diff --git a/wiki/pages/attributions.html b/wiki/pages/attributions.html
index 677056f629ae4fd1f9f5296485998320035d6078..012dd471a0e022c0cad9f02ce2dbea0dbe210476 100644
--- a/wiki/pages/attributions.html
+++ b/wiki/pages/attributions.html
@@ -53,7 +53,7 @@
<section>
<h3>Instructor Support</h3>
- <ul class="ul-mark mark-diamond2 l-start l-top-05">
+ <ul class="ul-mark mark-diamond2 l-start l-top-05 text-justify">
<li>Haining Wang: Expert coaching for dry team in text writing</li>
<li>Meirong Chen : wet team coach for lab and data analysis</li>
</ul>
@@ -61,11 +61,10 @@
<section>
<h3>Human Practice Support</h3>
- <ul class="ul-mark mark-diamond2 l-start l-top-05">
- <li>Pro. Huimei Shan, Doctor of Engineering, China University of Geosciences.</li>
- <li>Pro. Shiyuan Li, Cathy Fortune Capital Investment Synthetic Biology Line Research Business Director.
- </li>
- <li>Pro. Wen Zhiqiang, the doctor of biochemical engineering from the Zhejiang University.</li>
+ <ul class="ul-mark mark-diamond2 l-start l-top-05 text-justify">
+ <li>Prof. Huimei Shan, College of Environmental Science and Engineering, Guilin University of Technology</li>
+ <li>Dr. Shiyuan Li, Cathy Fortune Capital Investment Synthetic Biology Line Research Business Director</li>
+ <li>Prof. Wen Zhiqiang, School of Food and Pharmaceutical Engineering, Nanjing Normal University, PhD. from Zhejiang University</li>
<li>Jinke Environment Co. LTD</li>
<li>Zhanjiang Zhengda ENT Hospital</li>
<li>Suzhou Industrial Park Qingyuan Huayan Water Co., Lt</li>
@@ -75,7 +74,7 @@
<section>
<h3>Collaboration</h3>
- <ul class="ul-mark mark-diamond2 l-start l-top-05">
+ <ul class="ul-mark mark-diamond2 l-start l-top-05 text-justify">
<li>Team De-PETer (Beijing_United) from Beijing on July 25th through an online conference.</li>
<li>Team Ø-HAS (Shanghai_Metropolis) from Shanghai on July 27th through an online conference.</li>
</ul>
diff --git a/wiki/pages/engineering.html b/wiki/pages/engineering.html
index cc7b9371b40bbafc1cc7bb9b1d9e3dea481d8de5..4176b850ca1e0e5dea0ea0f4eb16f22024191afa 100644
--- a/wiki/pages/engineering.html
+++ b/wiki/pages/engineering.html
@@ -10,57 +10,148 @@
<h1 class="content-header">Engineering Success</h1>
<section>
- <h2>Introduction</h2>
+ <h2>Overview </h2>
<p>
- As more and more human activities and industries develop at an unprecedentedly rapid speed, huge amounts of
- heavy metal pollutants are released into the environment, contaminating the earth. Heavy metal ions such as
- Cadmium (Cd), Chromium (Cr), Lead (Pb), Mercury (Hg), and metalloids like Arsenic (As) cannot be degraded or
- absorbed by organisms. As a result, these ions can build up, create chronic toxicity, and manifest
- bioaccumulation up the trophic level.
+ Feruloyl esterase (FAE, EC 3.1.1.73), also known as cinnamate esterase or cinnamic acid hydrolase, is an
+ extracellular carboxylesterase that mainly degrades cell wall polysaccharides or arabinose in pectin or an
+ ester bond between galactose or hydroxycinnamic acid. Our project aims to couple the concentration of heavy
+ metals in the sample to be tested with the color in the reaction solution through the catalysis of FAE to
+ produce a color product, so as to visually display the concentration of metal ions in the sample. Therefore,
+ we designed a plasmid pHY300PLK-Pveg-Biosensor for constitutive expression FAE in <i>Bacillus subtilis to
+ test the enzymatic activity of FAE</i> to provide enzyme data for our biosensor.
