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

 
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<h3>★  ALERT! </h3>
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<p>This page is used by the judges to evaluate your team for the <a href="https://2018.igem.org/Judging/Medals">medal criterion</a> or <a href="https://2018.igem.org/Judging/Awards"> award listed below</a>. </p>
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<p> Delete this box in order to be evaluated for this medal criterion and/or award. See more information at <a href="https://2018.igem.org/Judging/Pages_for_Awards"> Instructions for Pages for awards</a>.</p>
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<h1>Improve</h1>
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<p>For teams seeking to improve upon a previous part or project, you should document all of your work on this page. Please remember to include all part measurement and characterization data on the part page on the Registry. Please include a link to your improved part on this page.</p>
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        }
  
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<h3>Gold Medal Criterion #2</h3>
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        #HQ_page th {
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<p><b>Standard Tracks:</b> Create a new part that has a functional improvement upon an existing BioBrick part. The sequences of the new and existing parts must be different. You must perform experiments with both parts to demonstrate this improvement.  Document the experimental characterization on the Part's Main Page on the Registry for both the existing and new parts. Both the new and existing Main Page of each Part’s Registry entry must reference each other. Submit a sample of the new part to the Registry.
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The existing part must NOT be from your 2018 part number range and must be different from the part documented in bronze #4.
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    <link rel="stylesheet" href="https://2018.igem.org/Template:BIT-China/css/common-style?action=raw&amp;ctype=text/css">
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    <link rel="stylesheet" href="https://2018.igem.org/Template:BIT-China/css/achieve-common-style?action=raw&amp;ctype=text/css">
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    <link rel="stylesheet" href="https://2018.igem.org/Template:BIT-China/css/project-common-style?action=raw&amp;ctype=text/css">
  
