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<!DOCTYPE html>
<html lang="en">

<head>
<meta charset="UTF-8">
<meta name="viewport" content="width=device-width, initial-scale=1.0">
<title>iDEC NEFU_China 2022</title>
<link rel="stylesheet" type="text/css" href="css/main_cssstyle.css">
<style>
header {
background: url("../../static/top/experiment.png");
min-height: 50%;
background-repeat: no-repeat;
background-size: cover;
}

.page-title {
padding-top: 10em;
padding-bottom: 1.5em;
}

/* footer的样式 */
.contact-icon {
display: flex;
align-items: center;
/*font-size: 3em;*/
color: white;
}

.contact-icon a {
color: white;
}

.contact-icon i {
font-size: 3em;
}
</style>
</head>

<body>
<a class="skip-main" href="#slide1-chapter1">Skip to main content</a>
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<!------------ 页面标题PAGE TITLE -------------------->
<div class="page-title">
<div class="page-title-content">
<h1>EXPERIMENTS</h1>
<!---Page title -->
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<div class="grid-normal">
<!--------------Introduction/Summary--------------------->
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<a class="main-nav-link" linkedidchapter="slide1-chapter1">Designing a dual selection system<p>
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<!------Chapter Two -->
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<a class="main-nav-link" linkedidchapter="slide1-chapter2">Directed evolution of PobR</a>
<!------Chapter Three -->
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<a class="main-nav-link" linkedidchapter="slide1-chapter3">Fluorescence assay and screening of
the PobR mutant library</a>
<!------Chapter Three -->
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<a class="main-nav-link" linkedidchapter="slide1-chapter4">The specificity and detection range
of PobR mutants responsive to new ligands </a>
<!------Chapter Three -->
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<a class="main-nav-link" linkedidchapter="slide1-chapter5">Modeling and docking</a>
<!------Chapter Three -->
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<!------------------------------ section 1------------------------------------>
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<div id="slide1-chapter1">
<h2>Designing a dual selection system</h2>
<p>We constructed a dual selection system based on plasmid
gYB2a-pobR<sup>WT</sup>-mCherry-codA-cmr. This plasmid mainly contains three parts:
</p>
<p>1. The <i>pobR</i> coding sequence (CDS) which can express PobR protein;</p>
<p>2. The cytosine deaminase(<i>codA</i>) gene which can express CDase protein;</p>
<p>3. An engineered operon consisting of the red fluorescent protein(<i>mCherry</i>)
and the chloramphenicol(Cm) resistant gene (<i>cmr</i>).</p>
<p>How this plasmid acts as a dual selection system is as follows:</p>
<p>1. Negative selection: Been activated by PobR<sup>WT</sup>, P<sub>pobA</sub>
activates the expression of CDase which is available for conversion of
exogenously added 5-FC to 5-FU, causing cell death;</p>
<p>2. Positive selection: Without no additional 5-FC, adding chloramphenicol and an
aromatic compound to cultivate <i>E. coli</i> cells will get a library of PobR
mutants. Strains with PobR<sup>mut</sup> that recognize the aromatic compound
are expected to grow in the LB agar medium.</p>
<p>To further understand each part, please <a href="../project/design.html">click
here</a> to view our design
part.</p>
<p>To constructed gYB2a-pobR<sup>WT</sup>-mCherry-codA-cmr, we first tried to
generate plasmid gYb2a-PobR-P<sub>pobA</sub>*2-mcherry-SacB-Cmr by Goldengate
assembly. However, when applying the plasmid
gYb2a-PobR-P<sub>pobA</sub>*2-mcherry-SacB-Cmr to achieve functions as a dual
screen system, the results are not ideal. </p>
<p>We obtain the two target fragments, gYb2a-PobR-P<sub>pobA</sub>*2-mcherry-Cmr and
CD by the method of PCR. Then the two fragments were ligated by using Goldengate
assembly and transformed into <i>E. coli</i> BW&#916;<i>codA</i> competent cells and
the plasmid gYB2a-pobR<sup>WT</sup>-mCherry-codA-cmr was constructed. </p>
<p>The result of DNA sequencing showed that our
gYB2a-pobR<sup>WT</sup>-mCherry-codA-cmr plasmid was constructed.</p>
</div>
<div id="slide1-chapter2">
<h2>Directed evolution of PobR</h2>
<p>Aiming to get strains that respond to different aromatic compounds by the dual
selection system, a large PobR mutant library which is able to include as many
mutants situations as possible is crucial. Therefore, we used error-prone PCR to
construct the PobR mutant library. </p>
<p>The generated library with highly random PobR mutants was transformed into
<i>E.coli</i> BW&#916;codA to obtain transformants containing mutant plasmids.
The PobR mutant library was transformed into BW&#916;codA competent cells and
transferred to M9 medium for culturing in shaking flasks. Ten clones were