</p>
</section>
<section>
- <h2>Design</h2>
+ <h2>Construct design </h2>
<p>
- The CadA gene is found in B. subtilis, where it functions to increase Cd resistance. This Cadmium Biosensor
- uses the promoter of the sequence, CadA Promoter, which enables transcription if cadmium is present.
+ The pHY300PLK-Veg-biosensor construct used to constantly express the feruloyl esterase in order to test the
+ enzymatic activity.
</p>
+ <div class="imager">
+ <img class="rw-65"
+ src="https://static.igem.wiki/teams/4288/wiki/engineering/t-fujian-united-engineering-03.jpg" alt="">
+ <span class="figure">
+ Figure 1. map of pHY300PLK-Veg-biosensor
+ </span>
+ </div>
+
+ <h3>1.1 The promotor veg fragment was amplified by PCR. </h3>
+ <div class="imager">
+ <img class="rw-25"
+ src="https://static.igem.wiki/teams/4288/wiki/engineering/t-fujian-united-engineering-04.jpg" alt="">
+ <span class="figure">
+ Figure 2. gel electrophoresis result of Veg fragment <br>
+ M: DNA ladder; lane1-2 promoter veg fragment
+ </span>
+ </div>
+ <p>
+ The promoter veg DNA fragment was amplified by primers Veg-F and Veg-R. The length of promoter Veg is 96 bp.
+ In addition, we used the super-fidelity Pfu DNA polymerase and its extension time of Pfu is 2 min/kb. The
+ extension time of PCR depends on the length of target DNA and DNA polymerase. Thus, the thermal cycler program
+ set as 20 seconds for extension time of PCR at 72℃. The PCR products were analyzed by 1.5% agar gel
+ electrophoresis. The electrophoresis result showed correct band in figure 2. The band of promoter Veg were
+ extract by gel extraction kit according to the protocol. The DNA fragment concentration was determined using
+ NanoDrop. The result showed the Veg fragment concentration is 3 ng/μl at a final volume of 40 μl.
+ </p>
+
+ <h3>1.2 The feruloyl esterase-pHY300PLK fragment was obtained by PCR</h3>
+ <div class="imager">
+ <img class="rw-25"
+ src="https://static.igem.wiki/teams/4288/wiki/engineering/t-fujian-united-engineering-05.jpg" alt="">
+ <span class="figure">
+ Figure3. gel electrophoresis result of feruloyl esterase-pHY300PLK fragment <br>
+ lane1: DNA ladder; <br>
+ lane2: feruloyl esterase-pHY300PLK fragment
+ </span>
+ </div>
+ <p>
+ The biosensor-pHY300PLK fragment was amplified by PCR using the primers pVector-F and pVector-R. The template
+ is biosensor-pHY300PLK plasmid which was extracted from E.coli Top10. Oving to the length of
+ biosensor-pHY300PLK fragment (5902 bp), we prolong the extension time of PCR for 340 seconds at 72℃ although
+ we used the same Pfu DNA polymerase. The electrophoresis result showed correct band in figure 3. The band
+ biosensor-pHY300PLK of were extract by gel extraction kit according to the protocol. Biosensor-pHY300PLK
+ concentration is 3 ng/μl at a final volume of 40 μl, which was measured by NanoDrop.
+ </p>
+
+ <h3>1.3 LB agar plate containing the single colonies of Veg-biosensor </h3>
<p>
- Feruloyl Esterase, a type of hydrolase, is responsible for catalyzing the 4-nitrophenyl ferulate (4NPF) into
- 4-Nitrophenol, the latter may be observed at the 410 nm wavelength, thus determined the enzyme activity.
+ The Veg fragment and pHY300PLK vector ligated using Gibson assembly method. Then, the recombinant plasmid was
+ transformed into the competent cells DH10. The bacteria spread to the selection plate with antibiotic and
+ incubated at 37 ℃ overnight.
