results.html 63.48 KiB
{% extends "layout.html" %} {% block title %}Results{% endblock %} {% block lead
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<div class="inp-title">
<p style="margin: 0 0 -10px 0">Results</p>
<p style="font-family: Chillax-Light; font-size: 1.5vw">2023 SDU-CHINA</p>
</div>
</div>
<div class="spacing-bar"></div>
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<div class="wrapper-inp bg-model">
<div class="row">
<div class="col-md-2 sidenav-wrapper">
<!-- 侧边菜单 -->
<div class="sidenav">
<a href="#intro">
<p><b>Introduction</b></p>
</a>
<a href="#01">
<p><b>01 Selection of QS system and bacterial strains</b></p>
</a>
<!-- <a href="#1.1">
<p><strong>Esa I/R QS system</strong></p>
</a>
<a href="#1.2">
<p><strong>Our strains and Previous work</strong></p>
</a> -->
<a href="#02">
<p><b>02 The characterization of Esa I/R QS system</b></p>
</a>
<!-- <a href="#2.1">
<p><strong>Design</strong></p>
</a>
<a href="#2.2">
<p><strong>Results</strong></p>
</a>
<a href="#2.3">
<p><strong>Analysis of results</strong></p>
</a> -->
<a href="#03">
<p>
<b
>03 Selection and characterization of stationary phase promoter</b
>
</p>
</a>
<!-- <a href="#3.1">
<p>
<strong
>Why we need stationary phase promoter and its advantages</strong
>
</p>
</a> <a href="#3.2">
<p>
<strong
>Selection and Characterization results of first batch promoters
</strong>
</p>
</a>
<a href="#3.3">
<p>
<strong
>Selection and Characterization results of second batch promoters
</strong>
</p>
</a>
<a href="#3.4">
<p><strong>Conclusion</strong></p>
</a> -->
<a href="#04">
<p>
<b>04 Characterization of Three-layer dynamic regulation system</b>
</p>
</a>
<!-- <a href="#4.1">
<p><strong>The results of characterization</strong></p>
</a>
<a href="#4.2">
<p><strong>Analysis</strong></p>
</a>
<a href="#4.3">
<p><strong>Conclusion</strong></p>
</a> -->
<a href="#05">
<p><b>05 Selection and Characterization of lysis gene</b></p>
</a>
<!-- <a href="#5.1">
<p><strong>Selection of lysis system</strong></p>
</a>
<a href="#5.2">
<p><strong>Plasmid construction</strong></p>
</a>
<a href="#5.3">
<p><strong>First characterization of lysis gene</strong></p>
</a>
<a href="#5.4">
<p>
<strong
>Second characterization of lysis gene and RBS of different
strength</strong
>
</p>
</a> -->
<a href="#06">
<p>
<b>06 Validation of the effectiveness of PHB synthesis genes</b>
</p>
</a>
<!-- <a href="#6.1">
<p><strong>Method</strong></p>
</a>
<a href="#6.2">
<p><strong>Analysis</strong></p>
</a> -->
<a href="#07">
<p><b>07 PHB fermentation analysis</b></p>
</a>
<!-- <a href="#7.1">
<p><strong>strains construction</strong></p>
</a>
<a href="#7.2">
<p><strong>Fermentation conditions</strong></p> </a>
<a href="#7.3">
<p><strong>Measurement</strong></p>
</a> -->
<a href="#F&E"
><p><b>Failures & Experiences</b></p></a
>
<a href="#ref"
><p><b>References</b></p></a
>
</div>
<div id="progress-container">
<div id="progress-bar"></div>
</div>
</div>
<!-- content -->
<div class="col-sm-9 wiki-content results">
<!-- intro -->
<div>
<div>
<ul class="gd-t1">
<a name="intro"> <br /></a>
<li><b>Introduction</b></li>
</ul>
</div>
<div class="row">
<p>
. On this page, we describe the most important results that portray
our journey while designing our project.
</p>
</div>
</div>
<!-- 01 -->
<div>
<div>
<ul class="gd-t1">
<a name="01"> <br /></a>
<li>01 Selection of quorum sensing system and bacterial strains</li>
</ul>
</div>
<!-- 1.1 -->
<div class="row">
<div>
<ul class="gd-t3">
<a name="1.1"> <br /></a>
<li>1.1 Esa I/R Quorum Sensing (QS) system</li>
</ul>
</div>
<p>
<b>Quorum Sensing</b>is a way for cells to regulate downstream gene
expression based on their own density. The concentration of the
signaling molecule - <b>AHL </b>- secreted by the cell increases as
the cell density increases. When the concentration of AHL reaches a
certain level, it can bind to the corresponding binding protein and
alter the expression of downstream genes.
</p>
<p>
<b>The Esa I/R system</b> is quite special from traditional QS
system. The EsaI/R QS system is homologous to the LuxI/R QS system
and originates the maize pathogen--Pantoea stewartii subsp.
stewartia. EsaR can act as
<b>both transcriptional activator and repressor</b>.
</p>
<div>
<img
src="https://static.igem.wiki/teams/4583/wiki/result/esa.jpg"
alt="Esa I/R system"
width="70%"
/>
</div> <figcaption><b>Fig.1 | </b>Esa I/R system</figcaption>
</div>
<!-- 1.2 -->
<div class="row">
<div>
<ul class="gd-t3">
<a name="1.2"> <br /></a>
<li>1.2 Our strains and Previous work</li>
</ul>
</div>
<p>
Our project builds on what has been done before. In 2020, Fei Gu et.
al succeeded in <b>redirecting the metabolic flow</b>of E. coli
using the Esa I/R system[1]. And they have applied the QS switch in
the production of PHB. The Stains that they used come from another
study[2]. Our project goes one step further. We have
<b>re-characterized the system </b>and
<b>added an auto-lysis system</b>.This constitutes
<b>a three-layer dynamic regulation model </b>that enables the
separation of the bacterial growth phase, the production phase and
the product-release phase. <b>L19 </b>and <b>L31 </b>were engineered
E. coli MG1655, which already have Esa R/I system in them. They have
different regulatory thresholds (Fig.2), and we chose L19 and L31 as
our strains.
