Our goal was to utilize E. coli engineered bacteria to produce the DMBT1 protein to inhibit the growth of Pseudomonas aeruginosa, so we constructed a protein expression system, and at the same time regulated the growth state of the engineered bacteria through a population sensing system, a toxin-antitoxin system, and an auto-inducible system to ensure that it performs its specific function under the appropriate conditions. Through this integrated approach, we aim to reduce the risk of Pseudomonas aeruginosa infections in contact lens users and to safeguard the safety and health of contact lens users.
Our validation experiments are divided into four segments, one is the protein expression system, the second is the population sensing system, the third is the hicA-hicB system, and the fourth is the self-induced system
The experiments are divided into plasmid extraction, plasmid transformation, plasmid sequencing,Corneal epithelial cell and Pseudomonas aeruginosa culture , and validation experiments for each of the four sections. The experiments are shown below.
1. Plasmid extraction
2. transformation
3. Plasmid sequencing
4. Corneal epithelial cell and Pseudomonas aeruginosa culture
5. validation: protein expression system
6. Validation: self-induction system
7. Validation: Population Induction System
8. Validation:Toxin-antitoxin system
1. Our various experimental and control groups had a total of 9 plasmids as follows, and their respective numbers and concentrations after bulk extraction are shown below.
We successfully transformed 2/3/6 and 4+5/7+8/6+7/6+8 plasmids after teamwork and experimental division of labor, and the experimental results are as follows
We obtained HCE-T primary cryopreserved cells from our company and cultured them in 5% CO2 incubator using DMEM complete medium supplemented with relevant growth factors and nutrients, and every 2~3 days, cells were passaged and some cells were frozen at -80s℃ for storage. Cells were digested and counted before use and used for cell spreading in 96-well plates. The cell morphology was normal and free of contamination during the culture process.
We cultured Pseudomonas aeruginosa in the BSL-2 laboratory by resuscitating and inoculating the purchased bacterial solution in LB broth medium and incubating it using a thermostatic shaker at 200 rpm with shaking at 37°C. Subsequently, the overnight solution was doubly diluted and then the plates were coated, and several dilutions of the plates were separately placed in a 37°C thermostatic incubator to culture the bacteria, and the plates in which 30-300 colonies were present were chosen to count the colonies.
When we designed the DMBT1 protein expression system, in order to reduce the difficulty of detecting the expression of the target protein, we fused the DMBT1 protein with the sfGFP fluorescent protein, and utilized the cytation3 fluorescent enzyme marker to take fluorescent photographs to detect whether the DMBT1 protein is expressed or not, and we obtained the following results of the pictures
We fused DMBT1 protein and sfGFP protein, took the E. coli bacterial fluid of pET30a-DMBT1-sfGFP, extracted the protein in the engineered bacteria by using the whole protein extraction reagent, and verified whether the DMBT1 protein was expressed or not by incubating with the antibody against sfGFP protein, and got the results in the figure below, and we got that the size of sfGFP was about 24kDa by checking the related papers. The size of sfGFP is about 24kDa, and we successfully verified the expression of DMBT1 protein by comparing the corresponding size of marker.
We wished to determine the cellular activity of HCE-T human corneal epithelial cells infiltrated by Pseudomonas aeruginosa by cck8 and inhibited the treatment of Pseudomonas aeruginosa using our production of DMBT1 protein.
