iGEM 2023 — Team rdfz-china

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{% block title %}Notebook{% endblock %}
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CONTENT

[Week 1: Experimental preparation](#a1)
[Week 2: Template extraction and plasmid construction begins](#a2)
[Week 3: Agarose gel electrophoresis, gel extraction, double digestion, and
recovery](#a3)
[Week 4: Preparation of competent cells](#a4)
[Week 5: Ligation and Transformation](#a5)
[Week 6: Validate and expand the successfully constructed plasmids extracted.](#a6)
[Week 7: FadL-enhanced uptake of long-chain fatty acids](#a7)
[Week 8: Enhancement of long-chain fatty acid degradation by FadD](#a8)
[Week 9: Enhanced absorption of long-chain fatty acids by co-expression of FadL
and FadD](#a9)
[Week 10: Western blot of phoA-hEGF-his fusion gene](#a10)
[Week 11: Impact of hEGF on the activity of 29T cells](#a11)
[Week 12 - Adaptation of Engineered Bacteria on BC Membrane](#a12)
[Week 13: Antibacterial effects of silver ions on Escherichia coli](#a13)

Week 1: Experimental preparation

Experimental plan: Prepare culture medium and experimental consumables, activate plasmid-carrying strains

Experimental procedure:

1. Use of a clean bench: Open the clean bench and place centrifuge tube stand, test tube rack, marker pen,
and alcohol for disinfection. Perform UV irradiation for 30 minutes.

2. Sterilization: Prepare Eppendorf tubes, PCR tubes, centrifuge tubes, and wrap them with gauze. Pack 2
boxes each of large, medium, and small tips, wrap the outer shell with parchment paper, and tie the tip boxes
with string/rubber bands. Use pre-prepared LB culture media to fill 20 test tubes and prepare 50 mL x 2
bottles of LB liquid culture medium. Sterilize the above items at 121℃ for 15 minutes (this process takes
about 1.5 hours). There was an experimental accident during the process of filling the liquid culture medium:
the medium spilled on the clean bench, and it has been properly cleaned afterwards.

3. Activation of strains: Including 1 tube of E. coli DH5α/empty vector 1, 1 tube of DH5α/carrying fadL, fadD
genes. 1 tube of E. coli DH5α/empty vector 2, 1 tube of DH5α/carrying phoA-hEGF-his gene. Open the clean
bench, ventilate it, place the sterilized LB test tubes in the bench, light the alcohol lamp, burn the test
tube mouth and stopper, add 100 μL of preserved bacterial solution to each tube, add 2.5 μL of Amp antibiotic
stock solution, and burn the test tube mouth and stopper again. Seal the test tube mouth, tie the test tubes,
and incubate overnight on a shaker (150 rpm, 37℃).

Week 2: Template extraction and plasmid construction begins

Experimental plan: Extract plasmids, PCR amplify target fragments

Experimental procedure:

1. Plasmid extraction: Extract 4 types of plasmids according to the plasmid extraction kit instructions:
DH5α/empty vector 1, DH5α/carrying fadL, fadD genes. DH5α/empty vector 2, DH5α/carrying phoA-hEGF-his gene.
After centrifuging the bacterial solution for 1 minute, collect the precipitate. Add p1 resuspension, invert
and mix with p2, invert and mix with p3, and centrifuge for 10 minutes. Add 500 μL of BL reagent to the
absorption column, discard the waste liquid, add the above centrifuged supernatant, wash twice with pw wash
solution, each time centrifuging for 1 minute. Add 50 μL of EB solution to the membrane of the absorption
column, centrifuge for 2 minutes. Use Nanodrop to measure the recovered DNA concentration and store the DNA at
-20℃. Most people successfully extracted the plasmids in this step.

![](https://static.igem.wiki/teams/4965/wiki/notebook/img1.jpg)
![](https://static.igem.wiki/teams/4965/wiki/notebook/img2.jpg)

2. Dilute primers: Centrifuge the lyophilized primers at 12000rpm for 1 minute, and dilute the primers to the
appropriate concentration by adding the corresponding amount of distilled water.

