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

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         </li>
 
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     </ul>
 
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     <a href="https://2018.igem.org/Team:BIT-China"><img id="imgA" class="imgA-new-pos" src="https://static.igem.org/mediawiki/2018/4/46/T--BIT-China--iGEM2018-A_img.png" /></a>
 
     <a href="https://2018.igem.org/Team:BIT-China"><img id="imgA" class="imgA-new-pos" src="https://static.igem.org/mediawiki/2018/4/46/T--BIT-China--iGEM2018-A_img.png" /></a>
 
     <!-- end -->
 
     <!-- end -->
  
 
     <div class="EXP-white-head"></div>
 
     <div class="EXP-white-head"></div>
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     <div class="EXP-title">
 
     <div class="EXP-title">
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         <a class="EXP-title-1" style="z-index:4;border-bottom-style: solid;text-decoration: none;color: #131313;">REGULATOR</a>
+
         <a class="EXP-title-1" style="z-index:4;border-bottom-style: solid;text-decoration: none;color: #131313;">OUTPUT</a>
 
     </div>
 
     </div>
  
−
     <div id="REG" class="EXP-content-container" style="z-index: 1;margin-top:calc(25vh - 30px);">
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     <div id="EXP" class="EXP-content-container" style="z-index: 1;margin-top:calc(25vh - 30px);">
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         <div id="REG0" class="cd-section">
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         <div id="EXP0" class="cd-section">
 +
        </div>
  
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            <div id="REG-1" class="cd-section">
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        <div id="EXP1" class="cd-section EXP-margin-toTitle">
−
                <div class="EXP-title-2 EXP-margin-Title2Up">
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−
                    <a style="text-decoration: none;color: #131313;">Overview</a>
+
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                </div>
+
  
−
                <div class="EXP-content-all">
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            <div class="EXP-margin-toTitle">
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                    <div class="EXP-content">
+
                <p class="EXP-content-p">To sense intracellular ROS content and express its changes quickly and
−
                        <p class="EXP-content-p">
+
                    intuitively, we constructed roGFP2-orp1 fusion protein and optimized it.
−
                            Previously we showed how we design the whole system, now we are going to tell you how we
+
                </p>
−
                            achieve them steps by steps:
+
−
                        </p>
+
−
                    </div>
+
−
                </div>
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             </div>
 
             </div>
  
−
             <div id="REG-2" class="cd-section ">
+
             <div class="EXP-title-2">
−
                <div class="EXP-title-2 EXP-margin-Title2Up">
+
                <a style="text-decoration: none;color: #131313;">Increase the sensitivity of roGFP2 to hydrogen
−
                    <a style="text-decoration: none;color: #131313;">regulator</a>
+
                    peroxide</a>
−
                </div>
+
            </div>
  
−
                <div class="EXP-content-all">
+
            <div class="EXP-content-all">
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                    <div class="EXP-content">
+
−
                        <p class="EXP-content-p">
+
−
                            To increase the accumulation of endogenous ROS in <i>Saccharomyces cerevisiae</i> and
+
−
                            to improve the tolerance of yeast to high levels of ROS, we optimized our yeast, which will
+
−
                            be shown as follow:
+
−
                        </p>
+
−
                    </div>
+
−
 
+
−
                    <div class="EXP-title-3">
+
−
                        <a style="text-decoration: none;color: #131313;">Increase the accumulation of endogenous ROS</a>
+
−
                    </div>
+
−
 
+
−
                    <div class="EXP-title-4">
+
−
                        <a style="text-decoration: none;color: #131313;">Overview:</a>
+
−
                    </div>
+
  
 +
                <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 literature searching, we selected two genes, <i>yno1</i> and <i>ndi1</i>, as candidates
+
                         We made codon optimization of roGFP2 gene sequences and constructed RoGFP2-Orp1 fusion protein
−
                         for overexpression to increase the accumulation of endogenous ROS. According to our data, both
+
                         to make roGFP2 more sensitive to the REDOX state of cells.
−
                        of these two genes can effectively increase the accumulation of endogenous ROS.
+
 
