iGEM 2022 — Team fujian-united

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{% block title %}Notebook{% endblock %}
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# Notebook

## 7.23

* Participated in a lecture on experiment procedures
* Familiarize with the construction of the designed plasmid
* Went through the local laboratory’s safety protocol and iGEM official safety guideline
* Familiarize with the operation of equipment

![](https://static.igem.wiki/teams/4288/wiki/notebook/t-fujian-united-notebook-01.jpg)

## 7.24

* Preparing pipette tips and both liquid/solid medium
* Disinfected pipette tips and the mediums
* Prepared the antibiotics
* Prepared petri dish with a solid medium
* Practiced the operation under aseptic conditions
* Inoculated bacteria with biosensor sequences and pHY300PLK backbone
* Cultivated the bacteria overnight at 37°C

![](https://static.igem.wiki/teams/4288/wiki/notebook/t-fujian-united-notebook-02.jpg)

## 7.25

* Processed plasmid extraction for the cultivated bacteria
* Prepared the enzyme restriction system for extracted plasmid (biosensor and pHY300PLK)
* Processed enzyme restriction for both biosensor sequence and pHY300PLK backbone
* Ran the gel electrophoresis for both plasmids
* Extracted the gel
* Ligate biosensor sequences and pHY300PLK through both DNA ligase and Gibson Assembly

![](https://static.igem.wiki/teams/4288/wiki/notebook/t-fujian-united-notebook-03.jpg)

## 7.26

* Transformed ligation product to the amplified bacteria E.coli DH5α
* Waited for DH5α colony to grow
* Researched for a heavy metal involved experiment (operation, safety, material handling)

![](https://static.igem.wiki/teams/4288/wiki/notebook/t-fujian-united-notebook-04.jpg)

## 7.27

* Inoculated the developed single colony of DH5α for tests
* Cultivated the bacteria overnight at 37°C
* Prepare the gradient concentrated heavy metal solutions

![](https://static.igem.wiki/teams/4288/wiki/notebook/t-fujian-united-notebook-05.jpg)

## 7.28

* Processed recombinant plasmid pHY300PLK-pVeg-Biosensor extraction for cultivated bacteria DH5α
* Prepared and processed the enzyme restriction for the recombinant plasmid
* Run the gel electrophoresis for identifying recombinant plasmid
* Transformed plasmids pHY300PLK-pVeg-Biosensor and pHY300PLK-pCadA-Biosensor to the bacteria Bacillus
  subtilis, respectively
* Cultivated the bacteria ArsA, ArsD, and ArsR overnight at 37°C

![](https://static.igem.wiki/teams/4288/wiki/notebook/t-fujian-united-notebook-06.jpg)

## 7.29

* Test the green fluorescence of the reaction product of bacteria and arsenic solution
* Waited for *Bacillus subtilis* colony to grow

![](https://static.igem.wiki/teams/4288/wiki/notebook/t-fujian-united-notebook-07.jpg)

## 7.30

* Cultivated the engineered *Bacillus subtilis* colony
* Express ferulic acid esterase protein in pHY300PLK-pVeg-Biosensor/*Bacillus subtilis*
* Test the protein expression of ferulic acid esterase in pHY300PLK- pCadA-Biosensor/*Bacillus
  subtilis* with cadmium and Zinc solution, respectively

![](https://static.igem.wiki/teams/4288/wiki/notebook/t-fujian-united-notebook-08.jpg)

## 7.31

* Detection of the enzymatic activity of ferulic acid esterase

![](https://static.igem.wiki/teams/4288/wiki/notebook/t-fujian-united-notebook-09.jpg)

## 8.1-8.5

* Data processing and analysis (shown in WiKi: Result)
* Take a group photo

![](https://static.igem.wiki/teams/4288/wiki/notebook/t-fujian-united-notebook-10.jpg)

## 8.6-8.15

* Functional test of cadmium biosensor (pHY300PLK-PcadA-Biosensor / *Bacillus subtilis*) and repeat at
  least three times
* Collect and analysis data
* Added to the experimental results of the wiki

![](https://static.igem.wiki/teams/4288/wiki/notebook/t-fujian-united-notebook-11.jpg)

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# Engineering Success

## Overview

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 *Bacillus subtilis to
test the enzymatic activity of FAE* to provide enzyme data for our biosensor.

