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| | </div> | | </div> |
| − | <div class="pageHeadline"><span>Improve</span></div> | + | <div class="pageHeadline"><span>Improvement</span></div> |
| | </div> | | </div> |
| | </section> | | </section> |
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| | <div class="side col-lg-3"> | | <div class="side col-lg-3"> |
| − | <nav class="dr-menu">
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| − | <h3 class="ml-5">Improve</h3>
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| − | <ul>
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| − | <li><a class="fa fa-plug" href="#1"> 1. Construction and Verification</a>
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| − | </li>
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| − | <li><a class="fa fa-plug" href="#2"> 2. Characterization of trp production</a>
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| − | <ul>
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| − | <li><a class="fa fa-plug" href="#2.1"> 2.1 Tryptophan determination</a></li>
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| − | <li><a class="fa fa-plug" href="#2.2"> 2.2 Tryptophan yield</a></li>
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| − | <li><a class="fa fa-plug" href="#2.3"> 2.3 Protein modelling</a></li>
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| − | </ul>
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| − | </li>
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| − | <li><a class="fa fa-plug" href="#3"> 3. Characterization of cell proliferation</a></li>
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| − | <li><a class="fa fa-plug" href="#4"> 4. Conclusions</a></li>
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| − | </nav>
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| | </div> | | </div> |
| | <div class="page xjtuText col-lg-8 col-12 justify-content-center"> | | <div class="page xjtuText col-lg-8 col-12 justify-content-center"> |
| − | <h1>Improvement</h1>
| |
| | <div class="row"> | | <div class="row"> |
| − | <div class="col-12 d-flex justify-content-center"> | + | <div class="col-12"> |
| − | <div class="highlightBox mt-5"> | + | < p class=" float-right nav mt-3"> This site has been archieved and moved to<a |
| − | <p>AroG (3-deoxy-7-phosphoheptulonate synthase, <a
| + | href="https://2021.igem.org/Team:XJTU-China/Improve"> |
| − | href="https://www.brenda-enzymes.org/enzyme.php?ecno=2.5.1.54">EC 2.5.1.54</a>,
| + | /Team:XJTU-China/Improve </a>. Sorry for the inconvenience caused.</p> |
| − | <a href="http://parts.igem.org/Part:BBa_K1060000">BBa_K1060000</a>), catalyzes the | + | |
| − | following reaction:<br> | + | |
| − | phosphoenolpyruvate(PEP) + D-erythrose-4-phosphate(E4P) + H<span
| + | |
| − | class="sub">2</span>O = 3-deoxy-D-arabino-hept-2-ulosonate
| + | |
| − | 7-phosphate (DAHP) + phosphate
| + | |
| − | </p>
| + | |
| − | <p>The reaction is a key branching point of the glycolysis and shikimate pathways.
| + | |
| − | Expression of
| + | |
| − | aroG can lead to more substrate into the shikimate pathway, which can improve the
| + | |
| − | yield of
| + | |
| − | downstream products as tryptophan, phenylalanine, tyrosine and benzazole <i>etc.</i>
| + | |
| − | </p>
| + | |
| − | <p>AroG-S211F, in which the serine at 211 was replaced by phenylalanine, has also been
| + | |
| − | reported
| + | |
| − | to be able to increase the production of downstream product in shikimate pathway.
| + | |
| − | However
| + | |
| − | the structural mechanism is unclear. And also it is not sure whether it can increase
| + | |
| − | the
| + | |
| − | production of our tryptophan. So In our project, aroG-S211F was overexpressed,
| + | |
| − | attempted to
| + | |
| − | improve the production of tryptophan. </p>
| + | |
| − | <p>An inducible circuit BBa_K3832008 containing lacUV5-controlled aroG S211F were
| + | |
| − | constructed to
| + | |
| − | characterize and measure the function of AroG-S211F in <i>E.coli</i> DH5alpha(Fig.
| + | |
| − | 1.1).
| + | |
| − | Firstly
| + | |
| − | the yield of tryptophan of mutant aroG and the native one respectively were detected
| + | |
| − | by PDAB
| + | |
| − | method modified by ourselves. Secondly, considering that the over-expression of aroG
| + | |
| − | will
| + | |
| − | significantly reduce the amount of substrate (glucose) entering the glycolysis
| + | |
| − | pathway, in
| + | |
| − | turn affecting the normal cell proliferation, the effect of aroG-S211F on the cell
| + | |
| − | proliferation was also tested by the comparison of growth rate of the wild-type
| + | |
| − | <i>E.coli</i> and
| + | |
| − | the engineered <i>E.coli</i> with aroG-S211F.
