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<h2 class="section-heading mb-4">
<span class="section-heading-lower"><strong>Results </strong></span>
</h2>
<span id="s1" class="section-heading-upper h3"><br />Overview</span>
<figure class="text-center">
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style="height: 150px;">
<img class="img-fluid mx-auto product-item-img mb-3 mb-lg-0 rounded"
src="https://static.igem.wiki/teams/5459/wiki/desc/b1006-23119.jpg"
alt="Transcriptional unit 1" />
</div>
<figcaption>
Fig1(a) Schematic map of Transcriptional unit 1 : BBa_J23119 - BBa_B0030 - cueR
- BBa_B1006.
</figcaption>
</div>
<div class="flex-fill">
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style="height: 150px;">
<img class="img-fluid mx-auto product-item-img mb-3 mb-lg-0 rounded"
src="https://static.igem.wiki/teams/5459/wiki/desc/b0015-190017.jpg"
alt="Transcriptional unit 2" />
</div>
<figcaption>Fig1(b) Schematic map of Transcriptional unit 2 : BBa_K190017 -
BBa_B0034 - mVenusNB -BBa_B0015. </figcaption>
</div>
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style="height: 150px;">
<img class="img-fluid mx-auto product-item-img mb-3 mb-lg-0 rounded"
src="https://static.igem.wiki/teams/5459/wiki/desc/b1006-promoter.jpg"
alt="Transcription unit 3" />
</div>
<figcaption>Fig1(c) Schematic map of Transcription unit 3: Promoter variants - RBS
variants - cueR - BBa_B1006</figcaption>
</div>
</div>
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<span id="s2" class="section-heading-upper h3"><br />Successful Construction of a Copper
Biosensor with Single Plasmid System</span>
<p>We built a plasmid with only the reporter system Fig.1a, which was transferred into E. coli.
This system utilizes E. coli's own CueR protein to sense copper ions and output a
fluorescent signal from mVenusNB. In order to better simulate the possible environment in a
realistic test, we incubated at room temperature of 25 degrees. Here we show the kinetic
curves at different copper ion concentrations (Fig. 2a), and we can see that the system
exhibits a significant difference from the blank group even at a low copper ion
concentration of 170nM within 10hrs. We selected the data points at 35hr for fitting the
induced response curve, and it can be found that the reporter system is basically saturated
at 250uM Cu2+ concentration(Fig. 2b). This work demonstrate that our design to construct a
copper biosensor using endogenous proteins is feasible, revealing the effects of the CueR of
chassis bacteria overlooked by previous work utilizing this promoter.</p>
<figure class="text-center">
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style="height: 300px;">
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src="https://static.igem.wiki/teams/5459/wiki/engineering/fig2a-time-a-u-curves.png" />
</div>
<figcaption>
Fig2(a) Time-A.U. curves are shown for transformation of transcriptional1 alone.
</figcaption>
</div>
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style="height: 300px;">
<img class="img-fluid mx-auto product-item-img mb-3 mb-lg-0 rounded"
src="https://static.igem.wiki/teams/5459/wiki/engineering/fig2b-response-curve-of-section-i-to-cu-ii.png" />
</div>
<figcaption>Fig2(b) response curve of Single Plasmid System to Cu(II).</figcaption>
</div>
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</figure>
<span id="s3" class="section-heading-upper h3"><br />Successful Construction and a Copper
Biosensor with Dual Plasmid System</span>
<span id="s31" class="section-heading-upper h4"><br />Higher CueR Expression Raises induced
change and lowers the leak expression</span>
<p>We then introduced another plasmid for high expression of CueR and carved out the response of
this system to different Cu concentrations under the same conditions. Comparing to the above
system response curves(Fig. 3a), it can be seen that the system with high expression of CueR
still has a better resolution for copper between 250uM-2mM, allowing the system to be used
in environments with higher concentrations of copper ions. The system also has lower
background leakage(3-fold lower than section I circuit) and higher induced FP
multiplicity(Fig. 3b, 4), making it less prone to false positives. Overall, the high
expression CueR improves the original copper biosensor</p>
<figure class="text-center">
<div class="image-container d-flex justify-content-between">
<div class="flex-fill d-flex flex-column">
<div class="img-wrapper d-flex align-items-center justify-content-center">
<img class="img-fluid product-item-img rounded"
src="https://static.igem.wiki/teams/5459/wiki/engineering/fig3a-time-a-u-curvesof-section-ii.png"
alt="Time-A.U. curves" />
</div>
<figcaption>
Fig3(a) Time-A.U. curves are shown for transformation of transcriptional1
</figcaption>
</div>
<div class="flex-fill d-flex flex-column">
<div class="img-wrapper d-flex align-items-center justify-content-center">
<img class="img-fluid product-item-img rounded"
src="https://static.igem.wiki/teams/5459/wiki/engineering/fig3b-response-curve-of-section-ii-to-cu-ii.png"
alt="Response curve" />
</div>
<figcaption>Fig3(b) Response curve of dual plasmid system to Cu(II)</figcaption>
</div>
</figure>
<span id="s31" class="section-heading-upper h4"><br />Increased CueR expression lowers the max
induced Fluorescence</span>
<p>It is also important to note that increasing CueR expression in our system was found to
simultaneously decrease maximal fluorescence expression(Fig. 4), contrary to expectations.