randomly picked to sequence their PobR CDS regions for the quality control,
which revealed diverse mutants with an average mutants rate of about 0.36%.
<div class="figure-container auto-margin">
</div>
</div>
<div id="slide1-chapter3">
<h2>Fluorescence assay and screening of the PobR mutant library</h2>
<p>Another part which is essential to get the new ligands specificities and
detection range of PobR<sup>mut</sup> is the screening part.</p>
<p>In the negative selection, the obtained strains were cultivated in liquid culture
supplemented by 4HB and 5-FC. In the initial negative selection, we used a
constant 4HB concentration of a relatively high level, 0.5 g/L, and then tested
different concentrations of 5-FC to inhibit both the pseudo-positive and
4HB-responsive strains. In this selection step, we first used 50 mg/L 5-FC, and
observed insufficient inhibition of the bacterial growth. Thus, we increased the
5-FC concentration to 200 mg/L, improving the selection effectiveness.</p>
<p>In the positive selection, we added seven aromatic compounds to LB agar medium
and cultivated <i>E. coli</i> cells harboring a library of PobR mutants in this
medium for strain selection. As for the aromatic compounds, we chose
phenylethanol (2-PE), mandelate (MA), 4-hydroxymandelate (HMA), phenylpyruvate
(PPA), 4-hydroxyphenylpyruvate (HPP), phenylacetaldehyde (PAld) and p-Coumaric
acid.</p>
<p>After culturing for 30 hours, a few pink colonies were picked and cultured in a
96 deep-well plate. Since the expression of the reporter gene <i>mCherry</i> was
positively correlated with responsiveness, all that required is to transfer the
pink colonies to LB media containing only Amp after activation and used 0.5 g/L
of the test ligands for initial characterization. At the same time, negative
control without the addition of test ligands was used to avoid a few biosensor
variants with a strong fluorescence response in the absence of any ligand. After
performing fluorescence measurement，we got some strains that are highly
responsive to aromatic compounds. </p>
<p>Besides, we used the dilution coating method to estimate the number of mutants
which are capable of aromatic compounds per screen. The selection capacity for
each compound was more than 900，000 clones (with at least four plates, the
original density of the two-round selected bacteria was 450,000 CFU/mL.)</p>
<p>Please <a href="../documentation/notebook.html">click here</a> to
view our notebook.</p>
</div>
<div id="slide1-chapter4">
<h2>The specificity and detection range of PobR mutants responsive to new ligands
</h2>
<p>We obtained several responsive strains, of which the fluorescence intensity was
1.5-fold higher than that of the negative control. To further evaluate the PobR
mutants obtained above, the second round of characterization experiments were
carried out to individually examine their responsiveness to each candidate
ligand. For each ligand, we selected a mutant strain with the highest responsive
profiles and plotted the curve for their ligand response. In total, 9 potential
biosensors were isolated after the second round of characterization, and all
these variants were sequenced to determine the mutations in their primary
sequences. Amino acids at positions 163, 177 and 234 are located near the ligand
binding pocket of PobR, and amino acid at position 40 is located in the DNA
binding domain. </p>
</div>
<div id="slide1-chapter5" style="margin: 0 0 8%;">
<h2>Modeling and docking</h2>
<p>To better understand and analyze the effects of amino acid substitutions on the
response of the PobR protein, we tried to use software to simulate the structure
of the protein and build a molecular docking between the PobR protein and its
inducer. </p>
<p>We first use Homologous Model website SWISS-MODEL to construct the PobR mutant
model using the PobR wild type as a template.</p>
<p>Secondly, we obtained the structure files of the ligands 4HB, 2-PE, MA, HMA,
PAld, HPP and PPA from the organic small molecule database Pubchem. For more
details you can <a href="https://pubchem.ncbi.nlm.nih.gov/">click here</a>.</p>
<p>Based on the structures of PobR monomer and ligands, the docking engine Autodock
is used to simulate molecular docking. Autodock search space coordinates were
set
as center_x = -4.672, center_y = 3.331, center_z = -2.213. Dimensions of the
search space were set as size_x = 40, size_y = 40, size_z = 40, and
exhaustiveness was set at 15. The 15 conformational conditions in a score based
on the lowest binding energy were listed as the docking results. To examine the
accuracy of our docking, we used Ligplus to check the interaction between the
small molecule and predicted protein receptors. Finally, the three-dimensional
schematics of the protein and its ligand were portrayed using PyMol Version
2.2.0.</p>
</div>
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