                     </p>
 
                     </p>
  
−
                    <div class="EXP-title-4">
+
                </div>
−
                        <a style="text-decoration: none;color: #131313;">Overview:</a>
+
−
                    </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">
 
                     <p class="EXP-content-p">
−
                         To determine whether overexpression of <i>ndi1</i> or <i>yno1</i> can increase the endogenous
+
                         <b>First</b>, we obtained the gene sequence of roGFP2 from the part:BBa_K2296006: Constitutive
−
                         ROS accumulation in yeast, we constructed an expression plasmid, based on pESC-Leu, in which
+
                         Promoter-RBS-roGFP2-Orp1 C82S and codon optimized it for our chassis organisms---yeast, in
−
                         the cloned <i>ndi1</i> or <i>yno1</i> is driven by GAL1 promoter. Thus the target gene can be
+
                         anticipation of better expression in yeast.
−
                        induced by galactose and repressed by glucose.
+
 
                     </p>
 
                     </p>
−
 
 
                     <figure class="EXP-Fig EXP-margin-toContentP">
 
                     <figure class="EXP-Fig EXP-margin-toContentP">
−
                         <img src="https://static.igem.org/mediawiki/2018/d/dc/T--BIT-China--ExperimentRegulatorFig1.png">
+
                         <img src="https://static.igem.org/mediawiki/2018/3/3a/T--BIT-China--ExperimentOutputFig1.png">
−
                         <figcaption></figcaption>
+
                         <figcaption><br></figcaption>
 
                     </figure>
 
                     </figure>
−
                    <figure class="EXP-Fig EXP-margin-toContentP">
 
−
                        <img src="https://static.igem.org/mediawiki/2018/f/f1/T--BIT-China--ExperimentRegulatorFig2.png">
 
−
                        <figcaption>Fig. 1,2 Transformed the plasmid into yeast</figcaption>
 
−
                    </figure>
 
−
 
 
                     <p class="EXP-content-p">
 
                     <p class="EXP-content-p">
−
                         After transformed the plasmid into <i>Saccharomyces cerevisiae</i>, we first measured the ROS
+
                         <b>Second</b>, we synthesized the codon-optimized roGFP2+linker sequence, obtained the sequence
−
                         production of yeast cells cultured in non-screening or screening media by Fluorescent
+
                         of Orp1 from the yeast genome and ligated them by OE-PCR. This enhances the specificity of
−
                         microplate reader. We mixed yeast cells with DCFH-DA, which can be oxidized by ROS to become a
+
                         roGFP2 for recognizing hydrogen peroxide and increases its sensitivity to H<sub>2</sub>O<sub>2</sub>.
−
                         strong green fluorescent substance DCF (dichlorofluorescein) that cannot penetrate the cell
+
                         After that, we completed the 82nd cysteine point mutation (C82S), which made our signal output
−
                         membrane.
+
                         more responsive.
 
                     </p>
 
                     </p>
−
 
 
                     <figure class="EXP-Fig EXP-margin-toContentP">
 
                     <figure class="EXP-Fig EXP-margin-toContentP">
−
                         <img src="https://static.igem.org/mediawiki/2018/b/bf/T--BIT-China--ExperimentRegulatorROSaccumulationFig3.png">
+
                         <img src=" https://static.igem.org/mediawiki/2018/2/20/T--BIT-China--ExperimentOutputFig2.png">
−
                         <figcaption></figcaption>
+
                         <figcaption>Fig.2 Orp1 protein,roGFP+Linker and fusion protein roGFP-Orp1 obtained by PCR.
−
                    </figure>
+
                            1.size of Orp1 (492bp) 2. size of roGFP+Linker (825bp) 3. size of roGFP2-Orp1 (1317bp)
−
                    <figure class="EXP-Fig EXP-margin-toContentP">
+
                         </figcaption>
−
                        <img src="https://static.igem.org/mediawiki/2018/c/ce/T--BIT-China--ExperimentRegulatorROSaccumulationFig4.png">
+
−
                         <figcaption>Fig. 3,4 results of ROS accumulation through overexpress <i>yno1</i> or <i>ndi1</i></figcaption>
+
 
