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| − | <a class="anchor" id="part-improvement"></a>
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| − | <h2 class="ml-3">Part improvement</h2>
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| − | <a class="anchor" id="introduction"></a>
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| − | <h3 class="ml-5">1. Introduction</h3>
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| − | <p>AroG (3-deoxy-7-phosphoheptulonate synthase, EC 2.5.1.54) is a key enzyme in the metabolism of
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| − | aromatic amino acids, catalysis following reaction:<br>
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| − | phosphoenolpyruvate + D-erythrose-4-phosphate+H2O =
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| − | 3-deoxy-D-arabino-hept-2-ulosonate-7-phosphate +phosphate</p>
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| − | <p>Wild aroG functions in the form of tetramer, which can be inhibited by its allosteric inhibitors
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| − | Phenylalanine (Phe). In order to increase the production of downstream products such as
| |
| − | tryptophan, Ser on site 211 of aroG was mutated to Phe to remove the inhibitory effect of Phe.
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| − | The CDS of the mutant (aroG-S211F, Part:BBa_3832000) is our improvement version of
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| − | Part:BBa_K1060000 which encodes wild-type aroG.</p>
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| − | <p>We used protein structure prediction tool (Alphafold2) to predict the structure of the mutant
| |
| − | aroG-S211F, and compared its ability to bind to substrates in the presence of Phe with that of
| |
| − | the wild type. </p>
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| − | <p>In our project, aroG-S211F is used to improve the production of tryptophan. Considering the
| |
| − | over-expression of aroG-S211F could significantly reduce the amount of substrate (glucose)
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| − | entering the glycolysis reaction, in turn affects the normal process of cell proliferation, the
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| − | expression of aroG is designed under strict control by toggle-switch circuit.</p>
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| − | <a class="anchor" id="construction"></a>
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| − | <h3 class="ml-5">2. Construction</h3>
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| − | <p>An inducible circuit (Part:BBa_K3832008) is constructed to characterize and measure the function
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| − | of aroG-S211F (Fig.2.1). LacUV5 promoter is used while LacI is also contained in our circuit as
| |
| − | a repressor. </p>
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| − | <div class="imgWrapper centerize">
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| − | <img src="https://static.igem.org/mediawiki/2021/f/fe/T--XJTU-China--POC-Fig2-1.png"
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| − | alt="Design of the inducible circuit for aroG-S211F" width="60%">
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| − | <span class="description"><strong>Fig. 2.1 Design of the inducible circuit for
| |
| − | aroG-S211F</strong></span>
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| − | </div>
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| − | <p>Both GolgenGate assembly and In-Fusion assembly are used to construct the circuit from basic
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| − | parts and insert into pET28a+ vector (In-Fusion assembly is done with the help of partner team
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| − | NWU-CHINA-A as our collaboration). </p>
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| − | <div class="card card-dark ml-5 mt-5 mb-5" style="width: 90%;">
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| − | <button class="btn btn-default" type="button" data-toggle="collapse" data-target="#GG"
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| − | aria-expanded="false" aria-controls="part">
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| − | Method (for Golden Gate Assembly)
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| − | </button>
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| − | <div class="collapse" id="GG">
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| − | <div class="card card-body card-dark">
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| − | <p>
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| − | <b>Reaction:</b><br>
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| − | Insert (purified PCR product)/ng= Length/bp × 1.08×102
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| − | Vector (purified PCR product)/ng= Length/bp × 2.16×102
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| − | BsaI-HFv2 (20U/ul) = 1 ul
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| − | T4 ligase (1000U/ul) = 1 ul
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| − | T4 ligase buffer (10×) = 2 ul
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| − | ddH20 = 20 ul
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| − | </p>
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| − | <p><b>Condition:</b></p>
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| − | <table class="ml-5 table table-striped table-light" style="width: 90%;">
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| − | <thead>
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| − | <tr>
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| − | <th scope="col">Temperature</th>
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| − | <th scope="col">Time</th>
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| − | <th scope="col">Cycle</th>
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| − | </tr>
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| − | </thead>
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| − | <tbody>
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| − | <tr>
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| − | <th scope="row">37 ℃</th>
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| − | <td>15 min</td>
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| − | <td rowspan="2">10</td>
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| − | </tr>
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| − | <tr>
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| − | <th scope="row">16 ℃</th>
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| − | <td>10 min</td>
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| − | </tr>
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| − | <tr>
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| − | <th scope="row">37 ℃</th>
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| − | <td>10 min</td>
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| − | <td>1</td>
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| − | </tr>
| |
| − | <tr>
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| − | <th scope="row">65 ℃</th>
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| − | <td>10 min</td>
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| − | <td>1</td>
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| − | </tr>
| |
| − | <tr>
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| − | <th scope="row">80 ℃</th>
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| − | <td>10 min</td>
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| − | <td>1</td>
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| − | </tr>
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| − | <tr>
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| − | <th scope="row" colspan="3">Store at 4 ℃</th>
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| − | </tr>
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| − | </tbody>
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| − | </table>
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| − | <p><b>Transformation: </b><br>
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| − | Using Trelief<span class="sub">TM</span> 5α Chemically Competent Cell (Tsingke
| |
| − | Biotechnology Co., Ltd.)
