Concept

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Welcome to the proof of concept page!

Here, we will show you how we implemented our project design in a wet laboratory, which utilizes a nitric oxide-induced genetic circuit to secrete fusion proteins, which are digested to release antimicrobial peptides with antimicrobial activity, and based on our experimental results, a matching hydrogel application was developed. We successfully implemented an E. coli-based antimicrobial peptide secretion system for the treatment of acne. You will see our experimental methods, data and analysis on this page, as well as our product prototypes and outlook.

overview

Our project aims to use synthetic biology methods to design a hydrogel capable of secreting antimicrobial peptides through E. coli for the treatment of acne. Acne is a common skin inflammatory disease mainly caused by bacterial infections such as Propionibacterium acne and Staphylococcus aureus. Currently, antibiotic drugs on the market are not effective in treating acne, and are prone to drug resistance and side effects.

In view of the above acne hazards and research status, we designed an antimicrobial peptide secretion system based on engineered bacteria. We finally chose antimicrobial peptides with antibacterial, anti-inflammatory and wound healing as our target molecules. And designed a plasmid containing a nitric oxide promoter to make it specific and transformed into E. coli. When the cell feels a certain concentration of nitric oxide signal, it will initiate expression. Then, we use V8 protease to treat the culture medium, cut off the cleavable check point, and release the antimicrobial peptide. Finally, we add the successfully converted E. coli to the finished hydrogel application, and add some other auxiliary substances to form a product that can continuously release the antimicrobial peptide and combine the treatment of acne.

Screening of antimicrobial peptides

Antimicrobial peptides are usually small peptides composed of short amino acid sequences, which have broad-spectrum antibacterial properties and low drug resistance, and some antimicrobial peptides also have obvious inhibitory effects on acne inflammation. By reviewing a large number of literatures, we studied their minimum inhibitory concentrations of Staphylococcus aureus, Propionibacterium acnes, and Escherichia coli, hemolytic properties, toxicity to cortical cells, advantages, isoelectric points, sources, and the latest research mechanisms. And the antimicrobial peptides were self-designed by the method of point mutation, and 9 plasmids were constructed on pET-32a (+) for follow-up experiments.

Plasmid construction and import

During our research, there are 9 antimicrobial peptides with different properties that need to be calculated for isoelectric points. These antimicrobial peptides all have good antimicrobial properties, but under laboratory conditions, we found that their effects can be further enhanced. To achieve this goal, we plan to add negative charges to these 9 antimicrobial peptides at suitable locations after calculating the isoelectric points. The purpose of this is to improve the stability and activity of the antimicrobial peptides so that they can function better in practical applications.

After the isoelectric point calculation is completed, we construct these negatively charged antimicrobial peptide sequences one by one on the pET-32a (+) vector. This vector has high expression efficiency in Escherichia coli, which can ensure the smooth realization of our research results. After the construction is completed, these vectors are converted into our engineered bacteria B834 (DE3) pLysS. This engineered bacteria has good expression performance and stability, which can ensure that Escherichia coli can smoothly produce the corresponding species of negatively charged antimicrobial peptides.

Overall, our research goal is to provide more effective antimicrobial solutions for practical applications by improving the structure of antimicrobial peptides and improving their inhibitory effect on bacteria.

Protein expression and purification

After a series of rigorous resuscitation activation and expansion operations, we successfully induced the engineering bacteria with 9 different plasmids to secrete the corresponding antimicrobial peptides. These antimicrobial peptides have good antibacterial properties and are an important result of our research. In order to obtain these antimicrobial peptides, we need to break up the engineered bacteria first, and then collect the lysate containing the antimicrobial peptides.

To ensure high-purity antimicrobial peptides are extracted, we purify the extract several times. This process involves removing cell debris, proteins and other impurities in order to obtain the target antimicrobial peptide. The purified extract will be used for subsequent experiments and analyses to validate our findings.

In addition, we also plan to conduct qualitative and quantitative analysis of antimicrobial peptides in different lysates to evaluate the expression level of engineered bacteria and the yield of antimicrobial peptides. This will help us further optimize experimental conditions and improve the yield and purity of antimicrobial peptides. During this process, we will continuously adjust and optimize experimental methods to ensure that our research can achieve better results.

Protein activity verification

First, we need to understand the definition of a fusion protein. A fusion protein is a novel protein formed by combining two or more protein molecules through genetic recombination techniques, with the functions and properties of their respective components. In the laboratory, scientists often use fusion proteins as research tools to explore protein functions and interactions.

