
                    <p class="EXP-content-p">
+
−
                        3. Use 1mL diluted hydrogen peroxide solution to suspend the precipitate.
+
−
                    </p>
+
−
                    <p class="EXP-content-p">
+
−
                        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
+
−
                    </p>
+
−
 
+
−
 
+
−
                    <p class="EXP-content-p">
+
−
                        <b>——Results and discussion</b>
+
 
                     </p>
 
                     </p>
  
 
                     <figure class="EXP-Fig EXP-margin-toContentP">
 
                     <figure class="EXP-Fig EXP-margin-toContentP">
−
                         <img src="">
+
                         <img src="https://static.igem.org/mediawiki/2018/0/05/T--BIT-China--ExperimentOutputFig9.png">
−
                         <figcaption></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.
−
                         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.
+
 
                     </p>
 
                     </p>
−
                    <figure class="EXP-Fig EXP-margin-toContentP">
+
           
−
                        <img src="">
+
−
                        <figcaption></figcaption>
+
−
                    </figure>
+
−
 
+
 
                     <p class="EXP-content-p">
 
                     <p class="EXP-content-p">
−
                         The ratio of the 488 nm (reduction peak) fluorescence value of the roGFP2-orp1 protein to the
+
                         Therefore, the redox of our roGFP2-Orp1 is reversible.
−
                        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.
+
 
                     </p>
 
                     </p>
  
−
                    <p class="EXP-content-p">
+
                </div>
−
                        To sense intracellular ROS content and express its changes quickly and intuitively, we
+
   
−
                        constructed roGFP2-orp1 fusion protein and optimized it.
+
−
                    </p>
+
  
 +
            </div>
  
−
                </div>
 
  
−
            </div>
 
  
 
             <div class="EXP-title-2">
 
             <div class="EXP-title-2">
−
                 <a style="text-decoration: none;color: #131313;">Increase the sensitivity of roGFP2 to hydrogen
+
                 <a style="text-decoration: none;color: #131313;">Codon optimization of roGFP2-Orp1</a>
−
                    peroxide</a>
+
 
             </div>
 
             </div>
 
             <div class="EXP-content-all">
 
             <div class="EXP-content-all">
  
 
                 <div class="EXP-title-3 EXP-margin-Title3Up">
 
                 <div class="EXP-title-3 EXP-margin-Title3Up">
−
                     <a style="text-decoration: none;color: #131313;">Overview</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 made codon optimization of roGFP2 gene sequences and constructed roGFP2-Orp1 fusion protein
+
                         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.
−
                        to make roGFP2 more sensitive to the REDOX state of cells.
+
−
                    </p>
+
−
 
+
−
                </div>
+
−
 
+
−
                <div class="EXP-title-3 EXP-margin-Title3Up">
+
−
                    <a style="text-decoration: none;color: #131313;">Specific methods:</a>
+
−
                </div>
+
−
                <div class="EXP-content">
+
−
                    <p class="EXP-content-p">
+
−
                        <b>First</b>, 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.
+
 
                     </p>
 
                     </p>
 
                     <figure class="EXP-Fig EXP-margin-toContentP">
 
                     <figure class="EXP-Fig EXP-margin-toContentP">
−
                        <img src="">
+
                            <img src="https://static.igem.org/mediawiki/2018/2/23/T--BIT-China--ExperimentOutputFig10.png">
−
                        <figcaption></figcaption>
+
                            <figcaption>Fig.10 Relationship Between R and DTT Concentration (After Codon Optimization)</figcaption>
 
                     </figure>
 
                     </figure>
−
                    <p class="EXP-content-p">
 
−
                        <b>Second</b>, 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 H<sub>2</sub>O<sub>2</sub>.
 
−
                        After that, we completed the 82nd cysteine point mutation (C82S), which made our signal output
 
−
                        more responsive.
 
−
                    </p>
 
 
                     <figure class="EXP-Fig EXP-margin-toContentP">
 
                     <figure class="EXP-Fig EXP-margin-toContentP">
−
                        <img src="">
+
                            <img src="https://static.igem.org/mediawiki/2018/0/0b/T--BIT-China--ExperimentOutputFig11.png">
−
                        <figcaption>Fig.1 roGFP2+Lingker and fusion protein roGFP-Orp1 obtained by PCR.</figcaption>
+
                            <figcaption>Fig.11 Relationship Between R and DTT Concentration (Before Odon Optimization)</figcaption>
 
                     </figure>
 
                     </figure>
−
                </div>
 
−
 
−
            </div>
 
−
 
−
            <div class="EXP-title-2">
 
−
                <a style="text-decoration: none;color: #131313;">Select the suitable promoter and verify the function
 