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<br><br>
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    <style>
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<b>Special Tracks:</b> Improve the function of an existing iGEM project (that your current team did not originally create) and display your achievement on your wiki.</p>
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        .table-head {
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            background-color: #BFBFBF;
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        .table-head table,
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        .table-body td {
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            height: 42px;
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        .PRO-title-4 {
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            font-family: 'kg_second_chances_solidRg';
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            font-size: 16px;
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            color: #131313;
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    </style>
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</head>
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<body>
 +
 +
            <ul id="left-nav">
 +
        <li>
 +
            <a>PROJECT</a>
 +
            <ul>
 +
                <li><a href="https://2018.igem.org/Team:BIT-China/Background">Background</a></li>
 +
                <li><a href="https://2018.igem.org/Team:BIT-China/Description">Description</a></li>
 +
                <li><a href="https://2018.igem.org/Team:BIT-China/Design">Idea & Design</a></li>
 +
            </ul>
 +
        </li>
 +
 +
        <li>
 +
            <a>EXPERIMENTS</a>
 +
            <ul>
 +
                <li><a href="https://2018.igem.org/Team:BIT-China/ExperimentsRegulator">Regulator</a></li>
 +
                <li><a href="https://2018.igem.org/Team:BIT-China/ExperimentsFeedback">Feedback</a></li>
 +
                <li><a href="https://2018.igem.org/Team:BIT-China/ExperimentsOutput">Output</a></li>
 +
                <li><a href="https://2018.igem.org/Team:BIT-China/Results">Results</a></li>
 +
            </ul>
 +
        </li>
 +
 +
        <li>
 +
            <a>MODELING</a>
 +
            <ul>
 +
                <li><a href="https://2018.igem.org/Team:BIT-China/Model">Overview</a></li>
 +
                <li><a href="https://2018.igem.org/Team:BIT-China/FluorescentProbesModel">Fluorescent Probe Model </a></li>
 +
                <li><a href="https://2018.igem.org/Team:BIT-China/H2O2DecompositionModel">H<sub>2</sub>O<sub>2</sub>
 +
                        Decomposition Model</a></li>
 +
 +
                <li><a href="https://2018.igem.org/Team:BIT-China/roGFP2-Orp1MichaelisEquationModel">roGFP2-Orp1
 +
                        Michaelis equation Model</a></li>
 +
            </ul>
 +
        </li>
 +
 +
        <li>
 +
            <a>HUMAN PRACTICES</a>
 +
            <ul>
 +
                <li><a href="https://2018.igem.org/Team:BIT-China/HPOverview">Overview</a></li>
 +
                <li><a href="https://2018.igem.org/Team:BIT-China/Human_Practices">Integrated Human Practices</a></li>
 +
                <li><a href="https://2018.igem.org/Team:BIT-China/Public_Engagement">Education & Public Engagement</a></li>
 +
                <li><a href="https://2018.igem.org/Team:BIT-China/Collaborations">Collaborations</a></li>
 +
            </ul>
 +
        </li>
 +
 +
        <li>
 +
            <a>NOTEBOOK</a>
 +
            <ul>
 +
                <li><a href="https://2018.igem.org/Team:BIT-China/Notebook">Lab Book</a></li>
 +
                <li><a href="https://2018.igem.org/Team:BIT-China/Protocols">Methodology / Protocols</a></li>
 +
                <li><a href="https://2018.igem.org/Team:BIT-China/Equipment">Material & Equipment</a></li>
 +
                <li><a href="https://2018.igem.org/Team:BIT-China/InterLab">Measurement / InterLab</a></li>
 +
                <li><a href="https://2018.igem.org/Team:BIT-China/Safety">Safety</a></li>
 +
            </ul>
 +
        </li>
 +
 +
        <li>
 +
            <a>ACHIEVEMENTS</a>
 +
            <ul>
 +
                <li><a href="https://2018.igem.org/Team:BIT-China/JudgingForm">Judging Form</a></li>
 +
                <li><a href="https://2018.igem.org/Team:BIT-China/Parts">Parts</a></li>
 +
                <li><a href="https://2018.igem.org/Team:BIT-China/Improve">Improve</a></li>
 +
                <li><a href="https://2018.igem.org/Team:BIT-China/Applied_Design">Applied Design</a></li>
 +
                <li><a href="https://2018.igem.org/Team:BIT-China/Demonstrate">Demonstrate</a></li>
 +
            </ul>
 +
        </li>
 +
 +
        <li>
 +
            <a>TEAM</a>
 +
            <ul>
 +
                <li><a href="https://2018.igem.org/Team:BIT-China/Team">Members</a></li>
 +
                <li><a href="https://2018.igem.org/Team:BIT-China/Attributions">Attributions</a></li>