## Construct design

The pHY300PLK-Veg-biosensor construct used to constantly express the feruloyl esterase in order to test the
enzymatic activity.

![](https://static.igem.wiki/teams/4288/wiki/engineering/t-fujian-united-engineering-03.jpg)

Figure 1. map of pHY300PLK-Veg-biosensor

### 1.1 The promotor veg fragment was amplified by PCR.

![](https://static.igem.wiki/teams/4288/wiki/engineering/t-fujian-united-engineering-04.jpg)

Figure 2. gel electrophoresis result of Veg fragment
M: DNA ladder; lane1-2 promoter veg fragment

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.

### 1.2 The feruloyl esterase-pHY300PLK fragment was obtained by PCR

![](https://static.igem.wiki/teams/4288/wiki/engineering/t-fujian-united-engineering-05.jpg)

Figure3. gel electrophoresis result of feruloyl esterase-pHY300PLK fragment
lane1: DNA ladder;
lane2: feruloyl esterase-pHY300PLK fragment

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.

### 1.3 LB agar plate containing the single colonies of Veg-biosensor

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.

![](https://static.igem.wiki/teams/4288/wiki/engineering/t-fujian-united-engineering-06.jpg)

Figure 4. LB agar plate for recombinant plasmid

### 1.4 colony PCR

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.

![](https://static.igem.wiki/teams/4288/wiki/engineering/t-fujian-united-engineering-08.jpg)

Figure5. colony PCR of Veg-biosensor recombination plasmid

### 1.5 Sequencing of Veg-biosensor recombination plasmid

The sequence alignment showed that there is no mutation or mismatch. Thus, we chose the No.3 for subsequent
assay.

![](https://static.igem.wiki/teams/4288/wiki/engineering/t-fujian-united-engineering-07.jpg)

Figure6. Sequence alignment

## Functional Test

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 *Bacillus
subtilis* 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:

feruloyl-polysaccharide + H2O = ferulate + polysaccharide

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.

![](https://static.igem.wiki/teams/4288/wiki/engineering/t-fujian-united-engineering-09.jpg)

Figure 7. The activity of ferulyol esterase in different culture time.

## Learn

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
*Bacillus subtilis* 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.

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# Experiments

This part contains plasmid construction and protein function test. We already have four plasmids including
pCadA-RpmG-Feruloyl esterase plasmid, ArsA-amilGFP, ArsD-amilGFP, and ArsR-amilGFP. In addition, we need to
construct pVeg-RpmG-Feruloyl esterase plasmid for detecting the cadmium and zinc.

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# Results

## 1. Construction of the Biosensor plasmids

We design two kinds of biosensor to detect the heavy metal cadmium, zinc and arsenic. For arsenic, our project
used the ArsD, ArsA, ArsR promotor fused amilGFP being its reporter protein. For cadmium and zinc, we utilize
the operon CadA to expresses reporter feruloyl esterase in *Bacillus subtilis*.

The first series of plasmids pUC57-ArsD-amilGFP, pUC57-ArsA-amilGFP, and pUC57-ArsR-amilGFP were a gift from
2021iGEM Team Shanghai\_United. The second series plasmid pHY300PLK-PcadA-Biosensor was handed over to
GenScript for synthesis. The synthesis report is as follows.

Based on the plasmid pHY300PLK-PcadA-Biosensor, we designed a plasmid pHY300PLK-Pveg-Biosensor for
constitutive expression feruloyl esterase in *Bacillus subtilis*. The difference between them is that the
cadmium ion-inducible promoter PcadA is replaced by a constitutive promoter Pveg.

![](https://static.igem.wiki/teams/4288/wiki/results/t-fujian-united-results-01.jpg)

Figure1. Gel electrophoresis results Veg fragment and pHY300PLK vector.
A. Lane M DNA ladder; Lane1, 2 Veg fragments, 97bp, correct;
B. Lane M DNA ladder; tLane1 pHY300PLK vector, 5902bp, correct.

