Catalog

Design

Acne: a chronic inflammatory disease

Our project aims to develop treatments for acne. Acne is a chronic inflammatory skin disease that affects the pilosebaceous glands and has certain disfiguring properties. It is characterized by comedones, papules, pustules, nodules, cysts and scars. It can occur in people of all ages, but the incidence rate is highest among teenagers (1). Acne that occurs in teenagers is generally acne vulgaris, also commonly known as whelk. The occurrence of acne is closely related to factors such as excessive sebum secretion, blockage of pilosebaceous gland ducts, bacterial infection, and inflammatory reaction (1, 2). The diversity of the acne skin microbiota is reduced, while the numbers of Propionibacterium acnes and Staphylococcus aureus are significantly increased (2, 3, 4).

There are many ways to treat acne at this stage, including topical treatments and oral medications. Topical treatments include the use of benzoyl peroxide. Oral drug treatment includes the use of medications such as oral antibiotics, oral contraceptives, and oral isotretinoin. These medications have certain side effects. For example, benzoyl peroxide may cause dry skin, flaking, and redness, and antibiotics may cause indigestion, diarrhea, and drug resistance (1, 2, 5). It is known that these treatments have side effects on the human body or are not targeted, so a new, effective, and precise treatment method is needed. Our team aimed to develop a system that could selectively kill acne-causing bacteria at sites of inflammation without damaging the normal skin microbiome.

Figure 1:Methods of treating acne and corresponding pitfalls

Our engineering chassis


We chose B834(DE3)pLysS. B834(DE3)pLysS is a derivative strain of B834(DE3). B834(DE3) is the parent strain of BL21(DE3) strain (BL21(DE3) is a derivative strain of B834(DE3) ). This strain has the following characteristics:

1.B834(DE3)pLysS is widely used in prokaryotic expression experiments of proteins and is a methionine auxotrophic strain.

2.B834(DE3)pLysS can express T7 RNA polymerase and E. coli RNA polymerase at the same time, which is suitable for protein expression of pET series plasmids, and the plasmid we selected is pET-32a(+).

3. The pLysS plasmid carried by B834(DE3)pLysS contains the gene for expressing T7 lysozyme, which can reduce the background expression level of the target gene but does not interfere with IPTG-induced expression. It is suitable for expressing toxic and non-toxic proteins.


Nitric oxide as an inflammatory marker

Our modified E. coli B834(DE3)pLysS is able to locally sense inflammation through a genetic circuit that responds to nitric oxide (NO) (6,7). It is unquestionable that acne is a chronic inflammatory disease involving the pilosebaceous glands, and nitric oxide (NO) levels are significantly increased at and near the site of skin inflammation (8). In addition, the molecule can diffuse through the bacterial cell wall, making it easier for specifically engineered bacteria to sense it. We plan to use the NO molecular mechanism to dynamically respond to changes in inflammation and automatically secrete antimicrobial peptides for treatment.

Since the final product is placed in a hydrogel, in order to improve the sensitivity of the NO sensor, we plan to optimize the NO sensor and use T7RNAP as an amplifier to improve expression. (9)

Figure 2:Schematic diagram of antimicrobial peptide secretion mechanism

It is also planned to use the CRISPR system for gene knockout, select different chassis engineering bacteria, transform the chassis engineering bacteria, and cooperate with amplifiers to achieve better results.

Figure 3:Schematic diagram of how to use CRISPR-Cas9

Choice of antimicrobial peptides

In particular, Propionibacterium acnes and Staphylococcus aureus, which are fungi that have been identified in recent studies on acne as having an important relationship with the onset of acne, will appear in acne due to changes in the skin environment into a state suitable for their growth. Multiply. After reviewing the literature, we learned that we screened the literature and conducted research based on previous research. After considering the sources, mechanisms of action, advantages and disadvantages of antimicrobial peptides, MICs against pathogenic bacteria and engineered bacteria, hemolysis, and toxicity to cortical cells, six of the antimicrobial peptides were selected for further screening and comparison. At the same time, the antimicrobial peptides were optimized and transformed, point mutations were carried out, and three antimicrobial peptides were transformed for comparative experiments, aiming to screen out the most suitable antimicrobial peptides. (10-12)

Figure 4:Mechanism of action of antimicrobial peptides

Create specific antibacterial peptides

Regarding the specific expression of our antimicrobial peptides in the skin, we plan to use the V8 enzyme cleavage site and a negatively charged peptide structure at one end of the antimicrobial peptide. The inactivation of the antimicrobial peptide caused by negative charge shielding can only be secreted by Staphylococcus aureus. Only after the V8 protease cleaves off the negatively charged part can it regain its activity and exert its antibacterial effect (13, 14). At the same time, we will not stop reading the literature to find other better proteases to make our project more complete and improved, and use experiments to verify it.