 +
                <li><a href="https://2018.igem.org/Team:BIT-China/Gallery">Gallery</a></li>
 +
            </ul>
 +
        </li>
 +
    </ul>
 +
 +
    <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 -->
 +
 +
    <img style="position:fixed;height:100vh;top:0;right:0;" src="https://static.igem.org/mediawiki/2018/0/09/T--BIT-China--iGEM2018-achievement-bg.png">
 +
 +
    <div id="PRO-content-all" class="content_container" style="margin-top:25vh;width: 50vw;">
 +
        <div id="JUD0" class="cd-section" style="position: relative;margin-top: calc(25vh - 30px);text-align: right;">
 +
            <a class="PT-title-1" style="border-bottom-style: solid;text-decoration: none;color: #131313;">IMPROVE</a>
 +
        </div>
 +
        <div id="PRO1" class="cd-section">
 +
            <div class="PRO-title-2">
 +
                <a style="text-decoration:none;color:#131313;">Introduction</a>
 +
            </div>
 +
 +
            <div class="PRO-content-all">
 +
                <div class="PRO-content">
 +
                    <p class="PRO-content-p">
 +
                        So, we set up this page specially describe the improved Part, <a href="http://parts.igem.org/Part:BBa_K2765043"
 +
                            target="_Blank" style="color:#131313;">BBa_K2765043</a>, which can produce redox-sensitive
 +
                        green fluorescent protein roGFP2-Orp1 and display intracellular ROS content by its fluorescence
 +
                        ratio.
 +
                    </p>
 +
 +
                </div>
 +
            </div>
 +
        </div>
 +
 +
        <div id="PRO2" class="cd-section">
 +
            <div class="PRO-title-2">
 +
                <a style="text-decoration:none;color:#131313;">Source</a>
 +
            </div>
 +
 +
            <div class="PRO-content-all">
 +
 +
                <div class="PRO-content">
 +
                    <p class="PRO-content-p">
 +
                        First, we obtained the gene sequence of roGFP2 from the part: BBa_K2296006: Constitutive
 +
                        Promoter-RBS-roGFP2-Orp1 C82S.
 +
                    </p>
 +
                    <p class="PRO-content-p">
 +
                        Second, we synthesized the codon-optimized roGFP2+linker sequence, obtained the sequence of
 +
                        <i>orp1</i> from the yeast genome and ligated them by OE-PCR, which enhanced the specificity and
 +
                        sensitivity of roGFP2 to H<sub>2</sub>O<sub>2</sub>.
 +
                    </p>
 +
                </div>
 +
            </div>
 +
        </div>
 +
 +
        <div id="PRO3" class="cd-section">
 +
            <div class="PRO-title-2">
 +
                <a style="text-decoration:none;color:#131313;">Codon Optimization</a>
 +
            </div>
 +
 +
            <div class="PRO-content-all">
 +
 +
                <div class="PRO-content">
 +
                    <p class="PRO-content-p">
 +
                        As shown, we mutated the cysteine at position 82 of the Orp1 protein to serine, which made our signal output
 +
                        more responsive and increased the protein expression of roGFP2-Orp1.
 +
                    </p>
 +
                    <figure class="PRO-Fig PRO-margin-toContentP">
 +
                        <img src="https://static.igem.org/mediawiki/2018/9/97/T--BIT-China--achievementimprovefig1.jpg">
 +
                        <figcaption></figcaption>
 +
                    </figure>
 +
 +
                </div>
 +
            </div>
 +
        </div>
 +
 +
        <div id="PRO4" class="cd-section">
 +
            <div class="PRO-title-2">
 +
                <a style="text-decoration:none;color:#131313;">Promoter Optimization</a>
 +
            </div>
 +
 +
            <div class="PRO-content-all">
 +
                <div class="PRO-content">
 +
                    <p class="PRO-content-p">
 +
                        According to this measurement principle, the concentration of roGFP2-Orp1 protein can influence
 +
                        the final signal output strength. So, 4 promoters, FBA1p, TEF2p, TEF1p, ENO2p, with different
 +
                        expression intensity were screened, because we wish this BioBrick Part can output signal with
 +
                        suitable intensity in different situation.
 +
                    </p>
 +
                </div>
 +
            </div>
 +
        </div>
 +
 +
        <div id="PRO5" class="cd-section">
 +
            <div class="PRO-title-2">
 +
                <a style="text-decoration:none;color:#131313;">Modeling Optimization</a>
 +
            </div>
 +
 +
            <div class="PRO-content-all">
 +
                <div class="PRO-content">
 +
                    <p class="PRO-content-p">
 +
                        Beyond these, we also did some dry-lab for this Part. Considering that fluorescence intensity
 +
                        of roGFP2-Orp1 can only characterize the relative level of intracellular ROS qualitatively, but