</p>
<div class="imager">
- <img class="rw-75"
- src="https://static.igem.wiki/teams/4288/wiki/engineering/t-fujian-united-engineering-01.jpg" alt="">
+ <img class="rw-50"
+ src="https://static.igem.wiki/teams/4288/wiki/engineering/t-fujian-united-engineering-06.jpg" alt="">
<span class="figure">
- Figure 1. The principle of the pET28a-Biosensor Function
+ Figure 4. LB agar plate for recombinant plasmid
+ </span>
+ </div>
+
+ <h3>1.4 colony PCR </h3>
+ <p>
+ We picked up 16 colonies for performing colony PCR. Colony PCR system comprised of primers pVeg-verf-up and
+ pVeg-verf-dn, Taq Master mix (DNA polymerase, buffer, loading, etc.), and colonies. The electrophoresis result
+ showed all selected colonies have correct band, figure 4. Thus, we sent No. 1, 3, 5, and 7 to DNA sequencing.
+ The M representative maker, 1-16 representative colonies containing the recombinant plasmid.
+ </p>
+ <div class="imager">
+ <img class="rw-65"
+ src="https://static.igem.wiki/teams/4288/wiki/engineering/t-fujian-united-engineering-08.jpg" alt="">
+ <span class="figure">
+ Figure5. colony PCR of Veg-biosensor recombination plasmid
+ </span>
+ </div>
+
+ <h3>1.5 Sequencing of Veg-biosensor recombination plasmid</h3>
+ <p>
+ The sequence alignment showed that there is no mutation or mismatch. Thus, we chose the No.3 for subsequent
+ assay.
+ </p>
+ <div class="imager">
+ <img class="rw-100"
+ src="https://static.igem.wiki/teams/4288/wiki/engineering/t-fujian-united-engineering-07.jpg" alt="">
+ <span class="figure">
+ Figure6. Sequence alignment
</span>
</div>
</section>
<section>
- <h2>Build</h2>
+ <h2>Functional Test</h2>
<p>
- Cadmium biosensor DNA fragment is commercially synthesized. It was digested with the enzymes EcoR1 and BamH1
- before assembly. Then, the DNA fragment inserted pET28a backbone vector (Left) which also obtained
- commercially. Map of pET28a-biosensor plasmid showed right panel in Figure 2. Meanwhile, the final vector
- verified by Sanger sequencing.
+ In order to confirm the protein expression and secret into the culture medium, the plasmid
+ pHY300PLK-Pveg-Biosensor which can consistently express feruloyl esterase(FAE) was transformed into <i>Bacillus
+ subtilis</i> and contand tested in different cultured time. Because FAE catalyzes the decomposition of the
+ substrate methyl ferulate, and the enzyme activity of FAE can be obtained by detecting the decline rate of
+ methyl ferulate at 340 nm. The chemical equation is as follow:
+ </p>
+ <p class="text-center">
+ feruloyl-polysaccharide + H<sub>2</sub>O = ferulate + polysaccharide
+ </p>
+ <p>
+ The results showed that the enzymatic activity of FAE increased with the prolongation of culture time,
+ indicating that the expression of enzyme increased with the increase of culture time.
</p>
<div class="imager">
- <img class="rw-100"
- src="https://static.igem.wiki/teams/4288/wiki/engineering/t-fujian-united-engineering-02.jpg" alt="">
+ <img class="rw-50"
+ src="https://static.igem.wiki/teams/4288/wiki/engineering/t-fujian-united-engineering-09.jpg" alt="">
<span class="figure">
- Figure 2. maps of pET28a and pET28a-biosensor
+ Figure 7. The activity of ferulyol esterase in different culture time.
</span>
</div>
</section>
<section>
- <h2>Discussion</h2>
+ <h2>Learn</h2>
<p>
- Our experiments showed that the pET-28a-Biosensor plasmid is able to detect Cadmium ions in the sample, and is
- quantifiable. It is potential to apply our engineered bacteria to detect cadmium ion in water samples.
+ We have already collected the enzyme activity data from our experiments. FAE has the effect of catalyzing
+ methyl ferulate to ferulate and polysaccharide. At the same time, since we detected the enzymatic activity of
+ the protein in the supernatant of the culture medium, this shows that our protein can indeed be expressed in
+ <i>Bacillus subtilis</i> and secreted to the outside of the cell, which verifies that our design idea is
+ correct. It
+ has laid a solid foundation for the cadmium-inducible promoter(Pcad) and the detection of cadmium ion
+ concentration.