                     </figure>
 
                     </figure>
  
−
                    <p class="EXP-content-p">
+
                </div>
−
                        Fluorescent microplate reader analysis showed that overexpression of either <i>ndi1</i> or <i>yno1</i>
+
−
                        may cause significant ROS production as revealed by green fluorescent substance DCF, and the
+
−
                        fluorescence intensity in <i>ndi1</i>-overexpressing cells was a liitle bit higher than tat in
+
−
                        <i>yno1</i>-overexpressing cells.
+
−
                    </p>
+
  
−
                    <p class="EXP-content-p">
+
            </div>
−
                        we performed three times in YPD medium and three times in SD-Leu-deficient medium. A time
+
−
                        period of 0h-48h was selected and detected once every four hours. After excluding some
+
−
                        experimental operational errors, we obtained the results in the figure.
+
−
                    </p>
+
  
 +
            <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">
−
                         It has been shown that external NADH dehydrogenases Ndi1 and Yno1 are involved
+
                         After optimizing the most important detector component roGFP2-orp1, we need to construct it
−
                         in the generation of intracellular oxidative stress, however, the results measured by the
+
                         into yeasts modified in regulator and feedback part. In order to make roGFP2-orp1 in a suitable
−
                         fluorescence microplate reader cannot exclude the interference of dead cells on OD600, we
+
                         redox state, we chose several promoters of different intensity and ligated them to roGFP2-orp1
−
                         therefore examined the ROS accumulation through flow cytometry.
+
                         through OE-PCR, then adding hydrogen peroxide to verify its function.
 
                     </p>
 
                     </p>
  
−
                    <figure class="EXP-Fig EXP-margin-toContentP">
 
−
                        <img src="https://static.igem.org/mediawiki/2018/5/52/T--BIT-China--ExperimentRegulatorFLOWcytometryFig5.png">
 
−
                        <figcaption>Fig.5 Results of ROS accumulation detection through flow cytometry</figcaption>
 
−
                    </figure>
 
  
−
                    <div class="EXP-title-4">
+
                </div>
−
                        <a style="text-decoration: none;color: #131313;">Replace the
+
−
                            promoter of <i>yno1/ndi1</i> in yeast genome</a>
+
−
                    </div>
+
  
 +
                <div class="EXP-title-3 EXP-margin-Title3Up">
 +
                    <a style="text-decoration: none;color: #131313;">Specific method:</a>
 +
                </div>
 +
                <div class="EXP-content">
 
                     <p class="EXP-content-p">
 
                     <p class="EXP-content-p">
−
                         In order to reduce the pressure on our engineered strains from the addition of plasmid and to
+
                         First, we obtain seven promoters of different intensity from Saccharomyces yeast genome through
−
                        prevent plasmid loss, we decided to introduce the gene circuit which increases the level of
+
                         enzyme digestion method. They are: FBA1,TEF1,TEF2,ENO2,PCK1,PDC1 and PGI1. <sup>[1]</sup>
−
                        endogenous ROS into the yeast genome. We chose to replace the <i>ndi1/yno1</i> promoter with
+
−
                         the galactose-inducible promoter GAL1p we used. Because the promoter of the endogenous <i>ndi1/yno1</i>
+
−
                        gene in yeast is a bidirectional promoter, we chose to insert the gal1 promoter upstream of the
+
−
                        <i>ndi1/yno1</i> gene by OE-PCR.
+
 
                     </p>
 
                     </p>
−
 
−
                    <figure class="EXP-Fig EXP-margin-toContentP">
 
−
                        <img src="https://static.igem.org/mediawiki/2018/2/28/T--BIT-China--ExperimentRegulatorreplacepromoterFig8.png">
 