3. PCR reaction:

(1) Amplify the target gene pheP, PAL, and two plasmid DNA fragments using high-fidelity enzyme Pfu. The
reaction system is as follows:

Pfu 0.5 μL

10x Pfu buffer 5 μL

Primer 1 1 μL

Primer 2 1 μL

Template (plasmid extracted in the previous step): 1 μL

dNTP Mixture 25 μL

Sterile water 20 μL

(2) Perform the following PCR amplification program:

98℃, 5 min

Repeat the following three steps for 30 cycles:

98℃ 10s

55℃ 5s

72℃ 1min/kb

72℃ 8 min

The plasmids, primers, and PCR products required for PCR are shown in the table below:

Week 3: Agarose gel electrophoresis, gel extraction, double
digestion, and recovery

Experimental plan: Agarose gel electrophoresis, gel extraction, double digestion, and recovery

Experimental procedure:

1. Agarose gel electrophoresis: Add the above PCR products to the prepared agarose gel, add 5 μL DL 5000 DNA
marker, and perform electrophoresis (120V, 20 min). After the electrophoresis is completed, observe using a
gel imaging system, cut the desired DNA gel, and place it in a 2 mL centrifuge tube for labeling.

![](https://static.igem.wiki/teams/4965/wiki/notebook/img3.png)
![](https://static.igem.wiki/teams/4965/wiki/notebook/img4.png)
![](https://static.igem.wiki/teams/4965/wiki/notebook/img5.png)

2. Gel extraction: Perform gel extraction on the PCR product DNA gel according to the instructions of the gel
extraction kit. Add 500 μL of equilibration buffer BL to the adsorption column CA2, centrifuge for 1 min,
discard the waste liquid in the collection tube, and place the adsorption column back into the collection
tube. Cut the single desired DNA band from the agarose gel and transfer it to a clean centrifuge tube. Add an
equal volume of solution PN to the gel block and incubate at 50°C until the gel block is completely dissolved.
Transfer the resulting solution to another adsorption column CA2, leave at room temperature for 2 min, and
centrifuge for 1 min. Add 600 μL of wash buffer PW (check if ethanol has been added before use) to the
adsorption column CA2, centrifuge for 1 min, repeat the wash twice, and air dry. Drop an appropriate amount of
elution buffer EB onto the middle position of the adsorption membrane and leave at room temperature for 2 min.
Centrifuge for 2 min and collect the DNA solution. Measure the recovered DNA concentration using a Nanodrop.

3. Double digestion: Perform double digestion on the extracted plasmid pET23b and amplified target DNA
fragments using EcoRI and XhoI. Use a 5x20 μL reaction. The 20 μL digestion system is as follows:

DNA 1 μg

EcoRI 1 μL

XhoI 1 μL

10xY buffer 4 μL

H2O up to 20 μL

Digest at 37°C for 4 h.

4. Agarose gel electrophoresis: Add the digested products to an agarose gel, add 5 μL DL 5000 DNA marker, and
perform electrophoresis (120V, 20 min). After electrophoresis, observe using a gel imaging system, cut the
desired DNA gel, and place it in a 2 mL centrifuge tube for labeling. The product was successfully extracted,
but the quantity is small. Proceed to a second round of gel extraction.

Week 4: Preparation of competent cells

Experimental plan: Activation of E. coli DH5α and Rosetta, preparation of competent cells

Experimental procedure:

1. Activation of bacterial strains: including Escherichia coli DH5α and Rosetta. Open the laminar flow hood,
ventilate, place sterilized LB test tubes on the bench, light an alcohol lamp, and burn the mouth of the test
tubes and the caps. Inoculate 100 μL of stock culture into each test tube, burn the mouth of the test tubes
and the caps again, seal the test tubes tightly, tie them together, and incubate overnight on a shaker (220
rpm, 37℃).

2. Preparation of competent cells:

(1) Open the laminar flow hood, place 1 mL, 200 μL, 5 mL, 2 mL, and 30 mL centrifuge tubes, and 2 x 50 mL LB
medium for UV irradiation. Pre-cool 0.1 M CaCl2 and 15% 0.1 M CaCl2 solution in a 4℃ refrigerator.

(2) Prepare DH5α and Rosetta competent cells as follows: Transfer the pre-activated DH5α and Rosetta bacteria
to a pre-cooled centrifuge at 4°C, centrifuge at 5000 g for 10 min, discard the supernatant, and add 800 μL of
solution.