</p>
</section>
</div>
diff --git a/wiki/pages/human-practices.html b/wiki/pages/human-practices.html
index 4871f321b318cdae370dc1e0d9526cabae92c387..6bf8da32f0dee586584035bdf62ff396fdd4eba1 100644
--- a/wiki/pages/human-practices.html
+++ b/wiki/pages/human-practices.html
@@ -505,7 +505,7 @@
src="https://static.igem.wiki/teams/4288/wiki/ihp/t-fujian-united-ihp-12.jpg"
alt="">
<span class="figure">
- Figure 12. Inside views of the
+ Figure 12. Inside views of the waterworks
</span>
</div>
<p>
@@ -611,6 +611,9 @@
<img class="rw-75"
src="https://static.igem.wiki/teams/4288/wiki/ihp/t-fujian-united-ihp-17.jpg"
alt="">
+ <span class="figure">
+ We were talking to the staff from Zhanjiang Zhengda ENT Hospital and taking notes
+ </span>
</div>
<p>
Firstly the staff introduced the harm of medical waste if it is not well disposed of. Since he has worked in
diff --git a/wiki/pages/improve.html b/wiki/pages/improve.html
index 3db7ec3d6c634a7e0f0c44b39c2deacaa66bf566..da805cd5de8a3e217e8e0e91dd61938bc1f8f737 100644
--- a/wiki/pages/improve.html
+++ b/wiki/pages/improve.html
@@ -11,6 +11,33 @@
<section>
<h2>Overview </h2>
+ <p>
+ Our composite part BBa_K4288005 was improved based on the existing part BBa_K174015 submitted by
+ iGEM09_Newcastle. In 2019, group iGEM_Gaston Day School combined this part with an RBS (B0036), RFP (E1010),
+ and a double terminator (B0014), and has shown that this promoter is sensitive to cadmium down to 0.25M
+ (p<0.01). Today, our team further improved the cadmium sensor by optimizing the sequence of cad promoter,
+ fusing a new signal peptide of RpmG which helps protein secret outside the cell, and combining a report
+ protein feruloyl esterase(FAE). Therefore, the new composite part BBa_K4288005 was developed.
+ </p>
+ <div class="imager">
+ <img class="rw-100"
+ src="https://static.igem.wiki/teams/4288/wiki/improvment/t-fujian-united-improvment-07.jpg"
+ alt="">
+ <span class="figure">
+ Figure 1. The DNA sequence difference between BBa_K4288005 and BBa_ K174015).
+ </span>
+ </div>
+ <p>
+ In order to prove the function of our new composite part cadA promoter-signal peptide of RpmG-feruloyl
+ esterase, we transformed the part in <i>Bacillus subtilis</i>, induced FAE protein expression with various
+ concentrations of cadmium nitrate, and detected the enzyme activity of FAE. The result shows that our
+ biosensor can detect the concentration of cadmium ions within 50μg/L, which broadens the concentration range
+ of biological detection of cadmium ions and takes a more important step towards practical application.
+ </p>
+ </section>
+
+ <section>
+ <h2>Introduction</h2>
<p>
As more and more human activities create accumulating impact, and as industries develop at an unprecedentedly
rapid speed, huge amounts of heavy metal pollutants are released into the environment, contaminating the
@@ -24,104 +51,59 @@
bodies, they can cause serious symptoms such as diseases in the kidney, the lung, the liver, the bone,
different types of cancers, and deficiency in embryonic neurodevelopment.
</p>
+ <p>
+ Therefore, it is urgent to develop a fast and convenient tool for the detection of heavy metals. Our project
+ designed a cadmium biosensor to detect the heavy metal cadmium.
+ </p>
+ </section>
+
+ <section>
+ <h2>Construction of Cadmium-biosensor plasmid</h2>
+ <p>
+ We utilize the operon CadA to expresses reporter feruloyl esterase in <i>Bacillus subtilis</i> and the plasmid
+ pHY300PLK-PcadA-Biosensor was handed over to GenScript for synthesis. The synthesis report is as follows.