−
                        <figcaption>Fig.6 Replace the promoter of <i>yno1/ndi1</i></figcaption>
 
−
                    </figure>
 
  
 
                     <p class="EXP-content-p">
 
                     <p class="EXP-content-p">
−
                         After successfully replacing the promoter of the yeast endogenous <i>ndi1/yno1</i> gene,
+
                         Second, we linked the promoter fragment to the previously constructed fragment roGFP2-orp1by
−
                         to obtain the overexpression effect, we tested the mRNA quantity. The results are as follows.
+
                         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="https://static.igem.org/mediawiki/2018/d/d3/T--BIT-China--ExperimentRegulatorqPCRFig6.png">
+
                         <img src="https://static.igem.org/mediawiki/2018/7/75/T--BIT-China--ExperimentOutputFig3.png">
−
                         <figcaption>Fig.7 qPCR result in original strain (C, in blue below), and in strain with <i>yno1</i>
+
                         <figcaption></figcaption>
−
                            overexpressed and <i>yca1</i> knockout (YY, in red below).</figcaption>
+
 
                     </figure>
 
                     </figure>
−
 
 
                     <figure class="EXP-Fig EXP-margin-toContentP">
 
                     <figure class="EXP-Fig EXP-margin-toContentP">
−
                         <img src="https://static.igem.org/mediawiki/2018/1/13/T--BIT-China--ExperimentRegulatorqPCRFig7.png">
+
                         <img src="https://static.igem.org/mediawiki/2018/8/89/T--BIT-China--ExperimentOutputFig4.png">
−
                         <figcaption>Fig.8 qPCR result in original strain (C, in blue below), and in strain with <i>yno1</i>
+
                         <figcaption></figcaption>
−
                            overexpressed and <i>ndi1</i> knockout (YN, in red below).</figcaption>
+
                    </figure>
 +
                    <figure class="EXP-Fig EXP-margin-toContentP">
 +
                        <img src="https://static.igem.org/mediawiki/2018/0/0c/T--BIT-China--ExperimentOutputFig5.png">
 +
                        <figcaption></figcaption>
 +
                    </figure>
 +
                    <figure class="EXP-Fig EXP-margin-toContentP">
 +
                        <img src="https://static.igem.org/mediawiki/2018/e/ef/T--BIT-China--ExperimentOutputFig6.png">
 +
                        <figcaption>Fig.3-6 Four promoters obtained by PCR</figcaption>
 
                     </figure>
 
                     </figure>
  
−
                    <p class="EXP-content-p">
+
                </div>
−
                        According to the result, we could find out that strain with <i>yno1</i> overexpressed and <i>yca1</i>
+
−
                        knockout had an obvious increase of mRNAs. However, we didn’t get the same result in ndi1
+
−
                        overexpressed strain. We believed this result showed that the overexpression of <i>ndi1</i> was
+
−
                        failed while the <i>yno1</i> one succeeded.
+
−
                    </p>
+
  
−
                    <div class="EXP-title-3">
 
−
                        <a style="text-decoration: none;color: #131313;">Improve the tolerance to high levels of ROS</a>
 
−
                    </div>
 
  
−
                    <div class="EXP-title-4">
+
            </div>
−
                        <a style="text-decoration: none;color: #131313;">Overview:</a>
+
−
                    </div>
+
  
−
                    <p class="EXP-content-p">
 
−
                        To block the response of yeast to ROS, we knocked out the yeast-derived yca1 gene. Because Yca1
 
−
                        is the only known yeast metacaspase, to demonstrate genetically that Ndi1 functions
 
−
                        independently of Yca1, we overexpressed Ndi1 in yca1 mutant background. Consistently,Ndi1
 
−
                        overexpression exacerbated cell death in the absence of Yca1
 
−
                    </p>
 
  
−
                    <div class="EXP-title-4">
 
−
                        <a style="text-decoration: none;color: #131313;">knocking out gene <i>yca1</i></a>
 