</p>
<div>
<img
src="https://static.igem.wiki/teams/4583/wiki/result/switching-od-of-different-strains.png"
alt="switching-od-of-different-strains"
width="70%"
/>
<figcaption>
<b>Fig.2 | </b>Switching OD of different strains[2]
</figcaption>
</div>
</div>
</div>
<!-- 02 -->
<div>
<div>
<ul class="gd-t1">
<a name="02"> <br /></a>
<li>02 The characterization of Esa I/R quorum sensing system</li>
</ul>
</div>
<!-- 2.1 -->
<div class="row">
<div>
<ul class="gd-t3">
<a name="2.1"> <br /></a>
<li>2.1 Design</li>
</ul>
</div>
<p>
We transformed pCL-PesaS-GFP (LVA) into E. coli L19 and L31 in
combination with pCL-PesaRwt-mkate, pCL-PesaRc-mkate, and
pCL-PesaRp-mkate, respectively. We repeatedly characterized the Esa
I/R QS system by detecting fluorescence intensity to reflect
promoter expression intensity. <b>Green fluorescence</b> was used to
reflect the transcriptional expression intensity of <b>PesaS</b>,
and <b>red fluorescence </b>represented the transcriptional
expression intensity of <b>PesaR</b>. In this way, the most suitable
models for growth and production stages were screened. Here are the
strains we constructed.
</p>
<figcaption>
<b>Table.1 | </b>Strains for QS switch characterization
</figcaption>
<div class="table-wrapper table-hw">
<table style="text-align: center"> <tr>
<th>ITEM</th>
<th>MEANING</th>
<th>CONNT</th>
<th>UNITS</th>
</tr>
<tr>
<td rowspan="3">L19</td>
<td rowspan="3">pCL-PesaS-GFP(LVA)</td>
<td>pCL-PesaRwt-mkate</td>
<td>L19SRw</td>
</tr>
<tr class="green">
<td>pCL-PesaRc-mkate</td>
<td>L19SRc</td>
</tr>
<tr>
<td>pCL-PesaRp-mkate</td>
<td>L19SRp</td>
</tr>
<tr class="green">
<td rowspan="3">L31</td>
<td rowspan="3">pCL-PesaS-GFP(LVA)</td>
<td>pCL-PesaRwt-mkate</td>
<td>L31SRw</td>
</tr>
<tr>
<td>pCL-PesaRc-mkate</td>
<td>L31SRc</td>
</tr>
<tr class="green">
<td>pCL-PesaRp-mkate</td>
<td>L31SRp</td>
</tr>
</table>
</div>
</div>
<!-- 2.2 -->
<div class="row">
<div>
<ul class="gd-t3">
<a name="2.2"> <br /></a>
<li>2.2 Results</li>
</ul>
</div>
<div>
<img
src="https://static.igem.wiki/teams/4583/wiki/result/the-results-of-qs-system-characterization.png"
alt="the-results-of-qs-system-characterization"
width="80%"
/>
<figcaption>
<b>Fig.3 | </b>The results of QS system characterization
</figcaption>
</div>
</div>
<!-- 2.3 -->
<div class="row">
<div>
<ul class="gd-t3">
<a name="2.3"> <br /></a>
<li>2.3 Analysis of results</li>
</ul>
</div>
<p>
Through preliminary characterization, we can clearly see that
<b
>PesaS and PesaR have a distinct sequential expression time
sequence at different stages</b
>. The <b>PesaS </b>transcript expression <b>peaked in the first 8 hours</b>, especially in the 4th-6th hours,
while PesaR peaked and stabilized after 10-12 hours (except for
PesaR-p).
</p>
<p>
We hoped that by comparing the before and after differences in the
expression of the two, we would be able to enter into the growth
mode during the propagation of the strains faster, and the
boundaries between the production mode and the growth mode would be
clearer, with a quicker and more complete transition, and the
results performed very well for both.
</p>
</div>
</div>
<!-- 03 -->
<div>
<div>
<ul class="gd-t1">
<a name="03"> <br /></a>
<li>
03 Selection and characterization of stationary phase promoter
</li>
</ul>
</div>
<!-- 3.1 -->
<div class="row">
<div>
<ul class="gd-t3">
<a name="3.1"> <br /></a>
<li>
3.1 Why we need stationary phase promoter and its advantages
</li>
</ul>
</div>
<p>
If artificial induction is used to make the bacteria lysed, the
inducer is not only <b>expensive</b>, but also produces
<b>strong toxicity</b> to the cells. If a general constitutive
promoter is used to express the cleavage gene, it will cause the
<b>bacterial metabolic burden</b> and <b>great growth pressure</b>.
</p>
<p>
So, we planned to use <b>stationary phase promoter</b> to control
the lysis gene. Bacteria undergo significant changes in their
physiological state after entering the plateau phase. The stationary
phase promoter endogenously produces more
<b>σ factors</b>
required for the stationary phase when bacteria enter the
exponential phase, and it can direct bacteriophage RNA polymerase to
transcribe specific genes during the stationary phase. Because the
signal required for the stationary phase promoter is produced
<b>endogenously</b>, it is not toxic to the bacteria. It is also
<b>dynamically regulated</b> and does not cause metabolic stress or
growth problems.
</p>
</div>
<!-- 3.2 -->
<div class="row">
<div>
<ul class="gd-t3">
<a name="3.2"> <br /></a>
<li>
3.2 Selection and Characterization results of first batch
promoters (Pflic, P1.1, P2.1, P3.1)
</li>
</ul>
</div>
<p>
We combined PACYC-Pflic-GFP, PACYC-P1.1-GFP, PACYC-P2.1-GFP, and
PACYC-P3.1-GFP plasmids (these plasmids were given by Xin Jin, our advisor) with pCL-PesaRwt, pCL-PesaRc, and pCL-PesaRp plasmids
(These plasmids were given by Fei Gu, our advisor), respectively,
and electrotransformed them into L19 and L31. In total,
<b>we constructed 24 strains</b> (Table 2)
</p>
<p>
We then used a Multi-Detection Microplate Reader (Synergy HT,
Biotek, U.S.) to detect the red (excitation at 585 nm and emission
at 640 nm) fluorescence and green (excitation at 485 nm and emission
at 528 nm) fluorescence.