![](https://static.igem.wiki/teams/4965/wiki/notebook/img6.png)

Week 5: Ligation and Transformation

Experimental Plan: Prepare solid agar plates with antibiotics, perform ligation reaction, transform the
ligation product

Experimental Procedure:

1. Preparation of antibiotic plates: Prepare LB agar medium (add 4.5 g agar to 300 mL LB liquid medium),
autoclave at 121℃ under high pressure for 15 minutes. Allow to cool to around 60℃, add 150 μL of 100 mg/mL
Ampicillin (Amp) antibiotic, shake well, and pour into disposable petri dishes (enough for approximately 10-15
plates).

2. Ligation reaction: Connect the vector and the target fragment through double enzyme digestion. The
ligation system is as follows (Note: this is an empirical system, the optimal volume ratio of the
vector/target fragment can also be calculated according to the instruction manual):

T4 DNA ligase 1 μL

10 x ligation buffer 2 μL

Linear plasmid 1 μL

Insert DNA 6 μL

Perform ligation at 16 ℃ for 1 hour using a PCR instrument. Obtain pET23b-insert and store the ligation
product at -20℃.

3. Transformation of the ligation product: Add 10 μL of the above ligation product to 100 μL competent cells,
incubate on ice for 30 minutes, heat shock at 42℃ for 60 seconds, incubate on ice for 5 minutes, add 1 mL LB
liquid medium, and incubate at 37℃, 150 rpm for 1 hour for recovery. In a laminar flow hood, take 100 μL of
the recovery solution and spread it onto an Ampicillin (Amp) resistant agar plate.

![](https://static.igem.wiki/teams/4965/wiki/notebook/img7.png)
![](https://static.igem.wiki/teams/4965/wiki/notebook/img8.png)

Week 6: Validate and expand the successfully constructed plasmids
extracted.

Experimental Plan: Colony PCR verification, expansion culture of positive clones, extraction of validated
plasmids and transformation

Experimental Procedure:

1. PCR verification: Label 6 colonies on each plate. Take half of the colonies as templates and perform
colony PCR using Taq enzyme. Primers are TF and TR. Follow the instructions for reaction system and program.
Prepare a 50 mL agarose gel and use DL2000 DNA marker for electrophoresis. Presence of correctly sized bands
indicates successful plasmid construction.

2. Expansion culture: Pick validated colonies and add them to 5 mL LB liquid medium, supplemented with 2.5 μL
Ampicillin (Amp) antibiotic, for expansion culture.

3. Plasmid extraction: Follow the instructions of the plasmid extraction kit to extract two types of
plasmids. After centrifuging the bacterial culture for 1 minute, collect the precipitate. Add p1 resuspension
solution, invert to mix, add p2, invert to mix, and add p3, invert to mix, then centrifuge for 10 minutes. Add
500 μL of BL reagent to the column, discard the flow-through, add the above centrifuged supernatant, wash
twice with PW washing solution, centrifuging for 1 minute each time. Add 50 μL of EB solution to the membrane
of the column, centrifuge for 2 minutes. Measure the concentration of the recovered DNA using Nanodrop and
store the DNA at -20℃.

4. Transformation: Transform the validated plasmid into expression-type Escherichia coli.

![](https://static.igem.wiki/teams/4965/wiki/notebook/img9.png)

Week 7: FadL-enhanced uptake of long-chain fatty acids

In this study, the fadL gene will be cloned into the pET23b vector to construct the recombinant plasmid
pET23b-fadL. The plasmid will then be transformed into Escherichia coli Rosetta. The transformed E. coli
Rosetta will be inoculated into LB medium containing antibiotics and cultured overnight. The bacterial culture
(2 mL) will be collected by centrifugation at 10,000 rpm for 1 minute after adjusting the optical density at
600 nm (OD600) to 1.

A control sample with empty plasmid and Rosetta will also be prepared. The bacterial pellets will be
resuspended in 2 mL of PBS buffer containing 100 μM palmitic acid (a long-chain fatty acid) and incubated for
1 hour to allow the bacteria to absorb the palmitic acid. The bacteria will then be centrifuged at 10,000 rpm
for 1 minute, and the supernatant (extracellular sample) will be collected.