−
                    </div>
 
  
−
                    <figure class="EXP-Fig EXP-margin-toContentP">
 
−
                        <img src="https://static.igem.org/mediawiki/2018/5/5b/T--BIT-China--ExperimentRegulatorknockoutyca1Fig1111.png">
 
−
                        <figcaption>Fig.9 knocking out gene <i>yca1</i></figcaption>
 
−
                    </figure>
 
  
−
                    <figure class="EXP-Fig EXP-margin-toContentP">
 
−
                        <img src="https://static.igem.org/mediawiki/2018/1/11/T--BIT-China--experimentregulatorverifyfig1.png">
 
−
                        <figcaption>Fig.10 Verify whether the gene is successfully knocked out</figcaption>
 
−
                    </figure>
 
  
−
                    <ul class="EXP-content-p">
 
−
                        <li>
 
−
                            1. Left homologous arm of <i>Δyca1</i>（500bp）
 
−
                        </li>
 
−
                        <li>
 
−
                            2: Right homologous arm of <i>Δyca1</i>（500bp）
 
−
                        </li>
 
−
                        <li>
 
−
                            3: wide-type
 
−
                        </li>
 
−
                        <li>
 
−
                            4: wide-type
 
−
                        </li>
 
−
                    </ul>
 
  
−
                    <p class="EXP-content-p">
 
−
                        Based on the result, we determined that there was no mistake in the process of knocking out <i>yca1</i>
 
−
                        gene and that the <i>yca1</i> gene was knocked out indeed.
 
−
                    </p>
 
  
−
                    <div class="EXP-title-4">
 
−
                        <a style="text-decoration: none;color: #131313;">verify whether knocking out gene <i>yca1</i>
 
−
                            can increase yeast tolerance to H<sub>2</sub>O<sub>2</sub></a>
 
−
                    </div>
 
  
 +
            <div class="EXP-title-2">
 +
                <a style="text-decoration: none;color: #131313;">Result & discussion</a>
 +
            </div>
 +
            <div class="EXP-content-all">
 +
 +
                <div class="EXP-title-3 EXP-margin-Title3Up">
 +
                    <a style="text-decoration: none;color: #131313;">The response of roGFP2-Orp1 to H<sub>2</sub>O<sub>2</sub></a>
 +
                </div>
 +
                <div class="EXP-content">
 
                     <p class="EXP-content-p">
 
                     <p class="EXP-content-p">
−
                         If the growth of <i>Δyca1</i> and <i>yno1-Δyca1</i> was better than that of WT and yno1</i>, it
+
                         According to literature<sup>[2]</sup>, roGFP2-Orp1 green fluorescent protein shows peak value
−
                        can be concluded that knocking out <i>yca1</i> gene can improve yeast tolerance to H<sub>2</sub>O<sub>2</sub>.
+
                         at 405nm (oxidation peak) and 488nm (reduction peak). Fluorescence ratio R (R=I408 / I488) is
−
                         We diluted seed liquid to OD600 at 2, which was Saccharomyces Cerevisiae of overexpressing yno1
+
                         uesed to the redox degree of roGFP2-Orp1. Therefore, we used different H<sub>2</sub>O<sub>2</sub>
−
                        gene and lacking the <i>yca1</i> gene <i> (yno1-Δyca1) </i> and strains of overexpressing yno1
+
                         concentrations (independent variable) to simulate the accumulation of ROS in cells and the
−
                         gene <i> (yno1) </i> as control group. And then adjust the concentration of H<sub>2</sub>O<sub>2</sub>
+
                         fluorescence ratio (dependent variable) to characterize the redox degree of roGFP2-Orp1, which
−
                         in the seed
+
                         means that the increase of fluorescence ratio R shows roGFP2-Orp1 is oxidized and the decrease
−
                         liquid to 0 mM, 1 mM, and 2 mM respectively. The yeasts were cultured for 16h. Take samples
+
                         shows reduction.
−
                         every 2-4h. Its OD600 was measured by the ultraviolet spectrophotometer. OD600 reflected a
+
−
                         growth of yeasts which can judge whether <i>yca1</i> knockout can improve yeast tolerance to
+
−
                        H<sub>2</sub>O<sub>2</sub>. <b>The results are shown as follow:</b>
+
 