</p>
<figcaption>
<b>Table.2 | </b>Plasmids combinations for first screening of
promoters
</figcaption>
<div class="table-wrapper table-pf mb-3">
<table style="text-align: center">
<tr>
<th>ITEM</th>
<th>MEANING</th>
<th>CONNT</th>
</tr>
<tr>
<td rowspan="12">L19</td>
<td rowspan="4">pCL-PesaRwt-mkate</td>
<td>PACYC-Pfic-GFP</td>
</tr>
<tr>
<td>PACYC-P1.1-GFP</td>
</tr>
<tr>
<td>PACYC-P2.1-GFP</td>
</tr>
<tr>
<td>PACYC-P3.1-GFP</td>
</tr>
<tr>
<td rowspan="4">pCL-PesaRp-mkate</td>
<td>PACYC-Pfic-GFP</td>
</tr>
<tr>
<td>PACYC-P1.1-GFP</td>
</tr>
<tr>
<td>PACYC-P2.1-GFP</td>
</tr>
<tr>
<td>PACYC-P3.1-GFP</td>
</tr>
<tr>
<td rowspan="4">pCL-PesaRc-mkate</td>
<td>PACYC-Pfic-GFP</td>
</tr>
<tr>
<td>PACYC-P1.1-GFP</td>
</tr>
<tr>
<td>PACYC-P2.1-GFP</td>
</tr>
<tr>
<td>PACYC-P3.1-GFP</td>
</tr>
<tr>
<td rowspan="12">L31</td>
<td rowspan="4">pCL-PesaRwt-mkate</td>
<td>PACYC-Pfic-GFP</td>
</tr>
<tr>
<td>PACYC-P1.1-GFP</td>
</tr> <tr>
<td>PACYC-P2.1-GFP</td>
</tr>
<tr>
<td>PACYC-P3.1-GFP</td>
</tr>
<tr>
<td rowspan="4">pCL-PesaRpt-mkate</td>
<td>PACYC-Pfic-GFP</td>
</tr>
<tr>
<td>PACYC-P1.1-GFP</td>
</tr>
<tr>
<td>PACYC-P2.1-GFP</td>
</tr>
<tr>
<td>PACYC-P3.1-GFP</td>
</tr>
<tr>
<td rowspan="4">pCL-PesaRc-mkate</td>
<td>PACYC-Pfic-GFP</td>
</tr>
<tr>
<td>PACYC-P1.1-GFP</td>
</tr>
<tr>
<td>PACYC-P2.1-GFP</td>
</tr>
<tr>
<td>PACYC-P3.1-GFP</td>
</tr>
</table>
<div>
<img
src="https://static.igem.wiki/teams/4583/wiki/result/2.png"
alt=""
width="80%"
/>
<img
src="https://static.igem.wiki/teams/4583/wiki/result/the-characterization-results-of-pfic-p1-1-p2-1-and-p3-1-in-l19.png"
alt="The characterization results of Pfic, P1.1, P2.1, and P3.1 in L19"
width="65%"
/>
<figcaption>
<b>Fig.5 | </b>The characterization results of Pfic, P1.1, P2.1,
and P3.1 in L19
</figcaption>
<img
src="https://static.igem.wiki/teams/4583/wiki/result/the-characterization-results-of-pfic-p1-1-p2-1-and-p3-1-in-l31.png"
alt="The characterization results of Pfic, P1.1, P2.1, and P3.1 in L31
"
width="70%"
/>
<figcaption>
<b>Fig.6 | </b>The characterization results of Pfic, P1.1, P2.1,
and P3.1 in L31
</figcaption>
</div>
<p>
The expression time was found
<b>to be close to that of PesaR</b>, with a maximum difference of
only 2-4 hours to peak (Fig.4). Also, expression of this promoter
is <b>very rapid</b>, with
<b>peak expression in less than 4 hours</b> (Fig5, Fig.6). This
means that by constructing an auto-lysis system with such a
stationary phase promoter, our engineered bacteria will
<b>only have a few hours to produce PHB</b>, not taking into
account the metabolic disruption and reduced yield caused by the
timing being too close to meet our expectations. : ( </p>
<p>
We also found that Pfic, P1.1, P2.1 and P3.1
<b>have different levels of expression</b> (Fig5, Fig.6)[3]. This
difference is particularly evident in L19 (Fig5, Fig.6).
</p>
</div>
</div>
<!-- 3.3 -->
<div class="row">
<div>
<ul class="gd-t3">
<a name="3.3"> <br /></a>
<li>
3.3 Selection and Characterization results of second batch
promoters (PYU3, PYU7, PYU16, and PYU92)
</li>
</ul>
</div>
<p>
So, by reading the scientific articles, we selected the late
stationary phase promoters of <b>PYU3, PYU7, PYU6</b> and
<b>PYU92</b>
for the second screening[4]. We combined them with PesaR and
characterized them. In this batch we also constructed 24 bacteria
(table 3).
</p>
<figcaption>
<b>Table.3 |</b> Plasmids combinations for second screening of
promoters
</figcaption>
<div class="table-wrapper table-pf">
<table style="text-align: center">
<tr>
<th>Strain</th>
<th>Plasmid 1</th>
<th>Plasmid 2</th>
</tr>
<tr>
<td rowspan="12">L19</td>
<td rowspan="4">pCL-PesaRwt-mkate</td>
<td>PACYC-PYU3-GFP</td>
</tr>
<tr>
<td>PACYC-PYU7-GFP</td>
</tr>
<tr>
<td>PACYC-PYU16-GFP</td>
</tr>
<tr>
<td>PACYC-PYU92-GFP</td>
</tr>
<tr>
<td rowspan="4">pCL-PesaRp-mkate</td>
<td>PACYC-PYU3-GFP</td>
</tr>
<tr>
<td>PACYC-PYU7-GFP</td>
</tr>
<tr>
<td>PACYC-PYU16-GFP</td>
</tr>
<tr>
<td>PACYC-PYU92-GFP</td>
</tr>
<tr>
<td rowspan="4">pCL-PesaRc-mkate</td>
<td>PACYC-PYU3-GFP</td>
</tr>
<tr> <td>PACYC-PYU7-GFP</td>
</tr>
<tr>
<td>PACYC-PYU16-GFP</td>
</tr>
<tr>
<td>PACYC-PYU92-GFP</td>
</tr>
<tr>
<td rowspan="12">L31</td>
<td rowspan="4">pCL-PesaRwt-mkate</td>
<td>PACYC-PYU3-GFP</td>
</tr>
<tr>
<td>PACYC-PYU7-GFP</td>
</tr>
<tr>
<td>PACYC-PYU16-GFP</td>
</tr>
<tr>
<td>PACYC-PYU92-GFP</td>
</tr>
<tr>
<td rowspan="4">pCL-PesaRp-mkate</td>
<td>PACYC-PYU3-GFP</td>
</tr>
<tr>
<td>PACYC-PYU7-GFP</td>
</tr>
<tr>
<td>PACYC-PYU16-GFP</td>
</tr>
<tr>
<td>PACYC-PYU92-GFP</td>
</tr>
<tr>
<td rowspan="4">pCL-PesaRc-mkate</td>
<td>PACYC-PYU3-GFP</td>
</tr>
<tr>
<td>PACYC-PYU7-GFP</td>
</tr>
<tr>
<td>PACYC-PYU16-GFP</td>
</tr>
<tr>
<td>PACYC-PYU92-GFP</td>
</tr>
</table>
<div>
<img
src="https://static.igem.wiki/teams/4583/wiki/result/ten-valid-combinations-of-late-stationary-promoter-characterization1.png"
alt="Ten valid combinations of late stationary promoter characterization "
width="80%"
/>
<img
src="https://static.igem.wiki/teams/4583/wiki/result/ten-valid-combinations-of-late-stationary-promoter-characterization2.png"
alt="Ten valid combinations of late stationary promoter characterization "
width="80%"
/>
<figcaption>
<b>Fig.7 | </b>Ten valid combinations of late stationary
promoter characterization
</figcaption>
</div>
<p>
From the 24 characterization results, the above ten combinations
with significant expression time differences were selected and
prepared for further screening (Fig. 7).