We post a potential explanation for this is that since the cell burden is limited, the
presence of a highly expressed CueR plasmid forced the intracellular anabolic flow to be
split between two exogenous proteins (mVenusNB and CueR) at the same time, which affects the
maximal expression of mVenus. </p>
<figure class="text-center">
<div class="image-container d-flex justify-content-between">
<div class="flex-fill d-flex flex-column">
<div class="img-wrapper d-flex align-items-center justify-content-center">
<img class="img-fluid product-item-img rounded"
src="https://static.igem.wiki/teams/5459/wiki/engineering/fig4-comparison-of-respose-curve.png"
alt="Time-A.U. curves" />
</div>
<figcaption>Fig4 The comparison of curve between the two section.</figcaption>
</div>
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</figure>
<span id="s4" class="section-heading-upper h3"><br />Fine-tuning the CueR expression contributes
to high-coverage copper receptor libraries</span>
<p>We randomly inserted Promoter and RBS libraries into the CueR 5' end to create a plasmid
library with different CueR expression intensities. After cotransforming this plasmid
library with the reporter plasmid, we obtained a Cu(II) biosensor library. We picked 96
strains and measured the kinetic of OD600 and fluorescence at 0uM and 500uM, respectively.
In this library, the fluorescence multiplicity before and after induction at 500uM ranged
from 5- to 10-fold(Fig.5), showing a broad range of the regulation. 8 of 96 strains had a
higher maximum fluorescence after induction than the two systems in sections I & II
(>35,000) under the same parameter conditions(Fig. 6). Our project demonstrates the
feasibility of tuning the CueR expression to parameterize the circuit and provides a
biosensor library for Cu(II) ions.</p>
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src="https://static.igem.wiki/teams/5459/wiki/engineering/fig5a-growth-curves-of-96-samples-without-indution.png" />
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style="height: 150px;">
<img class="img-fluid product-item-img rounded"
src="https://static.igem.wiki/teams/5459/wiki/engineering/fig5b-bar-plot-of-96-samples-without-indution.png" />
</div>
</div>
<div class="flex-fill d-flex flex-column">
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style="height: 150px;">
<img class="img-fluid product-item-img rounded"
src="https://static.igem.wiki/teams/5459/wiki/engineering/fig5c-heatmap-of-96-samples-without-indution.png" />
</div>
</div>
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<figcaption style="margin-top: -120px;">Figure 5. a, shows the expression levels of 96 different experimental groups in
the absence of copper induction, the horizontal coordinate is time and the vertical
coordinate is the relative fluorescence intensity (RFU) obtained by dividing the A.U
value after removing the background by the OD value, which reflects the local expression
levels of different Promoter RBS combinations, and the individual experimental data and
graphs of each set of experiments are shown in the Appendix. b, shows the histogram of
RFU peaks for 96 different experimental groups in the absence of copper induction. c,
shows the heatmap of RFU peaks for 96 different experimental groups in the absence of
copper induction.</figcaption>
</figure>
<br /><br />
<figure class="text-center">
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<div class="flex-fill d-flex flex-column">
<div class="img-wrapper d-flex align-items-center justify-content-center"
style="height: 150px;">
<img class="img-fluid product-item-img rounded"
src="https://static.igem.wiki/teams/5459/wiki/engineering/fig6a-growth-curves-of-96-samples-with-indution.png" />
</div>
</div>
<div class="flex-fill d-flex flex-column">
<div class="img-wrapper d-flex align-items-center justify-content-center"
style="height: 150px;">
<img class="img-fluid product-item-img rounded"
src="https://static.igem.wiki/teams/5459/wiki/engineering/fig6b-bar-plot-of-96-samples-with-indution.png" />
</div>
</div>
<div class="flex-fill d-flex flex-column">
<div class="img-wrapper d-flex align-items-center justify-content-center"
style="height: 150px;">
<img class="img-fluid product-item-img rounded"
src="https://static.igem.wiki/teams/5459/wiki/engineering/fig6c-heatmap-of-96-samples-with-indution.png" />
</div>
</div>
</div>
