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<!DOCTYPE html>
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<title>Proof of Concept</title>
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<div>Proof of Concept</div>
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<p>In order to develop a proof of concept for our project as a whole, we come up with the following ideas. These
experiments and prototypes may increase the credibility of our concept, making it more in line with realistic
scenarios.</p>
<article class="article">
<h1>1. The three-enzyme complex can effectively degrade PET plastic sheets, with improved degradation efficiency
compared with single enzyme reaction.</h1>
<p>The enzyme complex we constructed can effectively degrade PET plastic, and the efficiency is higher than that
of single enzyme (RIDD-PETase) treatment, which introduced in our engineering success. The increase in
degradation efficiency can prove the feasibility of our concept. After 7 days of incubation with the crude
enzyme solution and PET plastic sheet (crystallinity 12%), the TPA yield reached 0.9016mM, instead of using
enzyme complex, only one part (RIDD-PETase) was used to process the TPA yield Only 0.4815mM (Figure 1).</p>
<div class="figures">
<div class="row">
<div class="col-12 col-md-8 mx-auto">
<img src="https://2021.igem.org/wiki/images/d/db/T--DUT_China--poc_1.svg" alt="">
</div>
</div>
</div>
<p class="caption text-md-center">Figure 1: The concentration of TPA product in each experiment group</p>
<p>At the same time, the results of our scanning electron microscope also proved the feasibility and
effectiveness of our concept of constructing enzyme complexes. For details, please refer to the <a
href="https://2021.igem.org/Team:DUT_China/Engineering" target="_blank">engineering
success</a>. The PET plastic sheet treated by medium was examined by scanning electron microscopy, and
the
results were in line with expectations. The plastic sheet treated with <i>E. coli BL21</i> medium or MD
medium has
almost no scratches or holes. The plastic sheet after PD treatment has some obvious scratches, while the
plastic sheet after PD-MD-hA treatment. The surface is covered with scratches, and at the same time, densely
packed with holes of various shapes. This scanning electron microscopy test more intuitively proved that the
three-enzyme complex we constructed has a better PET plastic degradation function than single-enzyme
degradation.</p>
<div class="figures">
<div class="row">
<div class="col-12 col-md-6">
<img src="https://2021.igem.org/wiki/images/a/a4/T--DUT_China--poc_2a.jpg" alt="">
<p>(a)</p>
</div>
<div class="col-12 col-md-6">
<img src="https://2021.igem.org/wiki/images/3/30/T--DUT_China--poc_2b.jpg" alt="">
<p>(b)</p>
</div>
</div>
<div class="row">
<div class="col-12 col-md-6">
<img src="https://2021.igem.org/wiki/images/b/b6/T--DUT_China--poc_2c.jpg" alt="">
<p>(c)</p>
</div>
<div class="col-12 col-md-6">
<img src="https://2021.igem.org/wiki/images/2/2d/T--DUT_China--poc_2d.jpg" alt="">
<p>(d)</p>
</div>
</div>
</div>
<p class="caption">Figure 2: Scanning electron microscopy (a) PET plastic sheets treated in <i>E. coli BL21</i>.
(b) PET
plastic sheets
treated in <i>E. coli BL21/pET28a-PD</i>. (c) PET plastic sheets treated in <i>E. coli BL21/pET28a-M</i>.