</p> <p>
In several of these combinations, we found, for example,
L19-PesaRwt-PYU7, that the two promoters peaked in their
respective expressions at times that
<b>differed by more than 10 hours</b>The results show that
<b
>the production and cleavage phases can be temporally
uncoupled</b
>Therefore, the characterization results have met our requirement
-
<b
>the production phase is separated from the product-release
phase</b
>. : )
</p>
</div>
</div>
<!-- 3.4 -->
<div class="row">
<div>
<ul class="gd-t3">
<a name="3.4"> <br /></a>
<li>3.4 Conclusion</li>
</ul>
</div>
<p>
In the end,
<b>the late steady state promoters PYU3, PYU7, PYU16 and PYU92</b>
were selected as our ideal promoters. We also selected
<b>ten combinations</b> that met our expectations to further build
the three-layer dynamic regulation system.
</p>
</div>
</div>
<!-- 04 -->
<div>
<div>
<ul class="gd-t1">
<a name="04"> <br /></a>
<li>
04 Characterization of Three-layer dynamic regulation system
</li>
</ul>
</div>
<p>
In the previous section, we first verified that
<b
>the Growth Phase is temporally decoupled from the Production
Phase</b
>, and then verified that
<b
>the Production Phase and the Product-release Phase are temporally
decoupled</b
>.
</p>
<p>
To be more scientific, we decided to <b>co-characterize </b> the
growth phase, the production phase and the product launch phase, and
to check that all three were <b>expressed in chronological </b>order
and <b>at the required time</b>.
</p>
<p>
Due to the certain red-green crosstalk problem during detection, we
constructed PACYC-PYU3-BFP, PACYC-PYU7-BFP, PACYC-PYU16-BFP,
PACYC-PYU92-BFP plasmids by ligating the blue fluorescent protein gene
BFP with PYU3,7,16,92 promoter (Fig.8, Fig9) in order to make the
results clearer and the overall expression time sequence. We
constructed 10 strains that each have 3 plasmids (table 4).
</p>
<div> <img
src="https://static.igem.wiki/teams/4583/wiki/result/genetic-circuit-of-pacyc-pyu3-bfp.png"
alt="Genetic circuit of PACYC-PYU3-BFP "
width="70%"
/>
<figcaption>
<b>Fig.8 | </b>Genetic circuit of PACYC-PYU3-BFP
</figcaption>
<img
src="https://static.igem.wiki/teams/4583/wiki/result/bfp-fragment.png"
alt="BFP fragment"
width="80%"
/>
<figcaption><b>Fig.9 | </b>BFP fragment</figcaption>
</div>
<figcaption>
<b>Table.4 | </b> Plasmids combinations of 3-plasmids characterization
</figcaption>
<div class="table-wrappper" >
<table class="table-pf" style="text-align: center; width: 100%">
<tr>
<th>Strain</th>
<th>Plasmid 1</th>
<th>Plasmid 2</th>
<th>Plasmid 3</th>
</tr>
<tr>
<td rowspan="6">L19</td>
<td>pCL-PesaSGFP (LVA)</td>
<td rowspan="3">pCL-PesaRwt-mkate</td>
<td>PACYC-PYU3-BFP</td>
</tr>
<tr>
<td>pCL-PesaSGFP (LVA)</td>
<td>PACYC-PYU7-BFP</td>
</tr>
<tr>
<td>pCL-PesaSGFP (LVA)</td>
<td>PACYC-PYU92-BFP</td>
</tr>
<tr>
<td>pCL-PesaSGFP (LVA)</td>
<td>pCL-PesaRwt Pmkate</td>
<td>PACYC-PYU7-BFP</td>
</tr>
<tr>
<td>pCL-PesaSGFP (LVA)</td>
<td rowspan="2">pCL-PesaRc-mkate</td>
<td>PACYC-PYU3-BFP</td>
</tr>
<tr>
<td>pCL-PesaSGFP (LVA)</td>
<td>PACYC-PYU7-BFP</td>
</tr>
<tr>
<td rowspan="4">L31</td>
<td>pCL-PesaSGFP (LVA)</td>
<td rowspan="2">pCL-PesaRwt-mkate</td>
<td>PACYC-PYU3-BFP</td>
</tr>
<tr>
<td>pCL-PesaSGFP (LVA)</td>
<td>PACYC-PYU92-BFP</td>
</tr>
<tr>
<td>pCL-PesaSGFP (LVA)</td>
<td rowspan="2">pCL-PesaRpmkate</td>
<td>PACYC-PYU7-BFP</td>
</tr>
<tr> <td>pCL-PesaSGFP (LVA)</td>
<td>PACYC-PYU16-BFP</td>
</tr>
</table>
</div>
<!-- 4.1 -->
<div class="row">
<div>
<ul class="gd-t3">
<a name="4.1"> <br /></a>
<li>4.1 The results of characterization</li>
</ul>
</div>
<div>
<img
src="https://static.igem.wiki/teams/4583/wiki/result/the-characterization-results-of-three-layer-dynamic-regulation-model1.png"
alt=""
width="80%"
/>
<img
src="https://static.igem.wiki/teams/4583/wiki/result/the-characterization-results-of-three-layer-dynamic-regulation-model2.png"
alt=" The characterization results of Three-layer dynamic regulation model"
width="80%"
/>
<figcaption>
<b>Fig.10 | </b> The characterization results of Three-layer
dynamic regulation model
</figcaption>
<img
src="https://static.igem.wiki/teams/4583/wiki/result/the-model-of-l19-pesarwt-pyu3.png"
alt="The model of L19-PesaRwt-PYU3"
width="70%"
/>
<figcaption>
<b>Fig.11 | </b>The model of L19-PesaRwt-PYU3
</figcaption>
</div>
</div>
<!-- 4.2 -->
<div class="row">
<div>
<ul class="gd-t3">
<a name="4.2"> <br /></a>
<li>4.2 Analysis</li>
</ul>
</div>
<p>
We can see from this that the stationary phase begins to be
expressed when the production phase is at its peak and the
separation of the production phase from the desired lysis phase has
been achieved. This means that
<b>the growth phase of the bacteria is barely affected</b> and
<b
>the bacteria have more than enough time for the production
phase</b
>. It was only <b>after 40 hours</b> that the bacteria entered the
expected lysis peak and lysed in droves.