In this experiment, the researchers first successfully purified the protein and then performed activity verification on the fusion protein. Activity verification is to ensure that the fusion protein can perform its intended function. To do this, they used V8 protease for cleavage, a commonly used enzymatic cleavage method that can effectively study the activity of the protein.

The activity of the fusion protein changed significantly before and after digestion. This is an important finding because it suggests that the activity of the fusion protein is affected by its structure and composition. Next, the researchers confirmed that the fusion protein has the property of inhibiting bacterial growth by observing the growth inhibitory effect of the fusion protein on Propionibacterium acne and Staphylococcus aureus.

In the experiments, they observed a distinct inhibition zone, which is due to the activity of the fusion protein. The inhibition zone is a visual experimental result that shows that the fusion protein has an effective signal reporter gene activity. This discovery has important implications for studying protein function and developing new antimicrobial drugs.

In conclusion, by cleavage using the V8 protease, the researchers found that the fusion protein has the property of inhibiting the growth of Propionibacterium acne and Staphylococcus aureus, forming a distinct bacteriostatic circle. This suggests that the fusion protein has effective signal reporter gene activity, providing strong evidence for further research on protein function and application.

Hemolysin secretion system

Hemolysin secretion is a common protein expression system based on the natural mechanism of extracellular protein secretion in Escherichia coli, which utilizes the bacterial secretion machinery to transport target proteins to the bacterial extracellular for the expression and secretion of proteins in Escherichia coli.

We bind our antimicrobial peptide to the secretion signaling sequence HlyAs, which is recognized by the secretion system and transports proteins through the complex through the bacterial intracellular plasma membrane. It is through the synergistic work of multiple components such as HlyB, HlyD, Tolc, etc. to transport the target protein from the bacterial cytoplasm to the extracellular.

Modification of nitric oxide promoter

It is known that changes in nitric oxide (NO) concentrations are proportional to changes in the severity of inflammation. That is to say, as acne becomes more severe, our inflammation will increase and the amount of NO secretion will increase. So we use the NO promoter as a response mechanism to dynamically regulate the secretion of antimicrobial peptides.

When a biosensor inputs a signal, sometimes due to different external conditions or signal sources, there are occasional situations where the strength of the output signal is not enough to be fully perceived. Our engineered bacteria will be placed inside the application, which may interfere with the activation of our NO sensor while improving safety performance. To solve this problem, we have designed a sophisticated amplifier system that takes advantage of the high activity of the T7 promoter and can expand the output signal to a predetermined multiple. We cleverly inserted the gene of the T7 RNA polymerase and the T7 promoter downstream of the sensor, creating a component we call an "amplifier". The key property of this amplifier is that it only activates in the presence of the T7 RNA polymerase, allowing the output signal to be enhanced.

Modification of engineered bacteria

Antimicrobial peptides have broad-spectrum antimicrobial, anti-inflammatory and wound healing-promoting functions. To enable our system to secrete specific antimicrobial peptides, we designed plasmids with nitric oxide promoters, ribosome binding check points, hemolysin genes, etc., and transformed into E. coli B834 (DE3) pLysS. When cells sense a certain concentration of nitric oxide signaling, the expression of fusion proteins is initiated and secreted into the culture medium. We then collected the culture medium and treated it with V8 protease, cutting off the cleavable check point and releasing the antimicrobial peptide.

Optimization of hydrogel materials

As a bio-based polymer, sodium alginate has been widely used in various controlled release systems. Among them, the guloronic acid block of sodium alginate is easily complexed with divalent calcium ions to form a gel structure. The hydrophobically modified alginate derivatives can effectively improve the loading rate of hydrophobic drugs. Colinet et al. reported a pH-sensitive, amphiphilic and biocompatible gel bead based on alginate-g-PCL for controlling the delivery of theophylline drugs with poor water solubility. The study found that the load efficiency of alginate-g-PCL/Ca~ gel beads was significantly higher than that of alginate/Ca2 + gel beads.