−
                    of the gene</a>
 
−
            </div>
 
−
            <div class="EXP-content-all">
 
−
 
−
                <div class="EXP-title-3 EXP-margin-Title3Up">
 
−
                    <a style="text-decoration: none;color: #131313;">Overview</a>
 
−
                </div>
 
−
                <div class="EXP-content">
 
 
                     <p class="EXP-content-p">
 
                     <p class="EXP-content-p">
−
                        After optimizing the most important detector component roGFP2-orp1, we need to construct it
+
                            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.
−
                        into yeasts modified in regulator and feedback part. In order to make roGFP2-Orp1 in a suitable
+
                         </p>
−
                        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>
+
  
 
                 </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;">Specific methods:</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">
−
                         <b>First</b>, we obtain seven promoters of different intensity from <i>Saccharomyces cerevisiae</i> genome
+
                         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.
−
                        through enzyme digestion method.They are:FBA1,TEF1,TEF2,ENO2,PCK1,PDC1 and PGI1. <sup>[2]</sup>
+
−
                    </p>
+
−
 
+
−
                    <p class="EXP-content-p">
+
−
                        <b>Second</b>, 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">
−
                         <img src="">
+
                         <img src="https://static.igem.org/mediawiki/2018/4/40/T--BIT-China--ExperimentOutputFig12.png">
−
                         <figcaption>Fig.2-5 Four promoters obtained by PCR</figcaption>
+
                         <figcaption>Fig.12 Contrast After Codon Optimization with Before Odon Optimization on Protein Expression
 +
                                <br>(The Promoter is TEF1)
 +
                                </figcaption>
 
                     </figure>
 
                     </figure>
 +
                    <figure class="EXP-Fig EXP-margin-toContentP">
 +
                            <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
 +
                                    <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>
  
−
                    <p class="EXP-content-p">
+
                        <figure class="EXP-Fig EXP-margin-toContentP">
−
                        <b>Third</b>, we designed and performed a series of functional verification experiments.
+
                             <img src="https://static.igem.org/mediawiki/2018/8/8d/T--BIT-China--ExperimentOutputFig15.png">
−
                    </p>
+
                            <figcaption>Fig.15 Contrast After Codon Optimization with Before Odon Optimization on Protein Expression
−
                    <p class="EXP-content-p">
+
                                     <br>(The Promoter is TEF2)                                                                   
−
                        <b>——Preparation of gradient hydrogen peroxide solution</b>
+
                                     </figcaption>
−
                    </p>
+
                         </figure>
−
 
+
−
                    <div class="table-head">
+
−
                        <table>
+
−
                             <colgroup>
+
−
                                <col style="width: 30%;" />
+
−
                                <col style="width: 30%;" />
+
−
                                <col style="width: 40%;" />
+
−
                            </colgroup>
+
−
                            <thead>
+
−
                                <tr style="font-size: 20px;">
+
−
                                    <th>Hydrogen peroxide gradient concentration</th>
+
−
                                    <th>10mM/L H<sub>2</sub>O<sub>2</sub></th>
+
−
                                    <th>PBS</th>
+
−
                                </tr>
+
−
                            </thead>
+
−
                        </table>
+
−
                    </div>
+
−
                    <div class="table-body">
+
−
                        <table>
+
−
                            <colgroup>
+
−
                                <col style="width: 30%;" />
+
−
                                <col style="width: 30%;" />
+
−
                                <col style="width: 40%;" />
+
−
                            </colgroup>
+
−
                            <tbody style="font-size: 16px;">
+
−
                                <tr>
+
−
                                    <td>0mM</td>
+
−
                                    <td>0uL</td>
+
−
                                    <td>10mL</td>
+
−
                                </tr>
+
−
 
+
−
 
+
−
                                <tr>
+
−
                                    <td>0.1mM</td>
+
−
                                    <td>100uL</td>
+
−
                                    <td>9.9 mL</td>
+
−
                                </tr>
+
−
 
+
−
                                <tr>
+
−
                                    <td>0.2mM</td>
+
−
                                    <td>200uL</td>
+
−
                                    <td>9.8 mL</td>
+
−
                                </tr>
+
−
 
+
−
                                <tr>
+
−
                                    <td>0.3mM</td>
+
−
                                    <td>300uL</td>
+
−
                                    <td>9.7 mL</td>
+
−
                                </tr>
+
−
 
+
−
                                <tr>
+
−
                                     <td>0.4mM</td>
+
−
                                     <td>400uL</td>
+
−
                                    <td>9.6 mL</td>
+
−
                                </tr>
+
−
 
+
−
                                <tr>
+
−
                                    <td>0.5mM</td>
+
−
                                    <td>500uL</td>
+
−
                                    <td>9.5 mL</td>
+
−
                                </tr>
+
−
 