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| − | Add 20 ul assembly product in 50 ul competent cell, ice bath for 30 min.
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| − | Heat shock 42℃ for 45 sec.
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| − | Ice bath for 2 min.
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| − | Recover in 1ml SOC medium, 37℃, 200rpm, for 1 hour.
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| − | Spread to LB plate with 50 ug/ml kanamycin, 37℃ for 10-16 hours.
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| − | </p>
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| − | </div>
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| − | </div>
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| − | </div>
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| − | <div class="imgWrapper centerize">
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| − | <img src="https://static.igem.org/mediawiki/2021/c/c3/T--XJTU-China--aroG.png" alt="AroG-S211F gel">
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| − | <span class="description"><strong>Fig. 2.2 The DNA agarose gel electrophoresis result of
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| − | AroG-S211F circuit, plasmid and PCR product. </strong>(a) The length of the circuit is
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| − | 2503bp (b) The length of the plasmid is 4738bp. And the two discrete bands are thought as
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| − | either open-coiled or super-coiled plasmids (c)The amplicon is expected to be 2526bp.
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| − | </span>
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| − | </div>
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| − | <p>The constructed plasmid of AroG-S211F are then subjected to PCR amplification to verify the
| |
| − | length of circuit, and the expected amplicon is 2526bp in length. Fig.2.2 shows the fragment of
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| − | the inducible circuit (panel a),
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| − | corresponding cloned vector (panel b) and the amplicon (panel c). The result suggests plasmid
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| − | obtained contains an insert with proper length identical to the circuit, thus indicating the
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| − | plasmid to be successfully constructed. The sequencing result (unpublished) also conforms to
| |
| − | this suggestion.</p>
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| − | <a class="anchor" id="measurement"></a>
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| − | <h3 class="ml-5">3. Measurement</h3>
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| − | <a class="anchor" id="RT-qPCR"></a>
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| − | <h4 class="ml-5">3.1 RT-qPCR</h4>
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| − | <p>RT-qPCR is used to detect the transcription of aroG-S211F as first step.</p>
| |
| − | <div class="card card-dark ml-5 mt-5 mb-5" style="width: 90%;">
| |
| − | <button class="btn btn-default" type="button" data-toggle="collapse" data-target="#RT"
| |
| − | aria-expanded="false" aria-controls="part">
| |
| − | Method
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| − | </button>
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| − | <div class="collapse" id="RT">
| |
| − | <div class="card card-body card-dark">
| |
| − | <p><b>Cultivation:</b> Using LB liquid medium, 37℃, 200rpm. <br>
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| − | <b>Inducing condition:</b> 1 mM IPTG, over 8 hours.<br>
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| − | <b>Total RNA extraction:</b> Using RNAsimple Total RNA Kit,DP419 (TIANGEN BIOTECH
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| − | (BEIJING) CO.,LTD.)<br>
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| − | <b>cDNA preparation:</b> Using Evo M-MLV RT Mix (Vazyme Biotech Co.,Ltd); template
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| − | concentration: 50ng RNA/ul; reaction condition: 37℃ 15min, 85℃
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| − | 15sec.<br>
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| − | <b>qPCR:</b> Using ChamQ SYBR qPCR Master Mix (Vazyme Biotech Co.,Ltd).<br>
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| − |
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| − | Relative Normalized Expression data is calculated by using the equation below,<br>
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| − | Relative Expression = 2<span class="sup">-[ΔC<span class="sub">t</span>(T)-ΔC<span
| |
| − | class="sub">t</span>(C)]</span><br>
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| − | where ΔC<span class="sub">t</span>(T) represents the difference between C<span
| |
| − | class="sub">t</span> value of target gene and internal
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| − | standard gene in treatment group; ΔC<span class="sub">t</span>(C) represents the
| |
| − | difference between C<span class="sub">t</span> value
| |
| − | of target gene and internal standard gene in negative control group.