(d) PET plastic
sheets treated in <i>E. coli BL21/pET28a-PD-MD-hA</i> medium.</p>
</article>
<article class="article">
<h1>2. Growth curve method proved that the engineered bacteria <i>E. coli BL21/pET28a-PD-MD-hA</i> has the
feasibility
of large-scale cultivation in industrial production.</h1>
<p>In order to obtain a large amount of protein, it is necessary to cultivate engineered bacteria at a
relatively high density. However, introducing <i>pET28a-PD-MD-hA</i> into engineered bacteria may slow down
the
growth and replication speed due to the load of plasmids, which increases the cost of the bacterial culture
process for the industrialized mass production of enzymes. Therefore, we decided to use 1L of LB medium to
cultivate different types of bacteria at 37 °C, and compare the growth curves of <i>E. coli
BL21/pET28a-PD-MD-hA</i>
and <i>E. coli BL21</i> (wild type) , <i>E. coli BL21/pET28a</i> to examine the growth of the engineered
bacteria we
constructed.</p>
<div class="figures">
<div class="row">
<div class="col-12 col-md-8 mx-auto">
<img src="https://2021.igem.org/wiki/images/f/f5/T--DUT_China--poc_3.svg" alt="">
</div>
</div>
</div>
<p class="caption">Figure 3: The growth curve of <i>E. coli BL21,</i> <i>E. coli BL21/pET28a</i> and E. coli
BL21/pET28a-PD-MD-hA(Using a microplate reader, it enters a plateau when OD600≈0.4)</p>
<p>Firstly, we cultured <i>E. coli BL21,</i> <i>E. coli BL21/pET28a</i> and <i>E. coli BL21/pET28a-PD-MD-hA</i>
in kana free medium
respectively and developed their growth curve to see whether the introduction of <i>pET28a-PD-MD-hA</i>
plasmid has
an impact on bacterial growth. As shown in Figure 3, the growth curve of <i>E. coli BL21/pET28a-PD-MD-hA</i>
exhibited virtually the same growth trend as <i>E. coli BL21</i> and <i>E. coli BL21/pET28a,</i> suggesting
that the plasmid
of <i>pET28a-PD-MD-hA</i> had no effect on the growth of <i>E. coli BL21</i>.</p>
</article>
<article class="article">
<h1>3. In order to facilitate enzyme separation and purification, or even in situ PET plastic degradation in
the
environment, we studied the signal peptides for bacterial secretion lay the foundation for subsequent
protein secretion work.</h1>
<p>PelB is a signal peptide using SecB-dependent pathway and has been commercially used in PET expression
systems (Figure 4). We initially planned to use pelB as signal peptide to achieve effective secretion of the
target protein, but the expected goal was not achieved in the experiment. The normal functioning of pelB is
closely related to the hydrophobicity of the subsequent 20 amino acids, and the RIDD and RIAD used in our
system do not meet its hydrophobicity requirements, which results in the failure of enzyme secretion.</p>
<div class="figures">
<div class="row">
<div class="col-12">
<img src="https://2021.igem.org/wiki/images/3/34/T--DUT_China--poc_4.jpg" alt="">
</div>
</div>
</div>
<p class="caption text-md-center">Figure 4: Principle of signal peptide of SecB-dependent pathway</p>
<p>In PETase expression system, SecB-dependent pathway is well-established, with a variety of signal peptides,
within which we can screen out those that can adapt to the hydrophobicity of RIDD and RIAD, such as PhoA,
OmpC, OmpF, OmpA, Endoxylanase, and MalE.</p>
<div class="figures">
<div class="row">
<div class="col-12">
<img src="https://2021.igem.org/wiki/images/f/ff/T--DUT_China--poc_5.png" alt="">
</div>
</div>
</div>
<p class="caption text-md-center">Figure 5: Some different types of signal peptides later teams can try</p>
</article>
<article class="article">
<h1>4. We have studied the compatibility of future PD-MD-hA secreting bacteria <i>E. coli
BL21/pET28a-PD-MD-hA (Secretory)</i>, with PET plastic degradation products in the fermenter, proving
suitable for industrial applications.</h1>
<p>If an engineered bacteria that can secrete PD-MD-hA is constructed, a possible application will be
co-culturing <i>E. coli BL21/pET28a-PD-MD-hA (Secretory)</i> with PET plastic in fermenters to achieve
biodegradation of plastic. However, in this way, we have to solve a problem: the final product of PET
plastic degradation, TPA and EG may have a negative impact on bacterial growth, so we have studied growth
curve of engineered bacteria under different concentrations of TPA and EG.</p>
<div class="figures">
<div class="row">
<div class="col-12 col-md-6">
<img src="https://2021.igem.org/wiki/images/1/12/T--DUT_China--poc_6a.svg" alt="">
<p>(a)</p>
</div>
<div class="col-12 col-md-6">
<img src="https://2021.igem.org/wiki/images/0/0d/T--DUT_China--poc_6b.svg" alt="">
<p>(b)</p>
</div>
</div>
</div>
<p class="caption">Figure 6: (a)EG with concentration of 0, 0.1%, 1.0%, 5.0% was added into the culture medium
of <i>E. coli BL21/pET28a-PD-MD-hA</i>; (b)TPA with content of 0, 0.1mM, 1.0mM, 10,0mM was added into the
culture medium
of <i>E. coli BL21/pET28a-PD-MD-hA</i>.</p>
<p>We need to verify whether the products of PET degradation ——TPA and EG inhibit or promote bacterial growth.
