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Meng-na-mei authored Oct 5, 2023
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</div>
<div class="chapters">
<div class="dropdown-menu cool-link">
<a class="chapter-nav " href="https://idec-teams.github.io/2023_NEFU_China/">
<a class="chapter-nav " href="../../static/home.html">
<div class="chapter-content">
<div class="chapter-title active ">
Home
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<a href="../../static/report.pdf" class="dropdown-page cool-link2" target="_blank">
Report
</a>
<a href="../../static/supplementary information.pdf" class="dropdown-page cool-link2"
target="_blank">
Supplementary information
</a>
</div>
</div>
<div class="dropdown-menu cool-link">
Expand Down Expand Up @@ -151,29 +155,42 @@ <h1>EXPERIMENTS</h1>
<aside class="sticky">
<div id="sub-nav" class="sub-navigation-content">
<div class="sub-navigation-chapter cool-link2">
<a class="main-nav-link" linkedidchapter="slide1-chapter1">Designing a dual selection system<p>
<a class="main-nav-link" linkedidchapter="slide1-chapter1">Construction of the hamS mutant library
by error-prone PCR, and the plasmids mainly contain 3 parts.
</a>
<!------Chapter Two -->
</div>
<div class="sub-navigation-chapter cool-link2">
<a class="main-nav-link" linkedidchapter="slide1-chapter2">Directed evolution of PobR</a>
<a class="main-nav-link" linkedidchapter="slide1-chapter2"> Construction of the gY9s-dual
T7-Trrnb-HmaS(Scpa1)-Bio177 plasmid, and the plasmid contains 2 modules.</a>
<!------Chapter Three -->
</div>
<div class="sub-navigation-chapter cool-link2">
<a class="main-nav-link" linkedidchapter="slide1-chapter3">Fluorescence assay and screening of
the PobR mutant library</a>
<a class="main-nav-link" linkedidchapter="slide1-chapter3"> Construction of mismatch sgRNAs mutant
library</a>
<!------Chapter Three -->
</div>
<div class="sub-navigation-chapter cool-link2">
<a class="main-nav-link" linkedidchapter="slide1-chapter4">The specificity and detection range
of PobR mutants responsive to new ligands </a>
<!------Chapter Three -->
<a class="main-nav-link" linkedidchapter="slide3-chapter1">
<li class="t">Construction of mismatch sgRNA Target
eGFP</li>
</a>
<!------Chapter Two -->
</div>
<div class="sub-navigation-chapter cool-link2">
<a class="main-nav-link" linkedidchapter="slide4-chapter1">
<li class="t">Construction of Polygenic mismatch
sgRNA</li>
</a>
<!------Chapter Two -->
</div>
<div class="sub-navigation-chapter cool-link2">
<a class="main-nav-link" linkedidchapter="slide1-chapter5">Modeling and docking</a>
<a class="main-nav-link" linkedidchapter="slide1-chapter4">Characterization of CRISPRi platform
</a>
<!------Chapter Three -->
</div>


<!-- 返回顶部箭头-->
<div id="to-top-arrow">
<a title="Back to top" href="#"><i class="fas fa-chevron-circle-up"></i></a>
Expand All @@ -188,23 +205,85 @@ <h1>EXPERIMENTS</h1>
<div class="main-body">
<div class="main-body-content">
<div id="slide1-chapter1">


<h2>Construction of the hamS mutant library by error-prone PCR, and the plasmids mainly
contain 3 parts</h2>

<p>1. The catalytic module consisted of <i>hmaS</i>
gene. The HmaS coding sequence (<i>hmaS</i>),
which can express HmaS protein, having
key function in the conversion of 4-hydroxyphenylpyruvate (HPP) to
4-hydroxymandelate (HMA). And we constructed a hmaS
mutant library by error-prone PCR.</p>
<p>2. The sensing module consisted of <i>mCherry</i> gene, which could transcribes red
fluorescent protein. The fluorescence
intensity is coupled to the enzyme activity.</p>
<p>3. The cytosine deaminase codA gene that converts 5-fluorocytosine (5-FC) to
5-fluorouracil (5- FU), which kills
bacteria. And the catalytic activity is coupled with the strength of the reporter

</div>
<div id="slide1-chapter2">


<h2>Construction of the gY9s-dual T7-Trrnb-HmaS(Scpa1)-Bio177 plasmid, and the plasmid
contains 2 modules.</h2>
<p>1. The catalytic module contains 2 parts, dual T7 promoter and hmas gene. The high
specificity of T7 RNA polymerase and
T7 promoters enabled the specific mutations during transcription. The HmaS coding
sequence (<i>hmaS</i>), which can express
HmaS protein, having key function in the conversion of 4-hydroxyphenylpyruvate (HPP)
to 4-hydroxymandelate (HMA). </p>
<p>2. The sensing module contains of <i>PobR</i> promoter and mCherry gene. The
<i>PobR</i> gene
encoding the allosteric transcription
factor <i>PobR</i> protein that specifically responds to 4-hydroxyphenylpyruvate
(HPP). And
<i>mCherry</i>
gene, which could
transcribe red fluorescent protein. The fluorescence intensity is coupled to the
enzyme activity. The cytosine deaminase
<i>codA</i> gene that converts 5-fluorocytosine (5-FC) to 5-fluorouracil (5- FU),
which
kills bacteria.
</p>
<div class="figure-container auto-margin">
</div>
</div>
<div id="slide1-chapter3">

<h2> Construction of mismatch sgRNAs mutant library</h2>
<p><b>Construction of mismatch sgRNA Target eGFP</b></p>
<p>1. dCas9: the codon GAT for Asp10 of the <i>cas9</i> gene was replaced by Ala and the
codon
CAC for His840 of the <i>cas9</i> gene was
replaced by Ala.</p>
<p>2. sgRNA targeted the coding sequence (+40 bp) of eGFP</p>
<p><b>Construction of Polygenic mismatch sgRNA</b></p>
<p>1. Replace the ori to obtain R6K-dCas9-eGFP as the base plasmid for subsequent
characterization. Replace sgRNA spacers to
individually target <i>pykF</i>, <i>tyrB</i>, <i>tyrR</i> and <i>pheA</i> in
R6K-dCas9-eGFP, and thus generated
R6K-dCas-pykF, R6K-dCas-tyrB,
R6K-dCas-tyrR, R6K-dCas-pheA.</p>
<p>2.These four sgRNA fragments were linked together by the Golden Gate method to
generate the R6K-dCas9-pykF-tyrB-tyrR-pheA
plasmid. Using degenerate primers in the amplifications of these sgRNAs to obtain
their mutated fragments, which were
subsequently subcloned into vectors to construct sgRNA-mutated plasmids leading to
the construction of the mutant
library.</p>

</div>
<div id="slide1-chapter4">

</div>
<div id="slide1-chapter5" style="margin: 0 0 8%;">

<h2> Characterization of CRISPRi platform
</h2>
<p>1. To assess the capacity of the CRISPRi system in repressing eGFP gene expression,
the R6K-dCas9-eGFP plasmid was
co-transformed with the reporter plasmid pYB1a-eGFP into E. coli BW25113 to induce
expression. Then measure Cell density
(OD600) and green fluorescence intensity by 96-well plate reader.</p>
<p>2. To test the effect of gene inhibition based on CRISPRi in HPP production process,
pATP and R6K-dCas9-sgRNA were
co-transformed into <i>E. coli </i>BWΔCD. The bacteria were collected by and further
cultivated at 30℃ for 24 h, HPP levels
were detected by HPLC.</p>
</div>
</div>
</div>
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</script>
</body>

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