 +
                        can’t give the absolute content of intracellular ROS quantitatively, we established the model
 +
                        based on Michaelis equation and some assumptions, which can convert fluorescence intensity of
 +
                        roGFP2-Orp1 into the intracellular H<sub>2</sub>O<sub>2</sub> concentration. As a result, the
 +
                        output signal of roGFP2-Orp1 has a more intuitive meaning and higher application value. <a href="https://2018.igem.org/Team:BIT-China/roGFP2-Orp1MichaelisEquationModel"
 +
                            target="_Blank" style="color:#131313;">(click here for more details)</a>
 +
                    </p>
 +
                </div>
 +
            </div>
 +
        </div>
 +
 +
        <div id="PRO6" class="cd-section">
 +
            <div class="PRO-title-2">
 +
                <a style="text-decoration:none;color:#131313;">Characterization and Measurement</a>
 +
            </div>
 +
 +
            <div class="PRO-content-all">
 +
                <div class="PRO-title-3 PRO-margin-Title3Up">
 +
                    <a style="text-decoration:none;color:#131313;">Measure the response of roGFP2-Orp1 to H<sub>2</sub>O<sub>2</sub></a>
 +
                </div>
 +
 +
                <div class="PRO-content">
 +
                    <p class="PRO-content-p">
 +
                        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=I405 / I408) is
 +
                        uesed to the redox degree of roGFP2-Orp1. Therefore, we used different <b>H<sub>2</sub>O<sub>2</sub>
 +
                            concentrations</b> (independent variable) to simulate the accumulation of ROS in cells and
 +
                        the <b>fluorescence ratio</b> (dependent variable) to characterize the redox degree of
 +
                        roGFP2-Orp1, <b>which means that the increase of fluorescence ratio R shows roGFP2-Orp1 is
 +
                            oxidized and the decrease shows reduction.</b>
 +
                    </p>
 +
                    <figure class="PRO-Fig PRO-margin-toContentP">
 +
                        <img src="https://static.igem.org/mediawiki/2018/c/c9/T--BIT-China--achievementimprovefig2.jpg">
 +
                        <figcaption>Figure.2 Experimental Group</figcaption>
 +
                    </figure>
 +
                    <p class="PRO-content-p">
 +
                        As Figure.2 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="PRO-Fig PRO-margin-toContentP">
 +
                        <img src="https://static.igem.org/mediawiki/2018/1/12/T--BIT-China--achievementimprovefig3.jpg">
 +
                        <figcaption>Figure.3 Control Group</figcaption>
 +
                    </figure>
 +
                    <p class="PRO-content-p">
 +
                        As Figure.3 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>
 +
 +
                </div>
 +
 +
                <div class="PRO-title-3 PRO-margin-Title3Up">
 +
                    <a style="text-decoration:none;color:#131313;">Verify redox reversibility of roGFP2-Orp1</a>
 +
                </div>
 +
 +
                <div class="PRO-content">
 +
                    <p class="PRO-content-p">
 +
                        Firstly, we made the cells almost be oxidation state by adding 1mM H<sub>2</sub>O<sub>2</sub>
 +
                        and observed the change of fluorescence ratio R (dependent variable) with time (independent
 +
                        variable).
 +
                    </p>
 +
 +
                    <figure class="PRO-Fig PRO-margin-toContentP">
 +
                        <img src="https://static.igem.org/mediawiki/2018/0/07/T--BIT-China--achievementimprovefig4.jpg">
 +
                        <figcaption>Figure.4 Verify Redox Reversibility of roGFP2-Orp1</figcaption>
 +
                    </figure>
 +
                    <p class="PRO-content-p">
 +
                        As Figure.4 shown, the fluorescence ratio R decreased slightly in the range of 0 to 20 min,
 +
                        because cell itself has the mechanism of scavenging ROS and H<sub>2</sub>O<sub>2</sub> will
 +
                        decompose spontaneously. At the 23 min, we added DTT (strong reducing agent) with the final
 +
                        concentration of 5mM. As a result, the fluorescence ratio R decreased significantly.
 +
                    </p>
 +
                    <p class="PRO-content-p">
 +
                        Therefore, the redox of our roGFP2-Orp1 is reversible.
 +
                    </p>
 +
 +
                </div>
 +
 +
                <div class="PRO-title-3 PRO-margin-Title3Up">
 +
                    <a style="text-decoration:none;color:#131313;">Verify codon optimization of roGFP2-Orp1</a>
 +
                </div>
 +
 +
                <div class="PRO-content">
 +
 +
                    <div class="PRO-title-4 PRO-margin-Title4Up">
 +