The bacterial pellets will be resuspended in PBS and sonicated at 75W for 5 minutes to dissolve the bacterial
particles and release the intracellular contents (intracellular sample). Lipids will be extracted from both
extracellular and intracellular samples using the following method: equal volumes of chloroform/methanol (2:1,
v/v) will be added to the samples, mixed thoroughly, and allowed to stand for 10 minutes to transfer the
lipids to the chloroform phase. The samples will then be centrifuged at 15,000 rpm for 10 minutes to separate
the chloroform and aqueous phases. The chloroform phase containing the lipids will be collected and
transferred to a new centrifuge tube.

The chloroform will be evaporated at 50°C in a fume hood and further dried for 30 minutes in a vacuum
centrifugal evaporator. The dried lipids will be dissolved in 200 μL of lipid assay buffer by vortexing for 5
minutes. Palmitic acid standards will be prepared, and the palmitic acid content in the samples will be
determined using a non-esterified fatty acid (NEFA) assay kit according to the manufacturer's instructions.
The palmitic acid content in the samples will be calculated based on a standard curve. The data will be
presented as mean ± standard deviation.

One-way analysis of variance (ANOVA) and Tukey's post hoc test will be used to determine the statistical
significance of differences, with a p-value less than 0.05 considered significant.

![](https://static.igem.wiki/teams/4965/wiki/notebook/img10.png)

Week 8: Enhancement of long-chain fatty acid degradation by FadD

The FadD gene will be synthesized and cloned into the pET23b vector for ligation, followed by transformation
into Escherichia coli Rosetta. The palmitic acid content in both intracellular and extracellular samples will
be determined using the method described above.

Expression of FadD resulted in a reduction of intracellular palmitic acid content.

![](https://static.igem.wiki/teams/4965/wiki/notebook/img11.png)

Week 9: Enhanced absorption of long-chain fatty acids by
co-expression of FadL and FadD

FadL and FadD will be co-expressed in the pET23b vector and transformed into Escherichia coli Rosetta. The
palmitic acid content in both intracellular and extracellular samples will be determined using the method
described above.

The results showed a significant decrease in total palmitic acid content with the co-expression of FadL and
FadD.

![](https://static.igem.wiki/teams/4965/wiki/notebook/img12.png)

Week 10: Western blot of phoA-hEGF-his fusion gene

The pET23b vector carrying the phoA-hEGF-his fusion gene will be transformed into Escherichia coli Rosetta
cells. The engineered bacteria will be inoculated into 50 ml of LB medium supplemented with ampicillin and
incubated overnight. The next day, the bacterial culture will be collected by centrifuging at 10,000 x g for 1
minute. The supernatant will be transferred to a new centrifuge tube as the extracellular fraction sample.

The cell pellets will be washed with PBS to remove residual culture medium and then sonicated (150W, 1s
sonication, 3s interval, a total of 20 minutes). The lysed cell suspension will be transferred to a new
centrifuge tube as the intracellular content sample.

The protein solution will be mixed with 5 times the volume of reducing protein sample buffer at a ratio of
4:1 and denatured in a boiling water bath for 15 minutes. The proteins on a 15% polyacrylamide gel will then
be separated at 120V using SDS-PAGE. Subsequently, the proteins on the SDS-PAGE gel will be transferred to a
PVDF membrane (0.2 micrometer, 30 minutes) under low-temperature conditions. The membrane will be blocked with
5% skim milk (TBST) at room temperature for 1 hour.

The membrane will then be incubated overnight at 4°C with a mouse-derived anti-His tag antibody (1:1000,
AH367, Beyotime). After washing the membrane with TBST three times for 10 minutes each, it will be incubated
at room temperature for 1 hour with a horseradish peroxidase-conjugated goat anti-mouse IgG (H+L) antibody
(1:1000 dilution, A0216, Beyotime). The membrane will be washed three times with TBST for 10 minutes each. ECL
A and B solutions will be mixed in a 1:1 ratio as instructed. After washing the PVDF membrane, it will be
slightly dried on absorbent paper. Then the membrane will be placed on a chemiluminescent imager and covered
with prepared ECL substrate, ensuring full immersion of the membrane. After 1 minute, the excess liquid will
be removed using absorbent paper, and the membrane will be placed in the chemiluminescent imager, starting the
chemiluminescent reaction according to the preset program.