</p>
</div>
<!-- 4.3 -->
<div class="row">
<div>
<ul class="gd-t3">
<a name="4.3"> <br /></a>
<li>4.3 conclusion</li>
</ul>
</div>
<p>
We built three different modules,<b>the growth module</b> ,<b
>the production module</b >
, and <b>the product release module</b>, which form our
<b>Three-layer dynamic regulation model</b>. we expressed them in
the same bacterium, and the results were very good.
</p>
</div>
</div>
<!-- 05 -->
<div>
<div>
<ul class="gd-t1">
<a name="05"> <br /></a>
<li>05 Selection and Characterization of lysis gene</li>
</ul>
</div>
<!-- 5.1 -->
<div class="row">
<div>
<ul class="gd-t3">
<a name="5.1"> <br /></a>
<li>5.1 Selection of lysis system</li>
</ul>
</div>
<div>
<ul class="table-text">
<li>
<p>
<b
>Lambda phage lysis gene cassette[5] (hereinafter referred
to as SRRz System)</b
>
</p>
</li>
</ul>
<p>
When <b>holin monomers</b> are isolated from antiholins that
inhibit holin function, their hydrophobic structural domains are
inserted into the cell membrane and then aggregate to form a more
ordered assembly in a pore as large as ~500 kDa, allowing proteins
to cross the cell membrane. Endolysins accumulated in the
cytoplasm can then be released into the periplasm to degrade
peptidoglycan in the cell wall and, in addition, the Rz/Rz1
complex of the λ phage promotes fusion of the inner membrane (IM)
and outer membrane (OM), which in turn pushes the OM away from the
murein layer, removing the final barrier.
</p>
</div>
<div>
<ul class="table-text">
<li>
<p>
<b
>Phi X174 phage lysis system[6] (hereafter referred to as
the E system)</b
>
</p>
</li>
</ul>
<p>
<b>Protein E</b> of phi174 is a hydrophobic membrane protein
consisting of 91 amino acids that itself lacks enzymatic activity.
It is hypothesized to function by triggering endogenous murein
hydrolase in the host to
<b>form a transmembrane structure</b>
that spans the inner and outer membranes.
</p>
</div>
</div>
<!-- 5.2 -->
<div class="row"> <div>
<ul class="gd-t3">
<a name="5.2"> <br /></a>
<li>5.2 Plasmid construction</li>
</ul>
</div>
<p>
We cloned the backbone part on the PACYC plasmid except the GFP
fragment by PCR using <b>the Gibson method</b>, leaving a short
homologous sequence with the lysis gene. At the same time, we
obtained the SRRz gene by company synthesis and cloned the SRRz gene
fragment containing the homologous arm of PACYC; the E gene fragment
containing the homologous arm of PACYC was cloned by PCR on Phi X174
plasmid, and the PACYC backbone fragment and the cleaved gene
fragment were seamlessly joined under the action of recombinase.
PACYC-PYU3, 7, 16, 92-SRRz and PACYC-PYU3, 7, 16, 92-E plasmids were
constructed (Fig. 12). All gene recombinations in the experiment
were performed by the Gibson method.
</p>
<div class="row">
<div class="col">
<img
src="https://static.igem.wiki/teams/4583/wiki/result/map-of-lysis-plasmid-construction-l.png"
alt="Map of lysis plasmid construction"
width="80%"
/>
</div>
<div class="col">
<img
src="https://static.igem.wiki/teams/4583/wiki/result/map-of-lysis-plasmid-construction-r.png"
alt="Map of lysis plasmid construction"
width="82%"
/>
</div>
<figcaption>
<b>Fig.12 | </b> Map of lysis plasmid construction
</figcaption>
</div>
</div>
<!-- 5.3 -->
<div class="row">
<div>
<ul class="gd-t3">
<a name="5.3"> <br /></a>
<li>5.3 First characterization of lysis gene</li>
</ul>
</div>
<p>
We transformed the constructed plasmids into L19 and L31 to observe
its lysis effect. The left side of the figure shows L19 containing
the cleavage plasmid, and the right side does not contain the
cleavage plasmid. After 1h of resting, it can be seen that
<b>the left side</b> has been <b>cleaved and clarified</b>, and
<b>the right side</b> is
<b>turbid, and the bacterium is sinking</b>
(Fig. 13).
</p>
<div>
<img
src="https://static.igem.wiki/teams/4583/wiki/result/comparison-of-lysis-effect.jpg"
alt="Comparison of lysis effect"
width="30%"
/>
<figcaption><b>Fig.13 | </b>Comparison of lysis effect</figcaption>
</div>
</div>
<!-- 5.4 -->
<div class="row">
<div>
<ul class="gd-t3"> <a name="5.4"> <br /></a>
<li>
5.4 Second characterization of lysis gene and RBS of different
strength
</li>
</ul>
</div>
<p>
Due to incomplete lysis in L19 and L31, we utilized the
<b>Gibson self-assembly method</b> and designed primers to add RBS
(BBa_B0031, BBa_B0032, BBa_B0033, and BBa_B0034) of different
intensities to the promoter regions of PYU3, PYU7, PYU16, PYU92 with
intensities of 0.010. 0.07,0.3, and 1 (Fig. 14). Due to time and
effort constraints, we did not succeed in constructing all plasmids
(Fig. 15).
</p>
<div>
<img
src="https://static.igem.wiki/teams/4583/wiki/result/genetic-circuit-of-lysis-system.png"
alt="Genetic circuit of lysis system"
width="70%"
/>
<figcaption>
<b>Fig.14 | </b>Genetic circuit of lysis system
</figcaption>
<img
src="https://static.igem.wiki/teams/4583/wiki/result/electrophoretic-validation-results.png"
alt="Electrophoretic validation results"
width="70%"
/>
<figcaption>
<b>Fig.15 | </b>Electrophoretic validation results
</figcaption>
</div>
<p>
Due to primer design problems, PCR extension length setting, or
assembly errors, etc., the RBS substitution was not completely done
successfully, so the plasmids verified to be working successfully in
the following table were used for the next experiments.