                     </p>
 
                     </p>
−
 
 
                     <figure class="EXP-Fig EXP-margin-toContentP">
 
                     <figure class="EXP-Fig EXP-margin-toContentP">
−
                         <img src="https://static.igem.org/mediawiki/2018/0/04/T--BIT-China--ExperimentRegulatorFinalRESULTSFig9.png">
+
                         <img src="https://static.igem.org/mediawiki/2018/4/4b/T--BIT-China--ExperimentOutputFig7.png">
−
                         <figcaption>Fig.11 Strain Growth Curve under 0mM H<sub>2</sub>O<sub>2</sub> Stress</figcaption>
+
                         <figcaption>Fig.7 Experimental Group</figcaption>
 
                     </figure>
 
                     </figure>
  
 +
                    <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.
 +
                    </p>
 
                     <figure class="EXP-Fig EXP-margin-toContentP">
 
                     <figure class="EXP-Fig EXP-margin-toContentP">
−
                         <img src="https://static.igem.org/mediawiki/2018/1/14/T--BIT-China--ExperimentRegulatorFinalRESULTSFig10.png">
+
                         <img src="https://static.igem.org/mediawiki/2018/1/11/T--BIT-China--ExperimentOutputFig8.png">
−
                         <figcaption>Fig.12 Strain Growth Curve under 1mM H<sub>2</sub>O<sub>2 Stress</figcaption>
+
                         <figcaption>Fig.8 Control Group</figcaption>
−
                    </figure>
+
−
 
+
−
                    <figure class="EXP-Fig EXP-margin-toContentP">
+
−
                        <img src="https://static.igem.org/mediawiki/2018/c/cb/T--BIT-China--ExperimentRegulatorFinalRESULTSFig11.png">
+
−
                        <figcaption>Fig.13 Strain Growth Curve under 2mM H<sub>2</sub>O<sub>2 Stress</figcaption>
+
−
                    </figure>
+
−
 
+
−
                    <figure class="EXP-Fig EXP-margin-toContentP">
+
−
                        <img src="https://static.igem.org/mediawiki/2018/b/ba/T--BIT-China--ExperimentRegulatorFinalRESULTSFig12.png">
+
−
                        <figcaption>Figure.14 The OD<sub>600</sub> of strains at 12h under 0mM, 1mM, and 2mM H<sub>2</sub>O<sub>2</sub>
+
−
                                Stress stress</figcaption>
+
 
                     </figure>
 
                     </figure>
  
 
                     <p class="EXP-content-p">
 
                     <p class="EXP-content-p">
−
                         As Figure.11~ Figure.14 shown, comparing the growth curves of <i>yno1</i> and <i>yno1</i>-∆<i>yca1</i>
+
                         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>
−
                        under H<sub>2</sub>O<sub>2</sub> stress with 0mM, 1mM and 2mM, we can see that, the growth of strain knocking out <i>yca1</i>
+
                         concentration and remained unchanged.
−
                         is always better than the strain without knocking out <i>yca1</i> after overexpressing <i>yno1</i>.
+
 
                     </p>
 
                     </p>
  
−
                    <p class="EXP-content-p">
 
−
                        It can be concluded that knocking out <i>yca1</i> can improve our engineering strain tolerance
 
−
                        to H<sub>2</sub>O<sub>2</sub>.
 