To construct the plasmid pHY300PLK-Pveg-Biosensor, the promoter Veg fragments and pHY300PLK vector were
amplified by PCR, respectively. As can be seen from the figure 1A, there was a specific band at the position
of 97bp, indicating that the PCR amplification of the veg fragment was successful. And the pHY300PLK vector
was also amplified successfully (figure 1B).

The DNA fragment Veg and pHY300PLK vector ligate using the Gibson assembly method. Then, the recombinant
plasmid was transformed into the competent cells DH10b. The bacteria spread to the selection plate and
incubated at 37 ℃ overnight.

![](https://static.igem.wiki/teams/4288/wiki/results/t-fujian-united-results-02.jpg)

Figure 2. The plate of recombinant plasmid pHY300PLK-Pveg-Biosensor.

We picked up 15 colonies for performing colony PCR. The M representative DNA ladder, lane 1-16 representative
colonies. The result showed all selected colonies have the correct band. Thus, we sent colonies No. 1, 3, 5,
and 7 to DNA sequencing.

![](https://static.igem.wiki/teams/4288/wiki/results/t-fujian-united-results-03.jpg)

Figure 3. colony PCR

The sequence alignment showed that there is no mutation or mismatch. Thus, we choose colony No.3 for
subsequent assay.

![](https://static.igem.wiki/teams/4288/wiki/results/t-fujian-united-results-04.jpg)

Figure4. Sequence alignment

## 2. Functional Test

### 2.1 Arsenic-biosensor

We have tested the ability of three biosensors for detecting the heavy metal arsenic. The plasmids were
transformed into *E. coli* and tested while in LB medium. The florescence intensity to reflect the GFP
concentration after the induction of arsenic ions in different concentration. Three promoters are involved in
the test and comparison: ArsA, ArsD, and ArsR. Under different time scales, the fluorescence intensities of
those promoters are shown in the following graphs:

![](https://static.igem.wiki/teams/4288/wiki/results/t-fujian-united-results-05.jpg)

Figure 5. GFP intensity in different concentaiton of As and cultivation time
The 0-hour data set as the control group.

There was no significant expression of GFP displayed at 1 hour regarding all three types of biosensor. This
result indicates that under the time period of 1 hour, the three biosensors are not able to induce the
expression of GFP although the medium contain increasing arsenic solutions concentration. However, starting
from 2 hours, the data demonstrated certain level of positive association amount the florescence intensity and
the arsenic concentration ranging from 10ug/L to 50ug/L. The higher concentration of arsenic might inhibit the
growth of bacteria, so the GFP intensity decreased. No remarkable difference of florescence intensity was
detected between 2 hours and 3 hours. At 2 hours, 50ug/L is the maximum florescence expression for ArsA and
ArsD, and for ArsR it was about 20ug/L.

In conclusion, the results of the fluorescence data show that our arsenic detection sensor can reflect the
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.

### 2.2 The Activity of Feruloyl Esterase expressed by promoter veg

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 *Bacillus
subtilis* 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:

feruloyl-polysaccharide + H2O = ferulate + polysaccharide

![](https://static.igem.wiki/teams/4288/wiki/results/t-fujian-united-results-06.jpg)

Figure 6. The activity of ferulyol esterase in different culture time.

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 *Bacillus subtilis* 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.

### 2.3 Cadmium-biosensor

We tested the ability of cadmium-biosensor for detecting the heavy metal cadmium. The plasmid
pHY300PLK-PcadA-Biosensor was transformed into *Bacillus subtilis*(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.

![](https://static.igem.wiki/teams/4288/wiki/results/t-fujian-united-results-07.jpg)

Figure 7. Plate of the transformants of pHY300PLK-PcadA-Biosensor/Bacillus subtilis.

![](https://static.igem.wiki/teams/4288/wiki/results/t-fujian-united-results-08.jpg)

Figure 8. The activity of Ferulyol Esterase in different concentaiton of Cd.
The data were collected in 0.5h.

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 *Bacillus subtilis* 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.

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