</p>
<figcaption><b>Table.5 | </b>Valid plasmids</figcaption>
<div class="table-wrapper table-pf">
<table style="text-align: center">
<tr>
<th>Strain</th>
<th>Plasmid 1</th>
<th>Plasmid 2</th>
<th>Plasmid 3</th>
<th>Lysis gene</th>
</tr>
<tr>
<td rowspan="4">L19</td>
<td>PACYC</td>
<td rowspan="4">PYU3</td>
<td>B0031</td>
<td>E</td>
</tr>
<tr>
<td>PACYC</td>
<td>B0032</td>
<td>E</td>
</tr>
<tr>
<td>PACYC</td>
<td>B0033</td>
<td>E</td>
</tr>
<tr>
<td>PACYC</td>
<td>B0034</td> <td>E</td>
</tr>
<tr>
<td rowspan="4">L19</td>
<td>PACYC</td>
<td rowspan="4">PYU7</td>
<td>B0031</td>
<td>E</td>
</tr>
<tr>
<td>PACYC</td>
<td>B0032</td>
<td>E</td>
</tr>
<tr>
<td>PACYC</td>
<td>B0033</td>
<td>E</td>
</tr>
<tr>
<td>PACYC</td>
<td>B0034</td>
<td>E</td>
</tr>
<tr>
<td rowspan="4">L19</td>
<td>PACYC</td>
<td rowspan="4">PYU16</td>
<td>B0031</td>
<td>E</td>
</tr>
<tr>
<td>PACYC</td>
<td>B0032</td>
<td>E</td>
</tr>
<tr>
<td>PACYC</td>
<td>B0033</td>
<td>E</td>
</tr>
<tr>
<td>PACYC</td>
<td>B0034</td>
<td>E</td>
</tr>
<tr>
<td rowspan="4">L19</td>
<td>PACYC</td>
<td rowspan="4">PYU7</td>
<td>B0031</td>
<td>E</td>
</tr>
<tr>
<td>PACYC</td>
<td>B0032</td>
<td>E</td>
</tr>
<tr>
<td>PACYC</td>
<td>B0033</td>
<td>E</td>
</tr>
<tr>
<td>PACYC</td>
<td>B0034</td>
<td>E</td>
</tr>
<tr>
<td rowspan="2">L19</td> <td>PACYC</td>
<td rowspan="2">PYU3</td>
<td>B0031</td>
<td>SRRz</td>
</tr>
<tr>
<td>PACYC</td>
<td>B0032</td>
<td>SRRz</td>
</tr>
<tr>
<td rowspan="2">L19</td>
<td>PACYC</td>
<td rowspan="2">PYU16</td>
<td>B0031</td>
<td>SRRz</td>
</tr>
<tr>
<td>PACYC</td>
<td>B0034</td>
<td>SRRz</td>
</tr>
<tr>
<td rowspan="3">L31</td>
<td>PACYC</td>
<td rowspan="3">PYU92</td>
<td>B0031</td>
<td>SRRz</td>
</tr>
<tr>
<td>PACYC</td>
<td>B0032</td>
<td>SRRz</td>
</tr>
<tr>
<td>PACYC</td>
<td>B0034</td>
<td>SRRz</td>
</tr>
</table>
</div>
<p>
Next, we transformed them to L19,L31 for
<b>characterization</b>:
</p>
<div>
<img
src="https://static.igem.wiki/teams/4583/wiki/result/the-results-of-lysis-system-characterization.png"
alt="The results of lysis system characterization"
width="70%"
/>
<figcaption>
<b>Fig.16 | </b>The results of lysis system characterization
</figcaption>
</div>
<p>
<b>PYU16-B0034-SRRz </b>and <b>PYU92-B0034-SRRz</b> were finally
selected as the ideal lysis systems (Fig. 16). They have the
advantages of <b>higher efficient lysis capacity</b> and
<b>low impact on strain growth</b>.
</p>
</div>
</div>
<!-- 06 -->
<div>
<div>
<ul class="gd-t1">
<a name="06"> <br /></a>
<li>06 Validation of the effectiveness of PHB synthesis genes</li>
</ul> </div>
<p>
<b>Nile red</b> has fluorescent properties, so it can be used as a
fluorescent dye for staining microplastics, lipids and proteins, etc.
It is commonly used in fluorescence microscopy and flow cytometry to
detect intracellular lipid droplets. Through Nile red staining and
fluorescence microscopy of bacterial cells containing PHB and non-PHB
lipid storage substances, Nile red is a good fluorescent stain for
<b>lipid substances</b> stored in bacterial cells with high
sensitivity[7].
</p>
<!-- 6.1 -->
<div class="row">
<div>
<ul class="gd-t3">
<a name="6.1"> <br /></a>
<li>6.1 Method</li>
</ul>
</div>
<p>
Nile red dye powder, prepared as a solution using dimethyl
sulfoxide, was added to the medium at a working concentration of 0.5
μg/ml and plates were poured. We set up two groups:
<b>the first group</b> used Nile Red plates, with PHB-producing
strains and non-PHB-producing strains delineated on the plates;
<b>the second group</b> used PHB-producing strains, delineated on
Nile Red and non-Nile Red plates. Observations were made using a UV
imager after 48 hours of incubation.
</p>
<div>
<img
src="https://static.igem.wiki/teams/4583/wiki/result/17.jpg"
alt="Nile Red Plate：PHB-producing strain vs. non-PHB-producing strain"
width="40%"
/>
<figcaption>
<b>Fig.17 | </b>Nile Red Plate：PHB-producing strain vs.
non-PHB-producing strain
</figcaption>
<img
src="https://static.igem.wiki/teams/4583/wiki/result/18.jpg"
alt="PHB-producing strains streaked on Nile red plate and non-Nile red plate control"
width="40%"
/>
<figcaption>
<b>Fig.18 | </b>PHB-producing strains streaked on Nile red plate
and non-Nile red plate control
</figcaption>
</div>
</div>
<!-- 6.2 -->
<div class="row">
<div>
<ul class="gd-t3">
<a name="6.2"> <br /></a>
<li>6.2 Analysis</li>
</ul>
</div>
<p>
From the figure, we can see that
<b>the PHB-producing strains can be observed to fluoresce</b>
while the non-PHB-producing strains cannot fluoresce under UV
irradiation. And Nile Red works very effectively because there is no
fluorescence on the non-Nile Red plate. This is proof that
<b>our PHBcab gene is effective</b>.