In this study, we used the Ugi multi-component method to graft hydrophobic groups and phenylboronic acid groups to the sodium alginate (Alg) skeleton to prepare amphiphilic phenylboronic alginate (Ugi-Alg-PBA). We successfully prepared composite gel beads by complexing the Alg/Ugi-Alg-PBA compound solution with calcium ions. The hydrophobic groups not only improve the water-insoluble drug loading rate of the gel beads, but also enhance the structural stability of the gel beads through the interaction of hydrophobic groups, inhibit the swelling of the gel and slow down the drug release rate. At the same time, the phenylboronic acid group and the cis-diol on the Alg skeleton form a dynamic B-O covalent bond in an alkaline environment, making the gel beads pH-sensitive and more stable in an alkaline environment.

We also used this material to make a hydrogel application, which is used to wrap the engineered bacteria. The engineered bacteria will secrete antimicrobial peptides, and through a three-layer structure - one hydrogel containing the bacteria, one hydrogel containing no substance, and one hydrogel containing tannin acid, the engineered bacteria will not leak, and the antimicrobial peptide can be secreted. At the same time, for more safety, we plan to add a nitrocellulose membrane to make sure that the bacteria will not touch the outside skin. This design provides new ideas for achieving controlled release of drugs.

Fragrance helps agriculture

In order to be more in line with entrepreneurial ideas and increase innovation, we will use big data and artificial intelligence technology to accurately analyze fragrance choices to meet the needs of more users. Hainan University has an agricultural base and grows mango, coconut, dragon fruit and other fruits. We plan to start experiments from a small scale, gradually expand and cooperate with farmers to promote agricultural development and achieve a win-win situation. An excellent product should be continuously innovated and optimized in terms of function and user experience. Considering that the application may produce odors due to drugs, media or combined treatment with traditional Chinese medicine, we believe that it is necessary to make in-depth improvements in odor research to ensure that the user's experience is not affected. At the same time, providing a variety of fragrance options can not only meet the individual needs of users, but also promote the development of my country's local economy and agricultural progress. In order to achieve sustainable economic growth in my country, we will cooperate with farmers to create a closed-loop industrial chain integrating planting, research and development, production and sales, promote the modernization of agriculture, and contribute to the realization of sustainable economic growth in my country.

Summary

1. After repeated experiments, the nitric oxide promoter is more sensitive than the previous good promoter elements after modification.

2. After the protein was successfully purified, we verified the activity of the fusion protein. After cleavage with V8 protease, the fusion protein inhibited the growth of Propionibacterium acne and Staphylococcus aureus, and a clear bacteriostatic circle appeared. This indicates that we successfully transformed the plasmid and purified the protein.

3. Add the successfully transformed Escherichia coli B834 (DE3) pLysS to the finished hydrogel application. The combined therapeutic substances we selected have been added to the hydrogel application for enhanced treatment of acne and repair of acne marks. Experiments have shown that the application has antibacterial effects on Propionibacterium acne and Staphylococcus aureus.

To sum up, we implemented the technology to accurately and quickly cut individual bases and produce fluorescent proteins in the wet laboratory, and based on the results we achieved in the wet laboratory, we developed a matching hardware bacterial automatic culture instrument that allows us to Industrially produce our strains. Therefore, we have successfully achieved the goal of treating acne with antimicrobial peptides and combination therapeutic substances secreted by engineered bacteria based on hydrogel application.

Outlook

After we successfully completed all the designs of the project, we achieved the goal of treating acne with antimicrobial peptides and combined therapeutic substances secreted by engineered bacteria based on hydrogel application. In the future, we will continue to study the physicochemical properties of new synthetic antimicrobial peptides based on natural antimicrobial peptides through genetic engineering, so as to achieve stronger antibacterial and anti-inflammatory ability without causing any damage to the human body and better protect human health and life safety; in terms of exploration, we plan to establish partnerships with local high and middle school institutions to create an extracurricular practice platform to stimulate interest in the exploration of synthetic biology, and we will further optimize, such as sensors applied to detection.

At the same time, we have more prospects in many aspects. Skin diseases are not limited to acne, and more people are suffering from other diseases, such as age spots, freckles, melasma, etc. We are committed to using this method to solve other diseases, so that more people are free from disease interference. We will also conduct more in-depth research on pathogenic bacteria, learn and combine other technologies, and use better and better methods to solve acne and other skin diseases.

We will not give up our skin communication platform. We will uphold the spirit of iGEM to let more people know about synthetic biology and their own skin problems. Let more people be interested in biology, deepen their own understanding, and communicate with each other to solve skin problems. Of course, after passing clinical research and national review, we will sell the designed packaging on it to help you solve the acne problem.