+ </p>
+
+ <object type="application/pdf"
+ data="https://static.igem.wiki/teams/4288/wiki/improvment/t-fujian-united-improvment.pdf"
+ style="width: 100%; height: 120vh; margin-top: 2rem">
+ </object>
</section>
<section>
- <h2>Construct design</h2>
+ <h2>Functional test</h2>
<p>
- The pHY300PLK-Veg-biosensor construct used to constantly express the feruloyl esterase in order to test the
- enzymatic activity.
+ We tested the ability of cadmium-biosensor for detecting the heavy metal cadmium. The plasmid
+ pHY300PLK-PcadA-Biosensor was transformed into <i>Bacillus subtilis</i>(Fig2.) and tested while in different
+ concentration of cadmium nitrate. The enzyme activity of feruloyl esterase(FAE) is to reflect the cadmium
+ concentration after the induction of cadmium ions. Under different cadmium concentration scales, the enzyme
+ activity of FAE is shown in the figure 3.
</p>
<div class="imager">
- <img class="rw-50" src="https://static.igem.wiki/teams/4288/wiki/improvment/t-fujian-united-improvment-01.jpg"
+ <img class="rw-50"
+ src="https://static.igem.wiki/teams/4288/wiki/improvment/t-fujian-united-improvment-08.jpg"
alt="">
<span class="figure">
- Figure 1. map of pHY300PLK-Veg-biosensor
+ Figure 2. Plate of the transformants of pHY300PLK-PcadA-Biosensor/Bacillus subtilis.
</span>
</div>
-
- <section>
- <h3>1.1 The promotor veg fragment was amplified by PCR. </h3>
- <div class="imager">
- <img class="rw-25"
- src="https://static.igem.wiki/teams/4288/wiki/improvment/t-fujian-united-improvment-02.jpg" alt="">
- <span class="figure">
- Figure 2. gel electrophoresis result of Veg fragment <br>
- M: DNA ladder; lane1-2 promoter veg fragment
- </span>
- </div>
- <p>
- The promoter veg DNA fragment was amplified by primers Veg-F and Veg-R. The length of promoter Veg is 96 bp.
- In addition, we used the super-fidelity Pfu DNA polymerase and its extension time of Pfu is 2 min/kb. The
- extension time of PCR depends on the length of target DNA and DNA polymerase. Thus, the thermal cycler
- program set as 20 seconds for extension time of PCR at 72℃. The PCR products were analyzed by 1.5% agar gel
- electrophoresis. The electrophoresis result showed correct band in figure 2. The band of promoter Veg were
- extract by gel extraction kit according to the protocol. The DNA fragment concentration was determined using
- NanoDrop. The result showed the Veg fragment concentration is 3 ng/μl at a final volume of 40 μl.
- </p>
- </section>
-
- <section>
- <h3>1.2 The feruloyl esterase-pHY300PLK fragment was obtained by PCR</h3>
- <div class="imager">
- <img class="rw-25"
- src="https://static.igem.wiki/teams/4288/wiki/improvment/t-fujian-united-improvment-03.jpg" alt="">
- <span class="figure">
- Figure3. gel electrophoresis result of feruloyl esterase-pHY300PLK fragment <br>
- lane1: DNA ladder; <br>
- lane2: feruloyl esterase-pHY300PLK fragment
- </span>
- </div>
- <p>
- The biosensor-pHY300PLK fragment was amplified by PCR using the primers pVector-F and pVector-R. The
- template is biosensor-pHY300PLK plasmid which was extracted from E.coli Top10. Oving to the length of
- biosensor-pHY300PLK fragment (5902 bp), we prolong the extension time of PCR for 340 seconds at 72℃ although
- we used the same Pfu DNA polymerase. The electrophoresis result showed correct band in figure 3. The band
- biosensor-pHY300PLK of were extract by gel extraction kit according to the protocol. Biosensor-pHY300PLK
- concentration is 3 ng/μl at a final volume of 40 μl, which was measured by NanoDrop.