The Western blot results showed a band at around 8 kDa in both the extracellular and intracellular fractions
of Escherichia coli Rosetta cells, confirming the expression and secretion of the phoA-hEGF fusion protein.
The signal peptide of phoA successfully directed the hEGF fusion protein to the periplasmic space. Future
studies may focus on optimizing protein yield and purification.

![](https://static.igem.wiki/teams/4965/wiki/notebook/img13.png)

ELISA results: The ELISA analysis also indicated that the phoA signal peptide successfully directed the hEGF
fusion protein.

![](https://static.igem.wiki/teams/4965/wiki/notebook/img14.png)
![](https://static.igem.wiki/teams/4965/wiki/notebook/img15.png)

Week 11: Impact of hEGF on the activity of 29T cells

The phoA-hEGF fusion gene was synthesized and cloned into the pET23b vector, which was then transformed into
Escherichia coli Rosetta. The culture supernatant and bacterial content samples were collected by
ultrasonication and centrifugation. Escherichia coli Rosetta containing only the empty vector was used as a
control. Human embryonic kidney 293T cells were seeded at a density of 5,000 cells per well in DMEM medium
containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, in a 96-well plate. The cells were
cultured at 37°C and 5% CO2 in a humidified environment until they reached 60-70% confluency. Then, 10μL of
bacterial content and filtered supernatant of the engineered bacterial strain were added to each well in DMEM
medium. After 24 hours of incubation, cell viability was detected using the CCK8 assay; specifically, 10μL of
CCK8 solution was added to each well and incubated at 37°C for 3 hours. The absorbance at 450nm was measured
using an enzyme-linked immunosorbent assay reader. The average absorbance (a) values and standard deviations
of each group were calculated, and the data were analyzed and plotted using GraphPad Prism. One-way ANOVA was
used for statistical analysis of the data, followed by Tukey's post hoc test. A p-value less than 0.05 was
considered statistically significant.

![](https://static.igem.wiki/teams/4965/wiki/notebook/img16.png)

Week 12 - Adaptation of Engineered Bacteria on BC Membrane

The bacterial cellulose (BC) membrane was cut into 1 cm x 1 cm fragments and washed repeatedly with sterile
PBS buffer (pH 7.4). E. coli Rosetta/p23b-phoA-hEGF was then inoculated into LB medium and cultured overnight
at 37°C. The bacterial culture was collected, washed with PBS buffer, and the bacterial concentration was
adjusted to OD600=1. Using a pipette, 100 μL of the bacterial culture was spread onto the BC membrane. BC
membranes without bacterial culture were used as negative controls. The BC membranes with the bacterial
culture were placed in sterile petri dishes, and an additional 100 μL of bacterial culture was directly spread
on the petri dish as a positive control. The petri dishes were covered with sterile lids and placed in a
constant temperature incubator for 3 hours at 37°C. Subsequently, the BC membranes containing bacterial
suspension were carefully removed using sterile forceps and immersed in 0.9 mL of Luria-Bertani (LB) medium. A
10-fold serial dilution of the bacterial suspension was performed, and the diluted bacterial suspension was
plated onto LB agar plates. The plates were incubated overnight at 37°C, and the formed colonies were counted
to determine the colony-forming units (CFU) per milliliter of the original bacterial suspension. The results
showed that E. coli Rosetta/p23b-phoA-hEGF can survive on the BC membrane. The negative control confirmed the
absence of contamination during the experiment. These results demonstrate that the BC membrane can support the
survival of E. coli Rosetta/p23b-phoA-hEGF, making it a suitable substrate for bacterial applications.

![](https://static.igem.wiki/teams/4965/wiki/notebook/img17.png)

To evaluate the permeability of engineered bacteria on bacterial cellulose (BC) membranes, we first cut the
BC membrane into 5 cm x 5 cm (depending on the actual experiment) fragments and washed them repeatedly with
sterile PBS buffer (pH 7.4). Then, E. coli Rosetta/p23b-phoA-hEGF was inoculated into LB medium and cultured
overnight at 37°C. The bacterial suspension was collected and washed with PBS buffer, and then the bacterial
concentration was adjusted to OD600=1. Using a pipette, 100 μL of the bacterial suspension was spread onto the
BC membrane, ensuring an even distribution. The BC membrane with the spread bacteria was placed in a sterile
Petri dish and incubated at 37°C for 24 hours. The BC membrane was then imprinted on LB solid medium on both
sides and incubated at 37°C for 12 hours. Finally, the growth of engineered bacteria colonies on the solid
medium was observed.

