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@Meng-na-mei
Meng-na-mei authored Oct 5, 2023
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Expand Up @@ -246,12 +246,13 @@ <h2>Construction of hmaS mutant library</h2>
was 100%.</p>
<div class="figure-container" style="width: 60%;margin: 0 auto;">
<img src="../../images/图片1.png" alt="">
<p>gY9s-HmaSmut-Bio177 mutant library plates</p>
<p>gY9s-HmaS<sup>mut</sup>-Bio177 mutant library plates</p>
</div>

<p><b>Figure 1.</b> A mutant library with 10⁵ clones was constructed by using a
low-fidelity DNA polymerase, determining the concentrations
of 0.02 mM Mn2+ and 0.5 mM Mg2+and the optimized Golden Gate assembly procedure.</p>
of 0.02 mM Mn<sup>2+</sup> and 0.5 mM Mg<sup>2+</sup>and the optimized Golden Gate
assembly procedure.</p>
</div>
<div id="slide1-chapter2">
<h2>Construction and Test of a Dual-Module Screening Platform</h2>
Expand All @@ -274,7 +275,8 @@ <h2>Construction and Test of a Dual-Module Screening Platform</h2>
condition.</p>
<div class="figure-container" style="width: 60%;margin: 0 auto;">
<img src="../../images/图片2.png" alt="">
<p>0.01 mg/L 5-FC 0.05 mg/L 5-FC 0.1 mg/L 5-FC </p>
<p>0.01 mg/L 5-FC</p>
<p>0.05 mg/L 5-FC 0.1 mg/L 5-FC </p>
</div>
<p><b>Figure 2.</b> Spread strains on selection plates containing 0.01, 0.05 and 0.1
mg/L of 5-FC to simulate the screening process under
Expand All @@ -298,15 +300,16 @@ <h2>Construction and Test of a Dual-Module Screening Platform</h2>
<div class="figure-container" style="width: 60%;margin: 0 auto;">
<img src="../../images/t10.jpg" alt="">
</div>
<p><b>Figure 3.</b>Graph of the correlation between 5-FC concentration in the medium and
<p><b>Figure 3.</b> Graph of the correlation between 5-FC concentration in the medium
and
HMA production. The orange bars represent HMA
levels and blue line depicts fluorescence intensity.</p>

<p>
<div class="figure-container" style="width: 60%;margin: 0 auto;">
<img src="../../images/t8.jpg" alt="">
</div>
<p><b>Figure 4.</b>Graph of HMA production by the bacteria harboring
<p><b>Figure 4.</b> Graph of HMA production by the bacteria harboring
HmaS<sup>WT</sup>and
HmaS<sup>V152G</sup>clones. With 9 mM HPP as the substrate for
whole-cell catalysis, HMA accumulation by the gY9s-HmaS<sup>V152G</sup>-Bio177 and
Expand Down Expand Up @@ -342,7 +345,7 @@ <h2>Modeling and Molecular Docking</h2>
<div class="figure-container" style="width: 60%;margin: 0 auto;">
<img src="../../images/图片4.jpg" alt="">
</div>
<p><b>Figure 6.</b>Simulated structures of HmaS<sup>V152G</sup>-HPP binding analyzed
<p><b>Figure 6.</b> Simulated structures of HmaS<sup>V152G</sup>-HPP binding analyzed
by Pymol
and Ligplus. Compared to HmaS<sup>WT</sup>
and mutant, HmaS<sup>V152G</sup> uses G152,
Expand All @@ -357,7 +360,7 @@ <h2>Engineering an E. coli chassis with High-HPP Production</h2>
Precursor
Supply</b>
</p>
<p>we constructed the pSB1c-aroG<sup>fbr</sup>-tktA-ppsA (<i>pATP</i>) plasmid to
<p>we constructed the pSB1c-aroG<sup>fbr</sup>-tktA-ppsA (pATP) plasmid to
overexpress the
phosphoenol-pyruvate synthase (<i>ppsA</i>) gene,
the transketolase (<i>tktA</i>) gene and aro<i>G<sup>fbr</sup></i>gene.
Expand Down Expand Up @@ -386,7 +389,7 @@ <h2>Engineering an E. coli chassis with High-HPP Production</h2>
<div class="figure-container" style="width: 60%;margin: 0 auto;">
<img src="../../images/图片5.tif" alt="">
</div>
<p><b>Figure 7.</b>Bar graph of the Inhibitory efficiency by the random
<p><b>Figure 7.</b> Bar graph of the Inhibitory efficiency by the random
double-mismatched eGFP-sgRNAs. Fi means fully interference,
transformed into the strain that containing the non-mutated R6K-dCas9-eGFP
plasmid, which had CA in the 7-8bp spacer of
Expand Down Expand Up @@ -422,7 +425,7 @@ <h2>Engineering an E. coli chassis with High-HPP Production</h2>
<div class="figure-container" style="width: 60%;margin: 0 auto;">
<img src="../../images/图片7.tif" alt="">
</div>
<p><b>Figure 8.</b>Bar graph of HPP production with different genes inhibited by
<p><b>Figure 8.</b> Bar graph of HPP production with different genes inhibited by
CRISPRi. The blue column represents the strain transformed
by pATP, the green column is for the strain transformed by pATP and
R6K-dCas9-sgRNA, and the orange columns indicate the
Expand Down Expand Up @@ -470,24 +473,22 @@ <h2>Evaluation of Metabolic Engineering and Directed Evolution</h2>
<div class="figure-container" style="width: 60%;margin: 0 auto;">
<img src="../../images/11.jpg" alt="">
</div>
<p><b>Figure 9.</b> Bar graph to compare relative HMA levels produced by different
strains. Solid black dots indicate the presence of the
plasmids, hollow black dots indicate their absence.
</p>
<p>Fermentation was carried out for a long time using the HMA5 and HMA6 clones.
Aliquots
were collected every two hours,
compared with HmaSWT, the HmaSV152G obtained through the screening, showed a
compared with HmaS<sup>WT</sup>, the HmaS<sup>V152G</sup> obtained through the
screening, showed a
4.73-fold increase in HMA production and
produced 3.63 g/L HMA after 24 h with a single supply of 20 g/L glucose
(Fig.
10).
</p>
<div class="figure-container" style="width: 60%;margin: 0 auto;">
<img src="../../images/t9.jpg" alt="">
</div>
<p><b>Figure 9.</b>Bar graph to compare relative HMA levels produced by different
strains. Solid black dots indicate the presence of the
plasmids, hollow black dots indicate their absence.
</p>
<div class="figure-container" style="width: 60%;margin: 0 auto;">
<img src="../../images/图片7.tif" alt="">
<img src="../../images/20.png" alt="">
</div>
<p><b>Figure 10.</b>Comparison of HMA production by the HMA5 and HMA6 strains. The
bacteria were fermented in shaking flasks for 24 h with a
Expand All @@ -506,6 +507,23 @@ <h2>Evaluation of Metabolic Engineering and Directed Evolution</h2>
<p style="font-size: 20px;">TOP</p>
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Expand Down Expand Up @@ -582,4 +600,4 @@ <h3 class="c-footer__heading">Contact info</h3>
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