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| | </div> | | </div> |
| | </div> | | </div> |
| − | <a class="anchor" id="1"></a>
| |
| − | <h2 class="ml-5 mt-5">1. Construction and Verification of aroG circuit</h2>
| |
| − | <p>aroG S211F gene was chemically synthetized by Genewiz and cloned into pET28a+ backbone by Golden
| |
| − | Gate assembly (BsaI). After transformed into <i>E.coli</i> DH5alpha, plasmid extraction and
| |
| − | electrophoresis, PCR amplification
| |
| − | and sequencing were conducted to confirm its correctness. The results are list in Fig. 1.1 </p>
| |
| − | <div class="imgWrapper centerize">
| |
| − | <img src="https://static.igem.org/mediawiki/2021/c/c3/T--XJTU-China--aroG.png" alt="Fig. 1.1"
| |
| − | width="70%">
| |
| − | <span class="description"><strong>Fig. 1.1 The DNA agarose gel electrophoresis result of
| |
| − | AroG-S211F circuit, plasmid and PCR product. </strong>(a) The length of the circuit is
| |
| − | 2503bp (b) The length of the plasmid is 4738bp. And the two discrete bands are thought as
| |
| − | either open-coiled or super-coiled plasmids (c)The amplicon is expected to be 2526bp.
| |
| − | </span>
| |
| − | </div>
| |
| − | <p>Meanwhile, the quantitatively assay by RT-qPCR was also performed to verified its mRNA level. As
| |
| − | shown in Fig. 1.2, the transcriptional level was increased about two folds after IPTG induction,
| |
| − | indicating the circuit was successfully constructed with functional aroG mutant. The basal
| |
| − | expression of aroG without IPTG induction can be observed due to one copy of native aroG in
| |
| − | <i>E.coli</i> genome.
| |
| − | </p>
| |
| − | <div class="imgWrapper centerize">
| |
| − | <img src="https://static.igem.org/mediawiki/2021/2/21/T--XJTU-China--improvement3.1.png" width="70%"
| |
| − | alt="Fig. 1.2">
| |
| − | <span class="description"><strong>Fig. 1.2 The relative mRNA level of aroG-S211F in DH5alpha
| |
| − | strain with Part:BBa_K3832008 inserted in pET28a+ vector.</strong></span>
| |
| − | </div>
| |
| − | <a class="anchor" id="2"></a>
| |
| − | <h2 class="ml-5 mt-5">2. Characterization the effect of AroG-S211F on tryptophan production</h2>
| |
| − | <a class="anchor" id="2.1"></a>
| |
| − | <h3 class="ml-5">2.1 Tryptophan can be easily determined by modified PDAB chromogenic method</h3>
| |
| − | <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">
| |
| − | Modified PDAB chromogenic 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>
| |
| − | <a class="anchor" id="2.2"></a>
| |
| − | <h3 class="ml-5">2.2 The yield of tryptophan was significantly improved in AroG-S211F strain
| |
| − | compared to native
| |
| − | AroG</h3>
| |
| − | <p>As shown in Fig. 2.1, compared with the <i>E.coli</i> harboring the blank vector and native aroG
| |
| − | gene
| |
| − | (BBa_K1060000), the yield of tryptophan in the engineered <i>E.coli</i> with aroG-S211F induced
| |
| − | by 1 mM
| |
| − | IPTG continuously increased in the 30 h cultivation (green triangle), reaching a maximal
| |
| − | productivity of 160 mg/ml per OD, while the blank controls slowly increased and maintained its
| |
| − | production at about 1200 min, arriving about 80 mg/ml per OD, half of the previous one (circle
| |
| − | and square). It is the same case in absent of IPTG (blue triangle), indicating the low leaky
| |
| − | expression of our circuit. In all, our circuit containing AroG-S211F can efficiently produce
| |
| − | tryptophan with the highest productivity of 160 mg/ml per OD, which can be further improved
| |
| − | under the control of toggle-switch. </p>
| |
| − | <div class="imgWrapper centerize">
| |
| − | <img src="https://static.igem.org/mediawiki/2021/2/2c/T--XJTU-China--improvement3.3.png" width="70%"
| |
| − | alt="Fig. 2.1">
| |
| − | <span class="description"><strong>Fig. 2.1 The tryptophan production curve of the engineering
| |
| − | <i>E.coli</i> with aroG-S211F and <i>E.coli</i> with native aroG.</strong></span>
| |
| − | </div>
| |
| − | <a class="anchor" id="2.3"></a>
| |
| − | <h3 class="ml-5">2.3 The structural mechanisms was elucidated by protein structure modeling</h3>
| |
| − | <p>To explain the concrete mechanisms of the promotion effect by AroG-S211F comparing wild-type
| |
| − | AroG, protein structure modeling is used to analyze the thermodynamics and structure of them.