Week 13: Antibacterial effects of silver ions on Escherichia coli

LB solid medium was prepared, and wild-type Escherichia coli was activated. 50 μL of bacterial suspension was
evenly spread on LB solid medium. The bacterial cellulose membrane was cut into fragments half the size of the
Petri dish and washed with sterile PBS repeatedly. It was then soaked in a 50 μM silver nitrate solution for 3
hours to fully adsorb silver ions onto the bacterial cellulose membrane. The silver ion-infused bacterial
cellulose membrane was then overlaid onto the LB solid medium inoculated with wild-type E. coli and placed in
a 37°C constant temperature incubator overnight. The next day, the Petri dishes were taken out, and the growth
of bacterial colonies was observed on the part covered by the silver ion-treated bacterial cellulose membrane
and the uncovered part. The number and size of the colonies were compared to evaluate the antibacterial
effects of silver ions on Escherichia coli.

![](https://static.igem.wiki/teams/4965/wiki/notebook/img18.png)

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{% block title %}Engineering Success{% endblock %}
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{{self.title()}}

[Overview](#a1)
[Cycle 1: Enhanced absorption of Long chain fatty acid](#a2)
[Design](#b1)
[Build](#b2)
[Test](#b3)
[Learn](#b4)
[Cycle 2: hEGF expression and function](#a3)
[Design](#b5)
[Build](#b6)
[Test](#b7)
[Learn](#b8)

Overview

The core of synthetic biology lies in the rational design and engineering of biological components, devices, or
systems. Similar to other engineering fields, synthetic biologists employ an iterative process of
design-build-test to successfully develop innovative biological systems. In this experiment, we iteratively
designed two circuits to express substances for acne treatment, and through construction and testing, we were
able to achieve the expected results.

Cycle 1: Enhanced absorption of Long chain fatty acid

Design

The accumulation of long chain fatty acid (LCFA) is one of the main factors leading to acne, we decided to
break down LCFA. Therefore, we decided to bioengineer both FadD and FadL gene into bacteria for expression. FadL
can bind to LCFA and allow it to enter the periplasmic space of EcN (the space between the cell wall and the
cell membrane), while FadD can activate β-oxidation of exogenous LCFA and regulate the expression of the
transcription factor FadR. The cooperative function of the two genes is believed to complete the function of
metabolizing LCFA.

![](https://static.igem.wiki/teams/4965/wiki/engi/img1.png)

Figure 1 Experimental design diagram

Build

![](https://static.igem.wiki/teams/4965/wiki/engi/img2.png)

Figure 2 Design of the fadL and fadD.

We used the RBS (BBa\_B0034) as the basis for expressing FadD and FadL gene to allow the gene successfully
combined with ribosome. (Figure 1) We used promoter T7 (BBa\_L719005) and terminator part BBa\_B0015 to express
FadD and FadL gene and transformed the plasmids into E. coli Rossetta Strain.We expressed FadD and FadL gene in
E.coli Rossetta strain with an none-bioengineered bacteria as control.

Test

By comparing the endocellular and extracellular palmitic acid amount with expressing either FadL or FadD, we
examined the successful expression and function of this complex part. However, based on the experiment our team
did last year, we made some ameliorations such as achieveing more accurate data through extraction step and
one-way analysis of variance and having a more strict quantitative result and analysis. From figure 3B, the
total concentration of Palmitic acid the treatment group with both FadL and FadD genes is significantly lower
than that of Negative control group indicating that the LCFA intake and metabolization are achieved. The three
experiments indicates that the system can achieve the goal of Palmitic acid intake and metabolization in vitro,
and both synthesized proteins involved in the process, FadL and FadD, are functional and effective.

![](https://static.igem.wiki/teams/4965/wiki/engi/img3.png)

Figure3 Gel image of the fadL and fadD.

![](https://static.igem.wiki/teams/4965/wiki/engi/img4.png)

Figure4 The result of expression of fadL and fadD (a,The briefed protocol of the
experiment. b,The resulting picture of comparing FadL/FadD only expression and FadL and FadD co-expression)

Learn

We have achieved preliminary success by enhancing the absorption of long chain fatty acids through the
construction of engineered strains. Next, we plan to synthesize human epidermal growth factor hEGF for the
treatment of acne.