</p>
</div>
</div>
<!-- 07 -->
<div> <div>
<ul class="gd-t1">
<a name="07"> <br /></a>
<li>07 PHB fermentation analysis</li>
</ul>
</div>
<!-- 7.1 -->
<div class="row">
<div>
<ul class="gd-t3">
<a name="7.1"> <br /></a>
<li>7.1 strains construction</li>
</ul>
</div>
<figcaption>
<b>Table.6 | </b>Strains for fermentation analysis
</figcaption>
<div class="table-wrapper table-pf">
<table style="text-align: center">
<tr>
<th>No.</th>
<th>Strains</th>
<th>Note</th>
</tr>
<tr>
<td><strong>Group1</strong></td>
<td>
L19: PesaS-B0034 (integrated into the genome),
pUC-PesaRwt-PHBcab, PACYC-PYU16-0034-SRRz；
</td>
<td rowspan="2">Treatment group</td>
</tr>
<tr>
<td><strong>Group2</strong></td>
<td>
L31: PesaS-B0034(integrated into the genome), pUC-PesaRwt-
PHBcab，PACYC-PYU92-0034-SRRz
</td>
</tr>
<tr>
<td><strong>Group3</strong></td>
<td>
L19: PesaS-B0034(integrated into the genome), pUC-PesaR-
PHBcab
</td>
<td rowspan="6">Control group</td>
</tr>
<tr>
<td><strong>Group4</strong></td>
<td>
L31: PesaS-B0034(integrated into the genome), pUC-PesaR-
PHBcab
</td>
</tr>
<tr>
<td><strong>Group5</strong></td>
<td>L19: pUC-Pcon- PHBcab</td>
</tr>
<tr>
<td><strong>Group6</strong></td>
<td>L31: pUC-Pcon- PHBcab</td>
</tr>
<tr>
<td><strong>Group7</strong></td>
<td>L19: pUC-Pcon- PHBcab, PACYC-PYU16-B0034-SRRz</td>
</tr>
<tr>
<td><strong>Group8</strong></td>
<td>L31: pUC-Pcon- PHBcab, PACYC-PYU92-B0034-SRRz</td>
</tr> </table>
<div>
<img
src="https://static.igem.wiki/teams/4583/wiki/result/stains-construction02.jpg"
alt="Stains construction"
width="40%"
/>
<figcaption><b>Fig.19 | </b>Stains construction</figcaption>
</div>
</div>
</div>
<!-- 7.2 -->
<div class="row">
<div>
<ul class="gd-t3">
<a name="7.2"> <br /></a>
<li>7.2 Fermentation conditions</li>
</ul>
</div>
<p>
Single colonies were transferred to 5mL of Luria−Bertani (LB) Broth
with appropriate antibiotics. And they were culturing at 220rpm/ min
and 37℃for 8h. 1% seeds were inoculated in M9 medium supplemented
with 22g/L glucose.
</p>
<figcaption><b>Table.7 | </b>1L M9 medium</figcaption>
<div class="table-wrapper table-pf">
<table style="text-align: center">
<tr>
<th>Material</th>
<th>Volume/Mass</th>
</tr>
<tr>
<td><strong>ddH2O</strong></td>
<td>1L</td>
</tr>
<tr>
<td><strong>Yeast extraction</strong></td>
<td>2g</td>
</tr>
<tr>
<td><strong>NaCl</strong></td>
<td>1g</td>
</tr>
<tr>
<td><strong>NH4Cl</strong></td>
<td>1g</td>
</tr>
<tr>
<td><strong>Na2HPO4·12H2O</strong></td>
<td>15.6g</td>
</tr>
<tr>
<td><strong>KH2PO4</strong></td>
<td>3g</td>
</tr>
</table>
<div>
<img
src="https://static.igem.wiki/teams/4583/wiki/result/sampling.jpg"
alt="Sampling"
width="30%"
/>
<figcaption><b>Fig.20 | </b>Sampling</figcaption>
</div>
</div>
</div>
<!-- 7.3 -->
<div class="row">
<div> <ul class="gd-t3">
<a name="7.3"> <br /></a>
<li>7.3 Measurement</li>
</ul>
</div>
<p>
Samples were taken at 3,7,11,14,24,30,38,48h for OD600 and glucose
concentrations, and PHB concentrations were measured at the same
time from the <b>11th hour</b> onwards (Fig. 15).
</p>
<ul class="table-text">
<li>
<p><b>Glucose</b></p>
</li>
</ul>
<p>
After diluting the samples 50-fold (Fig. 16), the glucose
concentration was measured using a glucose tester and glucose was
replenished to 20 g/L at 14h and 25.5h of fermentation. Using our
hardware can easily complete this work. The raw data is in the
Notebook.
</p>
<div>
<img
src="https://static.igem.wiki/teams/4583/wiki/result/dilution-of-samples-for-glucose-detection02.jpg"
alt="Dilution of samples for glucose detection"
width="30%"
/>
<figcaption>
<b>Fig.21 | </b> Dilution of samples for glucose detection
</figcaption>
</div>
<ul class="table-text">
<li>
<p><b>OD600</b></p>
</li>
</ul>
<p>
Using spectrophotometer to measure OD600(Fig. 17). The raw data is
in the notebook.
</p>
<div>
<img
src="https://static.igem.wiki/teams/4583/wiki/result/spectrophotometer.jpg"
alt="Spectrophotometer"
width="40%"
/>
</div>
<figcaption>
<b>Fig.22 | </b>SpectrophotometerSpectrophotometer
</figcaption>
</div>
<ul class="table-text">
<li>
<p><b>PHB</b></p>
</li>
</ul>
<p>
To test the PHB content, we took 2.85mL of culture solution for each
group and added 0.15mL of chloroform (5%). Mix by gently inverting up
and down, then centrifuge at 3400xg for 8 minutes at 4°C. Remove the
chloroform-PHB phase (lower layer) in a glass vial with a pipette gun.
To it, 150ul of sulfuric acid was added, 850ul of methanol was added,
1000 µl of chloroform was added, and oil bath was used for 1 h. It was
taken out, cooled down to room temperature and then 1ml of dd water
was added, and the shaker was shaken for 30 s. It was left to stand
for more than 30 min for layering, and 80ul-150ul of the chloroform
layer was taken into the gas-phase vial. Determination was carried out
by <b>gas chromatography</b>[8].
</p> <div>
<img
src="https://static.igem.wiki/teams/4583/wiki/result/gas-chromatography.jpg"
alt="Gas chromatography"
width="60%"
/>
<figcaption><b>Fig.23 | </b>Gas chromatography</figcaption>
</div>
<p>
However, when we tested it, the results showed that
<b>there was no PHB</b>. Since we had previously performed a
step-by-step validation to ensure that each part could function
properly, we suspected that there was a problem with the method of
extracting PHB or the method of conducting the test.
</p>
<p>
In order to verify our conjecture, we took 1mL of the 48h culture
solution and centrifuged it, then removed the supernatant and took a
small amount of the bacterium and observed it under the fluorescence
microscope (Fig. 19 and Fig. 20). We found that in Group 3 bacteria,
<b>there were many glowing red particles</b>, which were intracellular
PHB (Fig. 20), while in Group 1 bacteria, there were few red
particles, which represented that most of the PHB had been released
(Fig. 19). This suggests that
<b
>our three-layer model is valid, it's the detection method that's
wrong</b
>.