| |
| − | </p>
| |
| − |
| |
| − | <p>From an energy perspective, our modeling results show that the mutant protein exhibits lower
| |
| − | binding free energy with the catalytic substrate in the presence of the inhibitor (Phe), that
| |
| − | is, it is able to bind more tightly and stably to the substrate, thus improving catalytic
| |
| − | efficiency. On the other hand, structural analysis also reflected that the binding tightness
| |
| − | between the mutated site and the inhibitor was reduced, which weakened its inhibitory effect.
| |
| − | </p>
| |
| − | <div class="imgWrapper centerize">
| |
| − | <img src="https://static.igem.org/mediawiki/2021/7/7e/T--XJTU-China--binding-energy.png" width="70%" alt="binding-energy">
| |
| − | <span class="description"><strong>Fig. 2.2 The binding energy of Phe with either AroG or AroG-S211F</strong></span>
| |
| − | </div>
| |
| − | <p>By the modeling result, the mutation (S211 to F211) in AroG is proposed to eliminate the
| |
| − | allosteric inhibition of phenylalanine, thus increasing the catalytic rate and downstream
| |
| − | product yield.</p>
| |
| − | <div class="row">
| |
| − | <div class="col-12">
| |
| − | <p class="float-right nav mt-3">For more detail of our protein modelling, see also our<a
| |
| − | href="https://2021.igem.org/Team:XJTU-China/protein_model">
| |
| − | Protein Modelling <span class="fa fa-wrench"></span></a></p>
| |
| − | </div>
| |
| − | </div>
| |
| − | <a class="anchor" id="3"></a>
| |
| − | <h2 class="ml-5 mt-5">3. Characterization the effect of aroG-S211F on cell proliferation</h2>
| |
| − | <p>The over-expression of aroG inhibits the glycolysis pathway, thus definitely affecting the cell
| |
| − | growth. So the effect of aroG-S211F on cell proliferation was also detected. The OD600 of
| |
| − | engineered <i>E.coli</i> and blank strain were continuously monitored, as shown in Fig. The
| |
| − | Logistic
| |
| − | equation was used to fit the growth curve, the obvious inhibitory effect of aroG expression on
| |
| − | cell proliferation was observed, especially with IPTG induction. The growth parameters K
| |
| − | (environmental capacity) and r (intrinsic growth rate) of different experimental groups was also
| |
| − | obtained from the fitting Logistic curve, and the parameter r decreased dramatically in
| |
| − | <i>E.coli</i>
| |
| − | with aroG-S211F induced by IPTG, indicating the increased doubling time of the cell.
| |
| − | </p>
| |
| − | <div class="imgWrapper centerize">
| |
| − | <img src="https://static.igem.org/mediawiki/2021/f/f2/T--XJTU-China--POC-Fig2-3.png" width="70%"
| |
| − | alt="Fig. 3.1">
| |
| − | <span class="description"><strong>Fig. 3.1</strong> (a) The population density of <i>E.coli</i>
| |
| − | was
| |
| − | measured at 600nm 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) shows the
| |
| − | growth parameters K (environmental capacity) and r (intrinsic growth rate) of different
| |
| − | experimental groups obtained from the fitting results in (a).</span>
| |
| − | </div>
| |
| − | <a class="anchor" id="4"></a>
| |
| − | <h2 class="ml-5 mt-5">4. Conclusions:</h2>
| |
| − | <p>A lacUV5 controlled-aroG S211F gene circuit was successfully constructed, and the overexpression
| |
| − | of aroG-S211F significantly improved the tryptophan production, with a highest productivity of
| |
| − | 160 mg/ml per OD. Protein structure modeling elucidate that the improvement may attribute to the
| |
| − | elimination of the allosteric inhibition of phenylalanine, thus increasing the catalytic rate
| |
| − | and downstream product yield. However, because of the inhibition on the glycolysis pathway of
| |
| − | aroG, the cell growth was obviously inhibited. The results confirmed our hypothesis that cell
| |
| − | proliferation and tryptophan production should be separated, and it has been designed to be
| |
| − | strictly controlled by toggle-switch circuit, in which cell proliferation (pykA gene
| |
| − | overexpression) and tryptophan production (aroG-S211F overexpression) was constructed in the two
| |
| − | arms of toggle-switch. (View our design on <b><a
| |
| − | href="https://2021.igem.org/Team:XJTU-China/Design">Team:XJTU-China/Design</a></b>).</p>
| |
| | </div> | | </div> |
| | <div class="col-lg-1"></div> | | <div class="col-lg-1"></div> |
