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

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.

Figure 1 Experimental design diagram
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.


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.

Figure3 Gel image of the fadL and fadD.
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)

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

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.

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.


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.

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.)

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.

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

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.


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.