<figcaption style="margin-top: -120px;">Figure 6. a, shows the expression levels of 96 different experimental groups with 500 uM Cu(II) induction, the horizontal coordinate is time and the vertical coordinate is the relative fluorescence intensity (RFU) obtained by dividing the A.U value after removing the background by the OD value, which reflects the local expression levels of different Promoter RBS combinations, and the individual experimental data and graphs of each set of experiments are shown in the Appendix. b, shows the histogram of RFU peaks for 96 different experimental groups with 500 uM Cu(II) induction. c, shows the heatmap of RFU peaks for 96 different experimental groups with 500 uM Cu(II) induction.</figcaption>
</figure>
<br /><br />
<figure class="text-center">
<div class="image-container d-flex justify-content-between">
<div class="flex-fill d-flex flex-column">
<div class="img-wrapper d-flex align-items-center justify-content-center"
style="height: 150px;">
<img class="img-fluid product-item-img rounded"
src="https://static.igem.wiki/teams/5459/wiki/engineering/fig7a-bar-plot-of-ratio.png" />
</div>
</div>
<div class="flex-fill d-flex flex-column">
<div class="img-wrapper d-flex align-items-center justify-content-center"
style="height: 150px;">
<img class="img-fluid product-item-img rounded"
src="https://static.igem.wiki/teams/5459/wiki/engineering/fig7b-heatmap-of-ratio.png" />
</div>
</div>
</div>
<figcaption style="margin-top: -120px;">Figure 7. a, barplot of induced fold-changes of fine-tuned CueR system. b, heatmap of induced fold-changes of fine-tuned CueR system.</figcaption>
</figure>
<p>We observed that all promoters in the promoter library exhibit a certain level of promoter
activity, indicating their significant role in transcriptional regulation. The initiation
strength of the promoters shows a degree of variability, with the lowest induction strength
being 5-fold, and the highest reaching up to 10-fold. To gain a deeper understanding of the
relationship between the activity of these promoters and their sequence characteristics, we
employed Sanger sequencing to determine the nucleotide sequences of these promoters.
Subsequently, based on the sequencing results, we conducted a correlation analysis between
the initiation strength of the promoters and their sequence features, and built a
mathematical model accordingly. The detailed construction process and parameter settings of
this model can be consulted on our provided "Model" page. Through this model, we aim to
reveal the specific mechanisms by which promoter sequence characteristics affect their
initiation activity, providing a theoretical foundation and experimental guidance for
subsequent research on gene expression regulation.</p>
<span id="s5" class="section-heading-upper h3"><br />Characterization of the impact of different
metal ions on the CueR-pCoA system</span>
<p>We assessed the impact of metal ions (Ca²⁺, Na⁺, K⁺) on the expression of mVenus NB and
binding of CueR in our E. coli system. A series of test conditions on 96 plate were prepared
and bacterial cultures were exposed to the metal ion solutions for a standardized period.
After treating the bacteria with metal ion solutions, we measured the fluorescence intensity
of mVenus NB in the bacterial cells for each metal ion treatment. We found that CueR is
highly specific to Cu2+ only.</p>
<figure class="text-center">
<div class="image-container d-flex justify-content-between">
<div class="flex-fill d-flex flex-column">
<div class="img-wrapper d-flex align-items-center justify-content-center">
<img class="img-fluid product-item-img rounded"
src="https://static.igem.wiki/teams/5459/wiki/results/res8.png" />
</div>
</div>
</div>
<figcaption style="margin-top: -10px;">Figure9. The response of CueR-pCoA system upon different Cu2+ concentrations over time.</figcaption>
</figure>
<span id="s6" class="section-heading-upper h3"><br />Understanding the effect of Cu2+
concentration on E.coli cell growth</span>
<p>We determined the impact of different Cu2+ concentrations on E.coli growth. E.coli containing
the CueR-pCoA system and E.coli with empty plasmid as control were cultured to exponential
growth stage and exposed to a series of Cu²⁺ solutions of different concentrations. The
florescent intensity curve over time was measured using microplate reader. We found that
concentration that <=12.5mM hinders the growth of bacteria. We can take this possibility
into account when working with data collected in the field.</p>
<figure class="text-center">
<div class="image-container d-flex justify-content-between">
<div class="flex-fill d-flex flex-column">
<div class="img-wrapper d-flex align-items-center justify-content-center">
<img class="img-fluid product-item-img rounded"
src="https://static.igem.wiki/teams/5459/wiki/results/res9.png" />
</div>
</div>
</div>
<figcaption style="margin-top: -10px;">Figure9. The response of CueR-pCoA system upon different Cu2+ concentrations over time.</figcaption>
</figure>
</div>
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