+
−
                                <tr>
+
−
                                    <td>0.6mM</td>
+
−
                                    <td>600uL</td>
+
−
                                    <td>9.4 mL</td>
+
−
                                </tr>
+
−
 
+
−
                                <tr>
+
−
                                    <td>0.7 mM</td>
+
−
                                    <td>700uL</td>
+
−
                                    <td>9.3 mL</td>
+
−
                                </tr>
+
−
 
+
−
                                <tr>
+
−
                                    <td>0.9 mM</td>
+
−
                                    <td>900uL</td>
+
−
                                    <td>9.1 mL</td>
+
−
                                </tr>
+
−
 
+
−
                                <tr>
+
−
                                    <td>1.2 mM</td>
+
−
                                    <td>1200uL</td>
+
−
                                    <td>8.8 mL</td>
+
−
                                </tr>
+
−
 
+
−
                                <tr>
+
−
                                    <td>1.4 mM</td>
+
−
                                    <td>1400uL</td>
+
−
                                    <td>8.6 mL</td>
+
−
                                </tr>
+
−
 
+
−
                                <tr>
+
−
                                    <td>1.7 mM</td>
+
−
                                    <td>1700uL</td>
+
−
                                    <td>8.3 mL</td>
+
−
                                </tr>
+
−
 
+
−
                            </tbody>
+
−
                         </table>
+
−
                    </div>
+
  
 
                     <p class="EXP-content-p">
 
                     <p class="EXP-content-p">
−
                         <b>——Culture and detection of bacterial liquid</b>
+
                         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.
 
                     </p>
 
                     </p>
 
                     <p class="EXP-content-p">
 
                     <p class="EXP-content-p">
−
                         1. CEN.PK2-1c-pESC-TRP, CEN.PK2-1c-pESC-TEF1-roGFP2-Orp1-CYC1t, CEN.PK2-1c-pESC-TEF1-roGFP2-Orp1(CORNELL)-CYC1t  were
+
                         Therefore, the codon optimization improved the roGFP2-Orp1 protein expression successfully.
−
                        inoculated into Synthetic Dropout Tryptophan Liquid Medium (SD-TRP) and cultured overnight for
+
−
                        32-36 hours.
+
 
                     </p>
 
                     </p>
−
                    <p class="EXP-content-p">
 
−
                        2. Add 2mL bacteria solution into the centrifuge tube, centrifuged 1min, and the supernatant
 
−
                        was discarded.
 
−
                    </p>
 
−
 
−
                    <p class="EXP-content-p">
 
−
                        3. Use 1mL diluted hydrogen peroxide solution to suspend the precipitate.
 
−
                    </p>
 
−
                    <p class="EXP-content-p">
 
−
                        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
 
−
                    </p>
 
−
                    <p class="EXP-content-p">
 
−
                        <b>——Results and discussion</b>
 
−
                    </p>
 
−
                    <figure class="EXP-Fig EXP-margin-toContentP">
 
−
                        <img src="">
 
−
                        <figcaption></figcaption>
 
−
                    </figure>
 
−
                    <p class="EXP-content-p">
 
−
                        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.
 
−
                    </p>
 
−
                    <figure class="EXP-Fig EXP-margin-toContentP">
 
−
                        <img src="">
 
−
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                        The ratio of the 488 nm (reduction peak) fluorescence value of the roGFP2-orp1 protein to the
 
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                        405 nm (oxidation peak) fluorescence value varies with the hydrogen peroxide concentration.
 
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                        Control: As the concentration of hydrogen peroxide changes, the fluorescence ratio of the
 
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                        CEN.PK2-1c-pESC-TRP reduction peak to the oxidation peak fluctuates between 5-6. Experimental group:
 
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                        The concentration of hydrogen peroxide was significantly reduced between 0mM and 0.6mM. When
 
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                        the concentration of hydrogen peroxide increases, the fluorescence value at 488 nm (reduction
 
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                        peak) decreases, and the fluorescence value at 405 nm (oxidation peak) increases, and the ratio
 
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                        decreases. The results indicate that the roGFP2-Orp1 protein responds to hydrogen peroxide.
 
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                <a style="text-decoration: none;color: #131313;">Sum of gene circuits design</a>
 
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                <a style="text-decoration: none;color: #131313;">Reference</a>
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                        [1][2] Kewen Wang,Xue Yin,Yu Wang,Yuhua Li,The selection of promoter and its application in the
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                        metabolic engineering of <i>saccharomyces cerevisiae</i>[J] Biotechnology bulletin 2018, 34(6):38-47
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Revision as of 22:52, 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)
Result & discussion
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=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.

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