- </p>
- </section>
-
- <section>
- <h3>1.3 LB agar plate containing the single colonies of Veg-biosensor </h3>
- <p>
- The Veg fragment and pHY300PLK vector ligated using Gibson assembly method. Then, the recombinant plasmid
- was transformed into the competent cells DH10. The bacteria spread to the selection plate with antibiotic
- and incubated at 37 ℃ overnight.
- </p>
- <div class="imager">
- <img class="rw-45"
- src="https://static.igem.wiki/teams/4288/wiki/improvment/t-fujian-united-improvment-04.jpg" alt="">
- <span class="figure">
- Figure 4. LB agar plate for recombinant plasmid
- </span>
- </div>
- </section>
-
- <section>
- <h3>1.4 colony PCR </h3>
- <p>
- We picked up 16 colonies for performing colony PCR. Colony PCR system comprised of primers pVeg-verf-up and
- pVeg-verf-dn, Taq Master mix (DNA polymerase, buffer, loading, etc.), and colonies. The electrophoresis
- result showed all selected colonies have correct band, figure 4. Thus, we sent No. 1, 3, 5, and 7 to DNA
- sequencing. The M representative maker, 1-16 representative colonies containing the recombinant plasmid.
- </p>
- </section>
-
- <section>
- <h3>1.5 Sequencing of Veg-biosensor recombination plasmid</h3>
- <p>
- The sequence alignment showed that there is no mutation or mismatch. Thus, we chose the No.3 for subsequent
- assay.
- </p>
- <div class="imager">
- <img class="rw-100"
- src="https://static.igem.wiki/teams/4288/wiki/improvment/t-fujian-united-improvment-06.jpg" alt="">
- <span class="figure">
- Figure6. Sequence alignment
- </span>
- </div>
- </section>
+ <div class="imager">
+ <img class="rw-65"
+ src="https://static.igem.wiki/teams/4288/wiki/improvment/t-fujian-united-improvment-09.jpg"
+ alt="">
+ <span class="figure">
+ Figure 3. The activity of Ferulyol Esterase in different concentaiton of Cd.
+ The data were collected in 0.5h.
+ </span>
+ </div>
+ <p>
+ As shown in the graph, the more the cadmium is, the more enzyme activity it has. While, when the concentration
+ of cadmium reaches 50μg/L, its enzyme activity value tends to be stable at about 0.35. We speculate that when
+ the cadmium ion concentration exceeds 50μg/L, the host <i>Bacillus subtilis</i> cannot express the protein
+ normally,
+ so the measured enzyme activity value is flat. This indicates that our current cadmium biosensor is suitable
+ for the detection of samples with cadmium ion concentration in the range of 0-50μg/L.
+ </p>
</section>
</div>
</div>
diff --git a/wiki/pages/notebook.html b/wiki/pages/notebook.html
index 543dbd105e39cb3488870d520a6d2f3d99cfccbe..9b559deb3972328638e2d7e6cf2050cae51ed63b 100644
--- a/wiki/pages/notebook.html
+++ b/wiki/pages/notebook.html
@@ -148,6 +148,21 @@
alt="">
</div>
</section>
+
+ <section>
+ <h2>8.6-8.15</h2>
+ <ul class="l-top-05">
+ <li>Functional test of cadmium biosensor (pHY300PLK-PcadA-Biosensor / <i>Bacillus subtilis</i>) and repeat at
+ least three times
+ </li>
+ <li>Collect and analysis data</li>
+ <li>Added to the experimental results of the wiki</li>
+ </ul>
+ <div class="imager">
+ <img class="rw-75" src="https://static.igem.wiki/teams/4288/wiki/notebook/t-fujian-united-notebook-11.jpg"
+ alt="">
+ </div>
+ </section>
</div>
</div>
</div>
diff --git a/wiki/pages/results.html b/wiki/pages/results.html
index 2b39a4bd731900ef2d3356ae2520dcd638acb33a..425423c6e374288ddb75fe9161916cd42d7c4693 100644
--- a/wiki/pages/results.html
+++ b/wiki/pages/results.html
@@ -85,6 +85,8 @@
<section>
<h2>2. Functional Test</h2>
+
+ <h3 class="upper">2.1 Arsenic-biosensor </h3>
<p>
We have tested the ability of three biosensors for detecting the heavy metal arsenic. The plasmids were
transformed into <i>E. coli</i> and tested while in LB medium. The florescence intensity to reflect the GFP
@@ -115,6 +117,64 @@
arsenic ion concentration in the sample during the reaction period of 2 hours and the detection concentration
range from 0 to 100μg/L, which is in line with the expectations of the project design.