Figure 5:Schematic diagram of the mechanism of action of specific antimicrobial peptides

hemolysin secretion system

Synthetic antimicrobial peptides usually include chemical synthesis and biosynthesis, and the biosynthesis method usually uses engineered bacteria to produce and lyse the engineered bacteria to obtain their secreted products for antimicrobial peptide treatment. This experiment designed the NO response mechanism to dynamically regulate the expression of antimicrobial peptides based on the severity of inflammation, which requires E. coli to secrete antimicrobial peptides to the outside. After consulting a large amount of literature and learning from the iGEM team in previous years, we chose the hemolysin secretion system to achieve the purpose of secreting proteins. (15)

The hemolysin secretion system is a commonly used protein expression system for expressing and secreting proteins in Escherichia coli. It is based on the natural mechanism of extracellular protein secretion of E. coli and uses the bacterial secretion machinery to transport the target protein outside the bacterial cell. It has the following advantages:

1. High expression level, the hemolysin secretion system can achieve high levels of protein expression, usually with higher expression levels than other expression systems such as E. coli expression systems.

2. Soluble expression, the hemolysin secretion system can dissolve the protein in the cell and release it into the culture medium through the secretion pathway, making the protein easy to purify and extract.

3. Maintain biological activity. The hemolysin secretion system is able to maintain the biological activity of the protein because the protein is correctly folded within the cell and undergoes correct post-modification through the cell secretion pathway.

Figure 6:Schematic diagram of the mechanism of action of the hemolysin secretion system

hydrogel part

In order to enable our engineered bacteria to receive NO signals on the skin surface and stably express antimicrobial peptides, the bacteria will not cause harm to our human body. We designed an alginate-based hydrogel with reference to Pluronic F127 hydrogel (16-18). The Ugi multi-component method was used to attach the hydrophobic group and phenylboronic acid group to the sodium alginate (AIg) skeleton to prepare amphiphilic phenylboronic acid-based alginate derivatives (Ugi-Alg-PBA). In order to simultaneously satisfy the growth of engineered bacteria, interact with the surrounding environment and inhibit their excessive reproduction, we will adopt a three-layer structure design idea. The three-layer structure includes an internal nutrient layer, a middle transition layer and an external inhibition layer. This design not only ensures that the engineered bacteria will not escape from the hydrogel matrix, but also ensures that the engineered bacteria can communicate with the external environment, receive NO signal molecules, and release antimicrobial peptides out of the hydrogel. (19,20) In order to prevent the leakage of engineering bacteria, we also added an additional layer of nitrocellulose to prevent engineering bacteria from contaminating the skin.

On the basis of hydrogel, we also consider combination therapy, which has the advantages of synergistic treatment mechanism, reduced drug dosage and enhanced therapeutic effect. We considered using some drugs that inhibit inflammatory factors in conjunction with the antibacterial effects of antimicrobial peptides to exert the synergistic antibacterial effect of the drugs to improve the efficacy. At the same time, it is planned to form a combined treatment with light therapy to achieve better therapeutic effects. (21,22)

Figure 7:Schematic diagram of hydrogel design

Safety

In terms of safety, we have dual designs for human safety and environmental safety. Use antibacterial tannic acid to block bacteria from infecting our skin (19). At the same time, you can also use nitrocellulose membrane to achieve the penetration of antibacterial peptides and NO and prevent the infiltration of E. coli (23), achieving double human protection. At the same time, we compared different sterilizing substances and finally chose titanium dioxide doped with silver (24). We plan to sell this as part of the product so that users can artificially sterilize it after use, so that bacteria cannot pollute the environment. And the engineering bacteria we use are auxotrophic bacteria, which are methionine-deficient strains and cannot produce this amino acid on their own. We will adjust the ratio of amino acids so that the bacteria can only survive for a certain period of time. Even if the user forgets to disinfect it after use, it will die on its own without polluting the environment, achieving double environmental protection.

Figure 8:4 safe designs

Fragrance and agricultural aid

As a product, in addition to considering the performance of the product itself, we also consider the user experience. It is known that the culture medium that provides a growth environment for bacteria has a certain odor, and our products are placed on the user's face, which may make the user feel uncomfortable, and our combined treatment will also have traditional Chinese medicine products that produce a medicinal smell. So we intended to use other fragrances to cover up the odor and make the user experience better. (25)

We use a variety of spices to improve the taste. After on-site inspections, literature reviews, teacher consultations and other methods, we combined Hainan’s local characteristics and referenced commonly used spices to decide on the agricultural aspect of self-production and sales to promote agricultural development and use coconut , mango, etc. as the basis to study the proportion of spices. At the same time, purchase commonly used spices such as peppermint oil and rose essential oil for comparative research to give users more choices. Using local characteristics to conduct product-related research will help drive the development of Hainan and drive the rural economy.

Figure 9:Fragrance selection diagram

Conclusion

Our system relies on a NO sensor and a negatively charged shielding module to target acne bacteria and prevent damage to normal skin microflora. To ensure specificity of expression, we will use a V8 protease site and a negatively charged peptide construct at the N-terminus of the peptide. In order to enable our engineered bacteria to receive NO signals on the skin surface and stably express antimicrobial peptides, we optimized the NO sensor and planned to perform gene knockout to transform a more suitable auxotrophic defective strain. For human safety and environmental safety, we designed a double protection mechanism and used amphiphilic sodium alginate to make a hydrogel, and finally formed a hydrogel.

Our project could improve quality of life for acne patients, reduce the burden of chronic inflammatory skin disease on society, and open up a new, promising field of research through the use of synthetic biology tools.

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