Cycle 2: hEGF expression and function

Design

Although E. coli Nissle 1917 can slowly secrete the hEGF growth factor, the secretion rate is relatively low,
which is not efficient enough for acne treatment. Therefore, we decided to add a phoA secretion tag to the hEGF
gene, facilitating the secretion of hEGF into the periplasmic space of E. coli. phoA is a signal peptide that
guides newly synthesized proteins towards the secretion pathway. hEGF is the gene responsible for synthesizing
hEGF, the human epidermal growth factor. We constructed a plasmid with fused phoA-hEGF gene, transformed it into
E. coli, and validated its production and secretion using ELISA and Western Blot.

Build

![](https://static.igem.wiki/teams/4965/wiki/engi/img5.png)

Figure 5 Design of the phoA-hEGF.

We used the old brick hEGF as the basis for fusing the phoA gene to get the phoA-hEGF fused gene. It was
synthesized and cloned into the pET23b vector with promoter T7 and terminator b0015, and transformed into E.
coli Rosetta Strain.

Test

2.1 Western blot proving the successful expression of hEGF

We first utilized western blot the examine the expression of hEGF. phoA-hEGF gene was bioengineered into the
bacteria and cultured overnight (time same as 1.1). When the bacteria collected, the supernatant was transferred
to a new centrifuge tube as extracellular component sample. The lysed cell suspension was transferred to a new
centrifuge tube as a sample of intracellular content. The protein solution was mixed, denatured separated and
thus transferred to a PVDF membrane. The membrane was then incubated overnight and rinsed. After washing the
membrane, we mixed ECL A and B solutions at a ratio of 1:1 and set aside. Then we place the membrane on the
chemiluminescence rack and covered it with the prepared ECL luminescent solution. After the reaction for 1
minute, the membrane is put into a chemiluminescence instrument to start the chemiluminescence reaction
according to the preset program.

Secondly, we utilized ELISA to further confirm the expression of hEGF. We set standard wells and sample wells
and added 50 μL of standard products of different concentrations to each standard well. Then, we set up blank
wells, which has no samples and enzyme label reagents, and sample wells. Afterwards, we added 40μl of sample
diluent to the well of the sample to be tested on the enzyme-labeled coated plate, and then added 10μl of the
sample to be tested. Then, we added the sample to the bottom of the well of the enzyme plate and mixed it. Next,
we added 100 μl of enzyme label reagent to each well, except blank wells. After setting the experimental and
comparative groups, we followed the protocol and when the reaction terminates, we measured the absorbance (OD
value) of each well at a wavelength of 450nm.

![](https://static.igem.wiki/teams/4965/wiki/engi/img6.png)

Figure 6 The result of hEGF ( a,the result of western blot c and d, the result of
ELISA )

From figure A, Western Blot results showed that there was a band of about 8 kDa in the extracellular and
cellular contents of the bacteria cells, indicating the expression and secretion of phoA-hEGF fusion protein.
The phoA signal peptide successfully guided the hEGF fusion protein into the periplasmic space.

From figure B and C, the level of concentration of pT7-hEGF group out of the cell is significantly lower than
that of the intracellular group, however, the level of concentration of pT7-phoA-hEGF group is somewhat the same
as the intracellular group, indicating that phoA signal peptide successfully guided the hEGF fusion protein into
the periplasmic space

2.2 hEGF effect on cell activity

Besides examining the expression of hEGF, we also testified the hEGF effect on cell activity. We bioengineered
phoA-hEGF gene into the bacteria and cultured overnight. Bacteria containing only the empty vector was used as a
control. Human embryonic kidney 293T cells were seeded in a 96-well plate and were cultured. The culture medium
was then replaced with 100 µL of the filtered supernatant, bacterial content, or fresh DMEM medium from the
engineered strain. After incubating for 24 hours, cell viability was assessed using the CCK8 assay. The
absorbance at 450 nm was measured using a microplate reader. (One-way ANOVA was used to analyze the statistical
differences in the data, followed by Tukey's post hoc test. A P-value of less than 0.05 was considered
statistically significant.)