Before the formal experiments began we conducted a part of the pre-experiment to explore the optimal incubation time and the number of cells inoculated in 96-well plates The results are shown below, where the optimal number of inoculated cells was obtained from the paper as 10,000 cells per null. The incubation time was about 2.5 hours
In addition, we performed bacterial enumeration of overnight P. aeruginosa bacterial fluids using plate colony counting, and obtained a P. aeruginosa density of approximately 9.6*10^8CFU/mL, with a counting petri dish as follows
We then centrifuged the Pseudomonas aeruginosa bacterial fluid after overnight incubation, centrifuged it in a centrifuge, discarded the supernatant and resuspended it in complete cell culture solution, and added DMBT1 protein extracted from the engineered bacteria for incubation, added it to a 96-well plate with the cells spread according to the ratio of bacteria/cells of 100/1, and incubated it overnight in an incubator, and then washed the 96-well plate with antibiotic-containing PBS to remove the Pseudomonas aeruginosa bacteria. Aeromonas aeruginosa was removed. Finally, complete cell culture medium containing cck8 was added and incubated in the cell culture incubator for 2.5 hours for measurement. The experimental group treated with DMBT1 and the control group without any treatment were set up, and the following results were obtained based on the analysis of the data results, which proved that DMBT1 could prevent P. aeruginosa from infesting the human corneal epithelial cells.
In order to compare the effect of hsp27 induction with IPTG induction, we set up three control groups and one experimental group with the same concentration, and put four tubes of bacterial fluids into the shaker at the same time to incubate, and detected the fluorescence intensity of the bacterial fluids every 1 hour, and then fitted them to make a curve after the data were obtained. The results are shown in the figure below, the fluorescence intensity indirectly reflected that the protein expression of the group adding 0.5 mmol IPTG was less than PET-21a-HSPB1(HSP27) group, while hsp27 was approximate to the protein expression of the group adding 1 mmol IPTG.
We extracted the proteins from the engineered bacteria using whole protein extraction reagent with pET-30a-DMBT1-sfGFP and pET28a-Hsp27 bacterial fluids and pET-30a-sfGFP and pET-28a-sfGFP bacterial fluids, respectively, and incubated with antibodies against sfGFP and Hsp27 proteins, respectively, to verify whether the Hsp27-SILEX self-inducible system was expressed or not. Hsp27-SILEX auto-inducible system is expressed or not, and the results are shown in the figure below. After checking the related papers, we found that the size of sfGFP is about 24kDa and the size of Hsp27 is about 28kDa, and the Hsp27-SILEX auto-inducible system was successfully verified by comparing with the corresponding size of the markers.
Hsp27 antibody results verification, the following results prove that Hsp27 protein is successfully expressed in the self-inducible system, and pET30-a-Hsp27 plasmid successfully expresses the protein gene of Hsp27, which proves that it can be expressed in the E. coli system.
In addition, we verified that Hsp27 protein could induce the expression of target protein genes on other plasmids using the sfGFP antibody results, and the analyzed images proved that Hsp27 protein promotes the expression of target genes successfully, based on the size of the sfGFP protein of about 24 kDa.
When we designed the group sensing system, in order to determine the strength of the group sensing corresponding to the downstream E-cleavage protein gene expression, we E-cleavage protein fused with the mCherry red fluorescent protein, and used the cytation3 fluorescence enzyme marker to take fluorescent photographs to detect whether the E-cleavage protein is expressed or not, so as to verify whether the group sensing system works or not, and we got the results of the pictures as follows.
We set up three experimental groups and one control group this time, respectively, we tested the growth curves of the population sensing plasmid again, placed in a shaker when all 4 tubes of bacterial solution had an OD600 value of 0.05, and measured the OD value every 2.5 hours, and the results are shown below.
With the increase of inducer concentration, the peak and decrease of E. coli concentration increased significantly compared to the control group, which proved the success of our improved colony induction system and had more obvious results.
we set up two groups of bacterial fluids i.e. control group (BL21 E. coli) as well as experimental group (hicA-hicB plasmid cotransformation fluids).The two groups were diluted to the appropriate concentration and coated plates were made.The plates of the control group were free of antibiotics whereas in experimental group due to plasmid having kanamycin resistance, the plates contained a certain concentration of kanamycin.The plates were divided into equal number of four groups and were placed into the incubator
The results showed that both experimental and control groups grew very well at 37°C, and the control group grew better at 23°C, while few colonies were found in the experimental group at 23°C, which proved that the dormant system basically worked, and after observing the results, the plates of the experimental group incubated at 23°C were put into the incubator at 37°C, and the number of colonies increased significantly after 16 hours, and the growth was good, which proved that the resuscitation system also worked. This proved that the resuscitation system was also effective.
