Providing valuable contributions to the future iGEM team and promoting knowledge sharing and teamwork are ideals and goals that deserve our utmost attention. We have inherited a lot of valuable experience from past theoretical guidance and operational practices. Following these excellent examples, we would like to expand our knowledge within the iGEM community to not only contribute to practical eye care, but also to provide information and clues for future iGEM teams to draw upon.
As technology continues to advance and the medical industry continues to grow, the application of contact lenses is gradually increasing. It combines comfort and convenience without affecting appearance, making more and more people choose contact lenses as a way to correct vision problems. With the frequent emergence of eye diseases, eye hygiene and health care have also become a topic of increasing importance to the public. Especially in the face of bacterial infections and inflammation caused by contact lens wear, which is a relatively new problem in the field of ophthalmology and rarely studied by the iGEM team in the past. One of our main contributions is to open up a new field of innovative strategies for the resolution of contact lens-induced diseases.
In the iGEM competition, our team designed a system that can help express and regulate the DMBT1 protein to inhibit pseudomonas aeruginosa by modifying the DMBT1 protein expressed in E. coli to bind to the flagellum of pseudomonas aeruginosa and inhibit the bacterial activity, and ultimately achieve the desired effect that pseudomonas aeruginosa's infectious ability to the corneal epithelium of the human cornea is greatly reduced (BBa_K4609070). In addition to this main protein expression system, we designed and constructed a population sensing system to effectively control the population density of E.coli (BBa_K4609072), and strictly regulated the growth state of E. coli with a toxin-antitoxin system (BBa_K4609073 and BBa_K4609055), which gave the instruction of dormancy or restoration to normal through the change of temperature (BBa_K4609054).
We designed and created the Hsp27-SILEX system to induce the expression of DMBT1 target protein in large quantities, replacing the commonly used IPTG inducer, alleviating the toxic side effects of chemical inducers and their inhibitory effect on the growth of E. coli, and it can be popularized and used in the future protein expression system of the iGEM team (BBa_K4609071). system is the first recombinant protein overexpression system that uses lac manipulator-inducible plasmids by self-induction without any medium adaptation, and we have made simple modifications to it to make it easy to induce while performing a wide variety of roles. Due to the lower metabolic burden, less variation in expression levels, and higher plasmid and expression stability of Hsp27 compared to other heat shock proteins,[1] it can be considered to be well suited for protein expression as a self-induction system.
The versatility of the Hsp27-SILEX system lies in the fact that, on top of its primary function of being able to induce efficient expression of target proteins, it is also able to exert the molecular chaperone properties of Hsp27 to promote the correct folding of recombinant proteins, and at the same time, it can effectively protect corneal epithelial cells, endow them with innate defenses and barriers, alleviate the tissue damage of corneal cells due to contact lens wear and promote repair healing (BBa_K4609056). [2]
In our hardware group work, the design of smart contact lens cases has made significant contributions within the field of eye health. Through sensor monitoring, automatic processing, and cloud connectivity, it guarantees the effectiveness of the user's care solution in real time, improves eye hygiene habits, and reduces the risk of infection, providing a safer and more convenient eye care experience for contact lens users. Meanwhile, our proposed bioreaction vessel design also brings new ideas for the automated production of DMBT1 protein.
In microbiological experiments performed in medical laboratories, bacterial colony counting plays a crucial role as a fundamental task in microbiological experiments and is often used to assess the growth and distribution of microorganisms. Traditional methods of colony counting usually involve placing a petri dish on a counting plate and then manually counting each colony one by one. This method, although simple, is time-consuming and prone to subjective operator error. Our modeling group proposes to develop a method to automatically count the number of colonies in photos of pseudomonas aeruginosa colonies using the powerful image processing capabilities of the OpenCV (Open Source Computer Vision Library) algorithm, in order to improve the accuracy and efficiency of the counting, and to achieve more convenient and efficient automated colony counting.
During the structural docking experiments of DMBT1 with pseudomonas aeruginosa flagellin FilC and pseudomonas aeruginosa type IV PilA, we proposed the idea of exploring the structural dockability of DMBT1 with pseudomonas aeruginosa flagellin FilC and pseudomonas aeruginosa type IV PilA by computational methods, and utilized the tools of AlphaFold2 and ClusPro, to achieve a more convenient and efficient automated colony counting. FilC and pseudomonas aeruginosa type IV flagellin PilA, so as to explore their spatial configuration secondary structure and docking effect more effectively and deeply.
The increase in population density leads to changes in their physiological and biochemical properties, displaying features that are not present in small numbers of organisms or in single organisms. We used data fitting to fit the discrete data obtained from experimental measurements of population densities to continuous functions or to match known data with more dense discrete equations. In addition to this, we have adopted the approach of constructing differential equation models, numerical simulations and parameter tuning to control the growth and dormancy of E.coli in the toxin-antitoxin system and the Flp-FRT system. Our data analysis and visualization provide a complete reference of experimental procedures and ideas for future iGEM teams.
[1] Fatemeh Sadat Shariati, Malihe Keramati, Vahideh Valizadeh, et al. Comparison of E. coli based self-inducible expression systems containing different human heat shock proteins. scientific Reports. 2021;11:4576.
[2] Yoo A, Park HM, Kang SS, Kim ES, Tchah H, Kim JY. RNA Interference-based Investigation of the Function of Heat Shock Protein 27 during Corneal Epithelial Wound Healing. j Vis Exp. 2016;(115):54280.