Since our project still involves enhancing the absorption of long chain fatty acid(LCFA), we utilized our results from last year. Continuely, we utilized Rosetta as our experimental basal microbiota since it is proved to have a higher success rate than the assumed E.coli Nessel 1917 in experiments. However, based on the experiment our team did last year, we made some ameliorations such as having more accurate data, strict quantity and analysis. In addition , we also designed the expression of functional hEGF fused with PhoA signal peptide and other optimized experiments.

We bioengineered FadD, FadL gene and co-expressed FadD and FadL into our bacteria Rossetta in three different experiments and then cultured the bacteria overnight. The bacteria culture was shake-nurtured for 24 hours with the optical density at 600 nm (OD600) adjusted to 1, and 2 mL of bacterial culture was collected by centrifugation. Rosetta containing empty plasmid was used as control. Then, the bacterial pellet was resuspended and incubated to take up the palmitic acid (a type of LCFA) and collected by centrifugation, and then the supernatant (extracellular sample) was collected. The bacterial pellet was resuspended lysed to release the intracellular contents (intracellular sample). Then, lipids were extracted from both extracellular and intracellular samples. The palmitic acid content in the samples was calculated based on the standard curve.

Figure.2 The result of fadL and fadD. (a.The successful expression of FadL and the function of LCFA intake b. The successful expression of FadD and the function of LCFA metabolism c. The cofunction of FadD and FadL)

A. From the figure, the total amount of palmitic acid in FadL group is lower than the negative control group, which the plasmid doesn’t contain FadL gene, indicating that LCFA intake and metabolization are achieved. Besides, the amount of palmitic acid in FadL group is higher than that in negative control group in intracellular area but lower in extracellular area, which shows successful LCFA intake.

B. From the figure, the total amount of palmitic acid in FadD group is lower than the negative control group, indicating that LCFA intake and metabolization are achieved. Besides, the amount of palmitic acid in FadD group is significantly lower than that in negative control group in intracellular area but higher in extracellular area, which shows successful LCFA metabolism.

C. From the figure, 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

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.

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.)

From figure 3, 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.

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.

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.

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

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.