In this project, we focused on building a comprehensive DMBT1 production system to deal with the potential Pseudomonas aeruginosa infections associated with contact lens use.
Through the design and validation of four key systems, we successfully realized the project's vision of providing contact lens users with a more comprehensive eye care and cleaning function to reduce the risk of eye infections and inflammation, thereby enhancing the user's eyewear experience.
First, we successfully constructed a protein expression system, and by monitoring the expression of green fluorescent protein, as well as validation experiments, we realized the efficient expression of DMBT1 protein and verified that it was effective in inhibiting bacteria. The establishment of this system provides a solid foundation for the large-scale production of DMBT1, which lays an important technical support for the subsequent spectacle cleaning process.
The introduction of the population sensing system enabled us to control the bacterial population density and trigger bacterial lysis when needed. Through the introduction of lysis genes and red fluorescence genes of phage and the measurement of growth curves under AHL induction, we successfully realized the function of the colony sensing system and verified its proper operation. This provides a key mechanism for maintaining a constant density of bacterial populations and ensures homeostasis of the entire system.
The role of the toxin-antitoxin system in the overall system is to keep the bacteria dormant and trigger their revival when needed. The successful realization of this system relies on the synergistic action of HicA toxin and HicB antitoxin, as well as the modulation of the temperature-sensitive promoter. We verified the normal expression of the HicA system by observing the growth of plate colonies at different temperatures and the change in the number of colonies before and after placing a 23 degree Celsius plate into a 37 degree Celsius case plate. This system provided us with precise control over the bacterial growth conditions and ensured the controllability of the whole system.
Finally, the introduction of the self-inducible system provided us with a new pathway for spontaneous expression of target proteins.The SILEX system is unique in that it does not require any media adaptation and makes protein self-induced expression possible through metabolic modifications driven by interactions with metabolic E. coli GAPDH enzymes. We basically affirmed its role as a tractable expression by its systematic comparison with IPTG. The successful application of this system provides an effective way for the spontaneous expression of DMBT1, while avoiding the toxic side effects that may be caused by the previous use of inducers such as IPTG. In addition, the introduction of the self-inducible system increased the stability of the whole system and ensured the continuous and efficient expression of DMBT1.
Taken together, the successful integration of these four systems provides a solid foundation for our comprehensive DMBT1 production system. The protein expression system ensures efficient expression of DMBT1, the population sensing system maintains a constant density of bacterial populations, the toxin-antitoxin system provides a precise means of growth state regulation, and the self-induced system makes spontaneous expression of DMBT1 possible
Our system is not just a simple combination of four independent parts, but rather, through careful design and commissioning, these parts are organically combined to form an integrated system with synergistic effects. The synergistic effect of these systems fulfills our initial experimental vision of building a comprehensive and efficient DMBT1 production system, providing an innovative and viable solution to our goal, which is expected to reduce the risk of Pseudomonas aeruginosa infection in contact lens users, and improve ocular health and experience. This will provide new strategies for preventing eye infections caused by Pseudomonas aeruginosa, bringing new innovations and advances to the field of eyewear care. Through this project, we validated our initial experimental concept and provided important technical support for future eye care product development.
This research is not only important for the fields of biopharmaceuticals and protein expression, but also contributes a strong practical example for the development of synthetic biology. The success of this project highlights the importance of interdisciplinary collaboration, combining microbiology, bioengineering, and ophthalmology for eye health, and its success brings new ideas to the field of eye care, laying a solid foundation for future research and innovation.
However, in order to apply it successfully in real production, further experiments and analyses are needed to ensure the feasibility and efficiency of the system, and more in-depth discussions may be required to clarify the interactions between the systems and the room for improvement. In the future, we can further optimize the parameters of the system to improve its efficiency and stability. Meanwhile, we can also consider applying this integrated system to actual contact lens care products to provide users with more comprehensive eye care and cleaning functions, and we look forward to bringing new progress and breakthroughs to this field.