</p>
+
+ <h3>2.2 The Activity of Feruloyl Esterase expressed by promoter veg</h3>
+ <p>
+ In order to confirm the protein expression and secret into the culture medium, the plasmid
+ pHY300PLK-Pveg-Biosensor which can consistently express feruloyl esterase(FAE) was transformed into <i>Bacillus
+ subtilis</i> and contand tested in different cultured time. Because FAE catalyzes the decomposition of the
+ substrate methyl ferulate, and the enzyme activity of FAE can be obtained by detecting the decline rate of
+ methyl ferulate at 340 nm. The chemical equation is as follow:
+ </p>
+ <p class="text-center">
+ feruloyl-polysaccharide + H2O = ferulate + polysaccharide
+ </p>
+ <div class="imager">
+ <img class="rw-65" src="https://static.igem.wiki/teams/4288/wiki/results/t-fujian-united-results-06.jpg"
+ alt="">
+ <span class="figure">
+ Figure 6. The activity of ferulyol esterase in different culture time.
+ </span>
+ </div>
+ <p>
+ The results showed that the enzymatic activity of FAE increased with the prolongation of culture time,
+ indicating that the expression of enzyme increased with the increase of culture time. At the same time, since
+ we detected the enzymatic activity of the protein in the supernatant of the culture medium, this shows that
+ our protein can indeed be expressed in <i>Bacillus subtilis</i> and secreted to the outside of the cell, which
+ verifies that our design idea is correct. It has laid a solid foundation for the cadmium-inducible
+ promoter(Pcad) and the detection of cadmium ion concentration.
+ </p>
+
+ <h3>2.3 Cadmium-biosensor </h3>
+ <p>
+ We tested the ability of cadmium-biosensor for detecting the heavy metal cadmium. The plasmid
+ pHY300PLK-PcadA-Biosensor was transformed into <i>Bacillus subtilis</i>(Fig7.) and tested while in different
+ concentration of cadmium nitrate. The enzyme activity of feruloyl esterase(FAE) is to reflect the cadmium
+ concentration after the induction of cadmium ions. Under different cadmium concentration scales, the enzyme
+ activity of FAE is shown in the figure 8.
+ </p>
+ <div class="imager">
+ <img class="rw-50" src="https://static.igem.wiki/teams/4288/wiki/results/t-fujian-united-results-07.jpg"
+ alt="">
+ <span class="figure">
+ Figure 7. Plate of the transformants of pHY300PLK-PcadA-Biosensor/Bacillus subtilis.
+ </span>
+ </div>
+ <div class="imager">
+ <img class="rw-65" src="https://static.igem.wiki/teams/4288/wiki/results/t-fujian-united-results-08.jpg"
+ alt="">
+ <span class="figure">
+ Figure 8. The activity of Ferulyol Esterase in different concentaiton of Cd.
+ The data were collected in 0.5h.
+ </span>
+ </div>
+ <p>
+ As shown in the graph, the more the cadmium is, the more enzyme activity it has. While, when the concentration
+ of cadmium reaches 50μg/L, its enzyme activity value tends to be stable at about 0.35. We speculate that when
+ the cadmium ion concentration exceeds 50μg/L, the host <i>Bacillus subtilis</i> cannot express the protein normally,
+ so the measured enzyme activity value is flat. This indicates that our current cadmium biosensor is suitable
+ for the detection of samples with cadmium ion concentration in the range of 0-50μg/L.
+ </p>
</section>
</div>
</div>