![](https://static.igem.wiki/teams/4965/wiki/engi/img7.png)

Figure 7 (cell growth examined by CCK8)

From figure 7, the supernatant and bacterial contents of the control group had less effect on the viability of
293T cells, with lower OD450 values. The supernatant and bacterial content of the engineered strain (Bacteria
carrying phoA-hEGF or hEGF) had a greater impact on the viability of 293T cells, with a higher OD450 value. The
supernatant of the engineering strain (phoA-hEGF) added with phoA secretion tag had the strongest promotion
effect on the viability of 293T cells, and the OD450 value was the highest. This suggests that phoA tag
contributes to the secretion of hEGF, thereby promoting the activity of 293T cells more effectively. The
supernatant of the engineering strain without phoA secretion tag (hEGF) promoted the activity of 293T cells
weakly, but still stronger than the control group. This suggests that hEGF itself can promote the activity of
293T cells, but adding a phoA tag can further enhance this effect. For the bacterial content, the engineered
strain with phoA secretion tag added (phoA-hEGF) and the engineered strain without phoA secretion tag (hEGF) had
a similar promotion effect on the viability of 293T cells, both of which were stronger than the control group.
Overall, these experimental results demonstrate that engineered strains (E. coli Rosetta carrying phoA-hEGF or
hEGF) can promote viability in 293T cells, which can be further enhanced by adding a phoA secretion tag.

**3.Bacteria survival on Bacterial Cellulose Membrane**

In order to examine our bacteria’s survival on our designed bacterial cellulose membrane, we established an
experiment as follow: Bacterial cellulose (BC) membranes were prepared and washed while the engineered gene (E.
coli Rosetta/p23b-phoA-hEGF) was cultured overnight. Then, 100 μL of the bacterial suspension was spread onto
the prepared BC membrane. BC membranes without bacterial suspension served as negative controls. The BC
membranes with bacterial suspension were placed in sterile culture dishes, and 100 μL of bacterial suspension
was directly spread onto the culture dish as a positive control. After certain culturing processes, the plates
were incubated overnight at 37°C, and the resulting colonies were counted to determine the colony-forming units
(CFU) per milliliter of the original bacterial suspension.

![](https://static.igem.wiki/teams/4965/wiki/engi/img8.png)

Figure 8 The survival of bacteria on BC membrane.

The results showed that the engineered bacteria (E. coli Rosetta/p23b-phoA-hEGF) can survive on BC membrane.
Negative controls confirmed that there was no contamination in the experiment. These results suggest that BC
membranes can support the survival of the engineered bacteria, making it a suitable substrate for bacterial
applications.

**4.Biosafty achieved by silver ion**

In order to achieve further function to kill P.acnes and relieve inflammation, RDFZ-CHINA iGEM searched and
eventually chose to incorporates Ag ion in to the patch. We’ve found that Ag ion has the ability to disturb the
metabolism, replication and broke the cell wall and membrane of a bacteria. Further experiment is as follow. LB
agar plates were prepared for bacterial culture. A 50 μL aliquot of wild-type E.coli was evenly spread onto the
LB agar plates. Bacterial cellulose (BC) membranes were cut into pieces approximately half the size of the
culture dishes and were thoroughly washed with sterile PBS. The BC membranes were then soaked in a 50 μM silver
nitrate solution for 3 hours to ensure adequate adsorption of silver ions. The silver-treated BC membranes were
placed onto the LB agar plates inoculated with wild-type E. coli and incubated overnight

![](https://static.igem.wiki/teams/4965/wiki/engi/img9.png)

Figure.9 The resulting medium of our experiment

From the resulting picture, the number of colonies of bacteria in the silver ion effected group is
significantly less than that of the control group(no silver ion added) and the size is much smaller.

Learn

We have proved that all parts of our project functions. We have demonstrated that our engineered bacteria
synthesized functional FadL and FadD proteins, successfully expressed functional hEGF combined with phoA signal
peptide , survival in bacterial cellulose membrane and that our proposed silver ion mechanism can prevent
possible escape of engineered bacteria to unintended environment.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block cover\_img %}https://static.igem.wiki/teams/4965/wiki/cover/experiments.jpg{% endblock %}
{% block page\_content %}{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

## Describe what your results mean

---

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

## Project Achievements

---

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

## Inspirations

---

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)

{% endblock %}