                        Determinate the DTT concentration making roGFP2-Orp1 completely reduced.
 +
                    </div>
 +
 +
                    <p class="PRO-content-p">
 +
                        We added three different concentrations of DTT, 0.5mM, 3mM and 5mM. When DTT was added, the
 +
                        roGFP2-Orp1 should be reduced and the fluorescence ratio R should decrease.
 +
                    </p>
 +
 +
                    <figure class="PRO-Fig PRO-margin-toContentP">
 +
                        <img src="https://static.igem.org/mediawiki/2018/e/e7/T--BIT-China--achievementimprovefig5.jpg">
 +
                        <figcaption>Figure.5 Relationship Between R and DTT Concentration <br>(After Codon Optimization)</figcaption>
 +
                    </figure>
 +
 +
                    <figure class="PRO-Fig PRO-margin-toContentP">
 +
                        <img src="https://static.igem.org/mediawiki/2018/d/d2/T--BIT-China--achievementimprovefig6.jpg">
 +
                        <figcaption>Figure.6 Relationship Between R and DTT Concentration <br>(Before Codon Optimization)</figcaption>
 +
                    </figure>
 +
 +
                    <p class="PRO-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.
 +
                    </p>
 +
 +
                    <div class="PRO-title-4 PRO-margin-Title4Up">
 +
                        Characterize roGFP2-Orp1 protein expression before and after codon optimization.
 +
                    </div>
 +
 +
                    <p class="PRO-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.
 +
                    </p>
 +
 +
                    <figure class="PRO-Fig PRO-margin-toContentP">
 +
                        <img src="https://static.igem.org/mediawiki/2018/a/a8/T--BIT-China--achievementimprovefig7.jpg">
 +
                        <figcaption>Figure.7 Contrast After Codon Optimization with Before Codon Optimization on Protein
 +
                            Expression
 +
                            <br>(The Promoter is TEF1p)
 +
                        </figcaption>
 +
                    </figure>
 +
 +
                    <figure class="PRO-Fig PRO-margin-toContentP">
 +
                        <img src="https://static.igem.org/mediawiki/2018/7/7e/T--BIT-China--achievementimprovefig8.jpg">
 +
                        <figcaption>Figure.8 Contrast After Codon Optimization with Before Codon Optimization on Protein
 +
                            Expression
 +
                            <br>(The Promoter is ENO2p)
 +
                        </figcaption>
 +
                    </figure>
 +
                    <figure class="PRO-Fig PRO-margin-toContentP">
 +
                        <img src="https://static.igem.org/mediawiki/2018/f/f1/T--BIT-China--achievementimprovefig9.jpg">
 +
                        <figcaption>Figure.9 Contrast After Codon Optimization with Before Codon Optimization on Protein
 +
                            Expression
 +
                            <br>(The Promoter is FBA1p)
 +
                        </figcaption>
 +
                    </figure>
 +
                    <figure class="PRO-Fig PRO-margin-toContentP">
 +
                        <img src="https://static.igem.org/mediawiki/2018/9/9a/T--BIT-China--achievementimprovefig10.jpg">
 +
                        <figcaption>Figure.10 Contrast After Codon Optimization with Before Codon Optimization on
 +
                            Protein Expression
 +
                            <br>(The Promoter is TEF2p)
 +
                        </figcaption>
 +
                    </figure>
 +
                    <p class="PRO-content-p">
 +
                        As Figure.7~Figure.10 shown, codon optimized roGFP2-Orp1 had higher expression protein than
 +
                        control group without codon optimization and wide-type without fluorescent protein.
 +
                    </p>
 +
                    <p class="PRO-content-p">
 +
                        Therefore, the codon optimization improved the roGFP2-Orp1 protein expression successfully.
 +
                    </p>
 +
 +
                </div>
 +
 +
                <div class="PRO-title-3 PRO-margin-Title3Up">
 +
                    <a style="text-decoration:none;color:#131313;">Reference</a>
 +
                </div>
 +
 +
                <div class="PRO-content">
 +
                    <p class="PRO-content-p">
 +
                        [1] Meyer A J, Dick T P. Fluorescent protein-based redox probes[J]. Antioxidants & redox signaling,
 +
                        2010, 13(5): 621-650.
 +
                    </p>
 +
 +
                    <p class="PRO-content-p">
 +
                        [2] Morgan B, Sobotta M C, Dick T P. Measuring EGSH and H<sub>2</sub>O<sub>2</sub> with
 +
                        roGFP2-based redox probes[J]. Free Radical Biology & Medicine, 2011, 51(11):1943-1951.
 +
                    </p>
 +
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Latest revision as of 00:32, 18 October 2018