</p>
<p>So, we will need to validate our results in a different way.</p>
<div>
<img
src="https://static.igem.wiki/teams/4583/wiki/result/group1-has-lysis-system-2.jpg"
alt=" Group1 (has lysis system)"
width="40%"
/>
<figcaption><b>Fig.24 | </b> Group1 (has lysis system)</figcaption>
<img
src="https://static.igem.wiki/teams/4583/wiki/result/group3-does-not-have-lysis-system-2.jpg"
alt="Group3 (does not have lysis system)"
width="40%"
/>
<figcaption>
<b>Fig.25 | </b>Group3 (does not have lysis system)
</figcaption>
</div>
<p>
We then added 3 ml of chloroform to 15 mL of culture medium at 48 h
and centrifuged it for 8 min at 4°C, which was used to extract
extracellular PHB. 100 μL of the chloroform layer was then taken, and
0.5 μL of Nile Red-stained chloroform layer (working concentration 0.5
μg/ml) was added and measured by fluorescence intensity using a
Multi-Detection Microplate Reader (Synergy HT, Biotek, U.S.) (Fig.
21).
</p>
<div>
<img
src="https://static.igem.wiki/teams/4583/wiki/result/using-fluorescence-intensity-to-measure-phb-content.png"
alt="Using fluorescence intensity to measure PHB"
width="70%"
/>
<figcaption>
<b>Fig.26 | </b>Using fluorescence intensity to measure PHB content
</figcaption>
<img
src="https://static.igem.wiki/teams/4583/wiki/result/results.png"
alt="results"
width="70%"
/>
<figcaption><b>Fig.27 | </b>results</figcaption> </div>
<p>
All of these results demonstrate that the extracellular PHB production
of the strains containing the three-layer regulatory system was
<b>much higher</b> than that of the strains without the addition of
the lysis system or without the use of the Quorum Sensing system to
regulate the metabolic flow.
</p>
</div>
<!-- Failures & Experiences: -->
<div>
<div>
<ul class="gd-t1">
<a name="F&E"> <br /></a>
<li>Failures & Experiences:</li>
</ul>
</div>
<p>1. Transformation：</p>
<p>
The failure rate of simultaneous transfer of two plasmids during
electroporation was significantly higher, which was later switched to
<b>one by one</b> with a higher success rate.
</p>
<p>
It is especially important to prepare receptor cells for
transformation plasmid experiments, and
<b>culturing them to the pre-exponential stage</b> (2 hours of
transfection) can improve transformation efficiency.
</p>
<p>
<b>The recovery culture should not be too long</b>. We have had
transformation failures due to too long recovery cultures.
</p>
<p>
E. coli can usually appear colonies in one day. When it appears to
take more than 24h to grow a colony,
<b>this colony may not be E. coli</b>. The first fermentation of this
experiment was re-tested for a second eletroporation and fermentation
analysis because the yeast colonies were picked resulting in no
fermentation product being detected in the end.
</p>
<p>
2. <b>The amount of bacterial solution</b> when applying the plate
<b>should not be too much</b>, it will result in growing into one
layer (all bacteria in this experiment are prone to grow into a layer,
except for the one containing a strong cleavage gene).
</p>
<p>
3. The strain needs to be
<b>re-cultured and preserved every 3 months</b>. If the time is too
long, the activity of the bacteria will be affected.
</p>
<p>
4. When using the <b>Gibson assembly method</b> for seamless ligation
of gene fragments, <b>try changing primers</b> if you encounter a
difficult ligation situation or if the pcr product is difficult to
obtain.
</p>
<p>
5. When extracting the plasmid, the incubation time needs to depend on
the situation. E. coli DH5α with <b>lysogeny plasmid</b> needs to
extract the plasmid <b>around 4 hours of growth</b>, otherwise
<b>the plasmid is lost</b> due to bacterial lysis resulting in large
amounts of content being discharged.
</p>
<p>
6. Our experiments required the measurement of extracellular PHB,
which did not work well despite being mentioned in some scientific
articles. We need to further explore the experimental conditions.
</p> </div>
<!-- reference -->
<div>
<ul class="gd-t3">
<a name="ref"> <br /></a>
<li>Refences</li>
</ul>
<div class="table">
<ul class="table-text" style="margin-left: 0">
<li><p>References</p></li>
</ul>
<img
id="table-re-switch"
src="https://static.igem.wiki/teams/4583/wiki/graphic-design/button-close.png"
alt="Fig.6 The formula we use to assess our web accessibility"
style="display: block; margin: 0 auto"
/>
</div>
<div id="table-re" class="table-wrapper hide">
<table class="table-fo" id="reference">
<tr>
<td>
<p>
1. Gu, F., et al., Quorum Sensing-Based Dual-Function Switch
and Its Application in Solving Two Key Metabolic Engineering
Problems. ACS Synth Biol, 2020. 9(2): p. 209-217.
</p>
</td>
</tr>
<tr>
<td>
<p>
2. Gupta, A., et al., Dynamic regulation of metabolic flux in
engineered bacteria using a pathway-independent quorum-sensing
circuit. Nat Biotechnol, 2017. 35(3): p. 273-279.
</p>
</td>
</tr>
<tr>
<td>
<p>
3. Jaishankar, J. and P. Srivastava, Strong synthetic
stationary phase promoter-based gene expression system for
Escherichia coli. Plasmid, 2020. 109: p. 102491.
</p>
</td>
</tr>
<tr>
<td>
<p>
4. Talukder, A.A., et al., RpoS-dependent regulation of genes
expressed at late stationary phase in Escherichia coli. FEBS
Lett, 1996. 386(2-3): p. 177-80.
</p>
</td>
</tr>
<tr>
<td>
<p>
5. Gao, Y., et al., Inducible cell lysis systems in microbial
production of bio-based chemicals. Appl Microbiol Biotechnol,
2013. 97(16): p. 7121-9.
</p>
</td>
</tr>
<tr>
<td>
<p> 6. Barrell, B.G., G.M. Air, and C.A. Hutchison, 3rd,
Overlapping genes in bacteriophage phiX174. Nature, 1976.
264(5581): p. 34-41.
</p>
</td>
</tr>
<tr>
<td>
<p>
7. Greenspan, P., E.P. Mayer, and S.D. Fowler, Nile red: a
selective fluorescent stain for intracellular lipid droplets.
J Cell Biol, 1985. 100(3): p. 965-73.
</p>
</td>
</tr>
<tr>
<td>
<p>
8. Borrero-de Acuña, J.M., et al., A novel programmable
lysozyme-based lysis system in Pseudomonas putida for
biopolymer production. Sci Rep, 2017. 7(1): p. 4373.
</p>
</td>
</tr>
</table>
</div>
</div>
</div>
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