−
                    </p>
 
  
−
                    <div class="EXP-title-3">
 
−
                        <a style="text-decoration: none;color: #131313;">Conclusions:</a>
 
−
                    </div>
 
  
 +
                </div>
 +
 +
                <div class="EXP-title-3 EXP-margin-Title3Up">
 +
                    <a style="text-decoration: none;color: #131313;">Redox reversibility of roGFP2-Orp1</a>
 +
                </div>
 +
                <div class="EXP-content">
 
                     <p class="EXP-content-p">
 
                     <p class="EXP-content-p">
−
                         Knock out <i>yca1</i> gene cannot increase the yeast tolerance to hydrogen peroxide, or even
+
                         Firstly, we made the cells almost be oxidation state by adding 1 mM H<sub>2</sub>O<sub>2</sub>
−
                         reduce the tolerance.
+
                         and observed the change of fluorescence ratio R (dependent variable) with time (independent
 +
                        variable).
 
                     </p>
 
                     </p>
  
 +
                    <figure class="EXP-Fig EXP-margin-toContentP">
 +
                        <img src="https://static.igem.org/mediawiki/2018/0/05/T--BIT-China--ExperimentOutputFig9.png">
 +
                        <figcaption>Fig.9 Verify Redox Reversibility of roGFP2-Orp1</figcaption>
 +
                    </figure>
 
                     <p class="EXP-content-p">
 
                     <p class="EXP-content-p">
−
                         It was observed that the growth rate and tolerance to hydrogen peroxide <i>Δyca1</i> were lower
+
                         As Figure.9 shown, the fluorescence ratio R decreased slightly in the range of 0 to 20 mins,
−
                         than those of WT which was contrary to our theoretical results.
+
                        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>
−
 
−
                    <div class="EXP-title-4">
 
−
                        <a style="text-decoration: none;color: #131313;">Summary of regulator part</a>
 
−
                    </div>
 
  
 
                     <p class="EXP-content-p">
 
                     <p class="EXP-content-p">
−
                         To summarize briefly, overexpress <i>ndi1/yno1</i> can accumulate ROS successfully, also,
+
                         Therefore, the redox of our roGFP2-Orp1 is reversible.
−
                        knocking out gene <i>yca1</i> can improve the tolerance to high level of ROS content.
+
 
                     </p>
 
                     </p>
 +
 
                 </div>
 
                 </div>
−
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            <p style="font-family:'kg_second_chances_solidRg';font-size:12px;margin: 0;padding: 0 0 3px;margin-top: 6px;">Contact</p>
 
−
            <p style="font-size:15px;margin: 0;padding: 0;margin-top: 4px;">Institute of
 
−
                Biotransformation and synthetic biosystem School of Chemistry and Chemical Engineering.
 
−
            </p>
 
−
            <p style="font-size:15px;margin: 0;padding: 0;margin-top: 4px;">
 
−
                Beijing Institute of Technology
 
−
            </p>
 
−
            <p style="font-size:15px;margin: 0;padding: 0;margin-top: 4px;">
 
−
                100081, Beijing
 
−
            </p>
 
−
            <p style="font-size:15px;margin: 0;padding: 0;margin-top: 4px;">
 
−
                Email:lichun@bit.edu.cn
 
−
            </p>
 
  
−
            <p style="font-size:15px;margin: 0;padding: 0;margin-top: 4px;color:  rgb(161, 161, 161);">
+
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                Copyright © 2018 BIT-China
+
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             </p>
+
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+
  
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 +
            <div class="EXP-title-2">
 +
                <a style="text-decoration: none;color: #131313;">Codon optimization of roGFP2-Orp1</a>
 +
            </div>
 +
            <div class="EXP-content-all">
  
−
    <script src="https://2018.igem.org/Template:BIT-China/js/jquery-min?action=raw&ctype=text/javascript"></script>
+
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−
    <script src="https://2018.igem.org/Template:BIT-China/js/copy-tendina?action=raw&ctype=text/javascript"></script>
+
                    <a style="text-decoration: none;color: #131313;">Determinate the DTT concentration making
−
    <script>
+
                        roGFP2-Orp1 completely reduced.</a>
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            speed: 400,
+
                    <p class="EXP-content-p">
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+
                        We added three different concentrations of DTT, 0.5 mM, 3 mM and 5 mM. When DTT was added, the
−
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Revision as of 23:55, 17 October 2018

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.

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