| |
| − | </p>
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| − | </div>
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| − | </div>
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| − | </div>
| |
| − | <p><b>Result:</b><br> As in Fig.3.1, expression of aroG-S211F under induction by IPTG is higher than
| |
| − | that in un-induced
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| − | group and negative control (DH5alpha with blank pET28a+ vector).</p>
| |
| − | <div class="imgWrapper ceterize">
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| − | <img src="https://static.igem.org/mediawiki/2021/2/21/T--XJTU-China--improvement3.1.png" alt="RTaroG"
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| − | width="60%">
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| − | <span class="description"><strong>Fig.3.1 The relative mRNA level of aroG-S211F in DH5alpha
| |
| − | strain with
| |
| − | Part:BBa_K3832008 inserted in pET28a+ vector.</strong></span>
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| − | </div>
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| − | <a class="anchor" id="characterization"></a>
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| − | <h4 class="ml-5">3.2 Characterization</h4>
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| − | <a class="anchor" id="growth-curve"></a>
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| − | <h5 class="ml-5">3.2.1 Growth Curve</h5>
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| − | <p>As is showed in Fig.3.2, by using the Logistic equation to fit the growth curve, the inhibitory
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| − | effect of aroG expression on cell proliferation was verified (as the parameter r decreased in
| |
| − | <i>E.coli</i> with aroG-S211F and induced by IPTG, which represents the reciprocal of the time
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| − | it takes
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| − | for the population to double). <br>
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| − | Cultivation condition: 20 ml LB medium, 37℃, 200rpm; Inoculation dose: 20 ul (0.1%)
| |
| − | </p>
| |
| − | <div class="imgWrapper ceterize">
| |
| − | <img src="https://static.igem.org/mediawiki/2021/f/f2/T--XJTU-China--POC-Fig2-3.png" alt="Fig. 3.2"
| |
| − | width="60%">
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| − | <span class="description"><strong>Fig. 3.2</strong> (a) The population density of <i>E.coli</i>
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| − | was
| |
| − | measured at 600 nm
| |
| − | by colorimetry. The scatter represents the result of the measurement. The Logistic equation
| |
| − | was used to fit the growth curve, and the fitting results were shown in the curve. (b) and
| |
| − | (c) respectively show the growth parameters K (environmental capacity) and r (intrinsic
| |
| − | growth rate) of different experimental groups obtained from the fitting results in
| |
| − | (a).</span>
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| − | </div>
| |
| − | <a class="anchor" id="tryptophan-production"></a>
| |
| − | <h5 class="ml-5">3.2.2 Tryptophan Production</h5>
| |
| − | <p>Fig. 3.3 shows the yield of tryptophan. The engineered <i>E.coli</i> with aroG-S211F induced by 1
| |
| − | mM
| |
| − | IPTG resents an increased production of tryptophan, comparing in absent of IPTG or aroG-S211F
| |
| − | (within blank pET28a+ vector).</p>
| |
| − | <p>Concentration of tryptophan is calculated by standard curve in Fig.3.4. Tryptophan production is
| |
| − | calculated by removing the concentration of blank medium, and normalized by divided by OD<span
| |
| − | class="sub">600</span>
| |
| − | (reflecting cell density).</p>
| |
| − | <div class="imgWrapper ceterize">
| |
| − | <img src="https://static.igem.org/mediawiki/2021/2/2c/T--XJTU-China--improvement3.3.png"
| |
| − | alt="Fig 3.3" width="60%">
| |
| − | <span class="description"><strong>Fig. 3.3 The relationship between tryptophan concentration in
| |
| − | culture
| |
| − | medium and culture time.</strong> The concentration of tryptophan is measured by PDAB
| |
| − | chromogenic
| |
| − | method. </span>
| |
| − | </div>
| |
| − | <div class="card card-dark ml-5 mt-5 mb-5" style="width: 90%;">
| |
| − | <button class="btn btn-default" type="button" data-toggle="collapse"
| |
| − | data-target="#ncharacterization" aria-expanded="false" aria-controls="part">
| |
| − | Method
| |
| − | </button>
| |
| − | <div class="collapse" id="ncharacterization">
| |
| − | <div class="card card-body card-dark">
| |
| − | <ol style="color: white; font-family: 'eras';">
| |
| − | <li>Freeze-thaw bacterial culture medium with suspension cells for over 3 times.
| |
| − | </li>
| |
| − | <li>Add 100 ul medium into 400 ul PDAB (p-dimethylaminobezaldehyde) solution (3
| |
| − | mg/ml in
| |
| − | 9 M solution of sulfuric acid). Then keep at 60℃ for 20 min.</li>
| |
| − | <li>Add 3 ul 0.5% (w/w) solution of sodium nitrite. Then keep at 60℃ for
| |
| − | 15min.
| |
| − | </li>
| |
| − | <li>Measure absorption under 590 nm wavelength (OD<span class="sub">590</span>).
| |
| − | </li>
| |
| − | </ol>
| |
| − | </div>
| |
| − | </div>
| |
| − | </div>
| |
| − | <p>The same method should be used to determine the OD<span class="sub">590</span> of a tryptophan
| |
| − | standard solution at a
| |
| − | known concentration to obtain a standard curve. Standard curve measured and used in our
| |
| − | experiment is as Fig.3.4.</p>
| |
| − | <div class="imgWrapper ceterize">
| |
| − | <img src="https://static.igem.org/mediawiki/2021/a/ab/T--XJTU-China--improvement3.4.png"
| |
| − | alt="Fig 3.4" width="60%">
| |
| − | <span class="description"><strong>Fig.3.4 Standard curve of measuring tryptophan tryptophan by
| |
| − | PDAB method.</strong></span>
| |
| − | </div>
| |
| | </div> | | </div> |
| | <div class="col-lg-1"></div> | | <div class="col-lg-1"></div> |