As shown in left part of Figure 6, EG with concentrations of 0%, 0.1%, 1% and 5% were added to the culture
medium respectively. After 19 hours of observation, the four growth curves were basically consistent, which
proved that EG had no significant effect on the growth of bacteria; On the other hand, in Figure 6 right, it
can be seen that <i>E. coli BL21</i> grew normally in the medium with low TPA content of 0, 0.1mM and 1mM.
However,
when the TPA content was too high(10mM), <i>E. coli BL21</i> did not grow. Obviously, high concentration of
TPA
will inhibit bacterial growth, so it is necessary to remove or further degrade TPA to avoid its high
concentration in the process of PET degradation in bacterial culture or in industrial production.</p>
</article>
<article class="article">
<h1>5. Kill switch for <i>E. coli BL21/pET28a-PD-MD-hA</i> and <i>E. coli BL21/pET28a-PD-MD-hA (Secretory)</i>
</h1>
<h2>①Kill switch for <i>E. coli BL21/pET28a-PD-MD-hA</i> and <i>E. coli BL21/pET28a-</i></h2>
<p>The internal killing switch was designed to rapidly eliminate Hetoul remained in the gastrointestinal tract
after the detection. This bacteria-lysing protein can destabilize bacterial cell membrane resulting in cell
death. Arabinose could be taken in through the digestive tract without being absorbed, which made it an
ideal inducer of internal killing switch.</p>
<p>Encoded by Phage Phi X 17,the internal killing switch is a combination of arabinose-induced promoter pBAD
(<a href="http://parts.igem.org/Part:BBa_K206000" target="_blank">BBa_K206000</a>), pBAD repressor AraC(<a
href="http://parts.igem.org/Part:BBa_I13458" target="_blank">BBa_I13458</a>) and toxic Protein E (<a
href="http://parts.igem.org/Part:BBa_K2500006" target="_blank">BBa_K2500006</a>).</p>
<div class="figures">
<div class="row">
<div class="col-12 col-md-8 mx-auto">
<img src="https://2021.igem.org/wiki/images/a/aa/T--DUT_China--poc_7.png" alt="">
</div>
</div>
</div>
<h2>②Kill switch for <i>E. coli BL21/pET28a-PD-MD-hA (Secretory)</i> in the environment</h2>
<p>In order to contain the possible contamination of genes in the environment, we've decided to adopted a
kill-switch system inspired by 2021 <a href="http://2021.igem.org/Team:Tongji_China" target="_blank">Tongji_China</a>
iGEM team.</p>
<div class="figures">
<div class="row">
<div class="col-12">
<img src="https://2021.igem.org/wiki/images/3/31/T--DUT_China--poc_8.png" alt="">
</div>
</div>
</div>
<p class="caption text-sm-center">Figure 8: Kill switch schematic diagram</p>
<p>The workflow of this kill-switch system involves three modes. During the propagation stage, the kill-switch
system is silenced, where only the TpiAB transporter is synthesized. Because of the presence of terminator
B0015, no toxin is produced.</p>
<p>When TPA is detected by the sensor system provided by a previous iGEM Team <a
href="http://2018.igem.org/Team:UMaryland" target="_blank">UMaryland</a>, suggesting the
degradation process has begun, the TphC transcriptional activator will be activated, thus initiating the
production of both the antitoxin and DRE recombinase. The recombinase will in turn cut the terminator B0015,
which leads to the permanent activation of the expression circuit of the toxin. However, under this
situation, both antitoxin and toxin are present in the system, so the cell itself will continue to thrive
and the degradation ensues.</p>
<p>After the detection of TPA, as long as the concentration of TPA reduces to a certain level, which means that
the degradation has finished or the cell is dispersed in the environment, the TphC activator will be turned
off, terminating the synthesis of antitoxin. Whereas the production of toxin is not regulated by TphC
activator and continues, the cell will be killed.</p>
<h2>Acknowledgements:</h2>
<p>We would like to thank Team <a href="http://2021.igem.org/Team:Tongji_China" target="_blank">Tongji_China</a>
for the inspiration of the system design. We also appreciate the previous work of Team <a
href="http://2018.igem.org/Team:UMaryland" target="_blank">UMaryland</a> on the TPA sensor system.
</p>
</article>
</section>
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