Introduction

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

Source

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

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

Codon Optimization

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

Promoter Optimization

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

Modeling Optimization

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

Characterization and Measurement
Measure the response of roGFP2-Orp1 to H2O2

According to literature[1], roGFP2-Orp1 green fluorescent protein shows peak value at 405nm (oxidation peak) and 488nm (reduction peak). Fluorescence ratio R (R=I405 / I408) is uesed to the redox degree of roGFP2-Orp1. Therefore, we used different H2O2 concentrations (independent variable) to simulate the accumulation of ROS in cells and the fluorescence ratio (dependent variable) to characterize the redox degree of roGFP2-Orp1, which means that the increase of fluorescence ratio R shows roGFP2-Orp1 is oxidized and the decrease shows reduction.

Figure.2 Experimental Group

As Figure.2 shown, the fluorescence ratio R of roGFP2-Orp1 increases with the increase of H2O2 concentration. And the fluorescence ratio is basically unchanged when the concentration of H2O2 exceeds 0.8 mM.

Figure.3 Control Group

As Figure.3 shown, the fluorescence ratio of wide-type was not affected by the change of H2O2 concentration and remained unchanged.

Verify redox reversibility of roGFP2-Orp1

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

Figure.4 Verify Redox Reversibility of roGFP2-Orp1

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

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

Verify codon optimization of roGFP2-Orp1
Determinate the DTT concentration making roGFP2-Orp1 completely reduced.

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

Figure.5 Relationship Between R and DTT Concentration
(After Codon Optimization)
Figure.6 Relationship Between R and DTT Concentration
(Before Codon Optimization)

As shown, when the concentration of DTT was 3 mM, the fluorescence ratio R was no longer decreased with the increase of the DTT concentration. At that time, the cells were in reduced state completely.

Characterize roGFP2-Orp1 protein expression before and after codon optimization.

To exclude the influence of individual differences, we made cell be in the same state of redox by adding 5Mm DTT. In that case, roGFP2-Orp1 proteins were in the complete reduction state. Fluorescence intensity can characterize protein expression and we used fluorescence / OD600 to approximate the expression protein of roGFP2-Orp1 in single cell.

Figure.7 Contrast After Codon Optimization with Before Codon Optimization on Protein Expression
(The Promoter is TEF1p)
Figure.8 Contrast After Codon Optimization with Before Codon Optimization on Protein Expression
(The Promoter is ENO2p)
Figure.9 Contrast After Codon Optimization with Before Codon Optimization on Protein Expression
(The Promoter is FBA1p)
Figure.10 Contrast After Codon Optimization with Before Codon Optimization on Protein Expression
(The Promoter is TEF2p)

As Figure.7~Figure.10 shown, codon optimized roGFP2-Orp1 had higher expression protein than control group without codon optimization and wide-type without fluorescent protein.

Therefore, the codon optimization improved the roGFP2-Orp1 protein expression successfully.

Reference

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

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

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