The theme of our project is to use synthetic biology methods to construct an E. coli system capable of producing antimicrobial peptides for the treatment of acne. Acne is a common inflammatory skin disease mainly caused by Propionibacterium acnes. At present, the main methods for treating acne include antibiotics, topical drugs, and laser, but they all have certain side effects and limitations.
Antimicrobial peptides are a type of small molecule peptides with broad-spectrum antibacterial activity. By designing nitric oxide promoters, etc., we have made antimicrobial peptides specific, which can effectively kill or inhibit the growth of Propionibacterium acnes. Harmless to human cells and normal flora. Therefore, we want to use the technology of synthetic biology to introduce antimicrobial peptide genes into E. coli so that it can express and secrete antimicrobial peptides under appropriate conditions, and make it into a hydrogel form for external treatment. Acne.
Cycle stage A
1. Design
Acne vulgaris is a chronic inflammatory disease of the pilosebaceous glands that is common in adolescents, in which excessive proliferation of Propionibacterium acnes is an important factor in aggravating the inflammatory response in acne. At this stage, there are many ways to treat acne, but they all have their corresponding disadvantages. Different from the single-target bactericidal principle of traditional antibiotics, antimicrobial peptides can destroy pathogens at multiple targets, which can greatly reduce the occurrence of drug-resistant bacteria. There are currently clinical cases of antimicrobial peptides being used in the treatment of pathogenic bacterial infections, wound healing, and cancer. After searching the information, we decided to use antimicrobial peptides to treat acne.
When acne occurs, it indicates an imbalance of the epidermal microbiota, a significant decrease in bacterial flora diversity, and abnormal fungal colonization. Probiotics are defined by the Food and Agriculture Organization of the United Nations and the World Health Organization as live microorganisms that are beneficial to host health when administered in sufficient amounts. We want to use probiotics to influence the composition of skin microorganisms, thereby restoring the epidermal microecology and skin barrier, while controlling and alleviating excessive inflammatory responses, and ultimately improving skin health.
2. Build and Test
By reviewing the literature, we compared various antimicrobial peptides against Propionibacterium acnes and selected several antimicrobial peptides for subsequent experiments. At the same time, after comparing the literature, we selected Staphylococcus epidermidis as the probiotic. Staphylococcus epidermidis has a positive effect in regulating skin immune response, maintaining skin barrier, antibacterial defense and promoting wound healing.
Click to view antimicrobial peptide screening PDF
Click to view probiotic screening PDF
3. Learn
Through further understanding, we found that our experimental design has two drawbacks. The first one is that the antimicrobial peptide has a broad-spectrum antibacterial property, which is likely to kill the probiotic we use to express it, and also kill other strains of the human body itself, which will be dangerous. The second point is that the number of probiotics implanted is difficult to control, while less research has been done, and Staphylococcus epidermidis may also cause diseases, such as skin problems and post-surgical infections caused by its imbalance.
Cycle stage B
1. Design
In response to its broad-spectrum antimicrobial properties, we are trying to use an antimicrobial peptide to target the Propionibacterium acnes bacteria, which is directly responsible for the development of acne. The intention is to find a substance secreted only by Propionibacterium acnes to target acne. To address the safety concerns of probiotics, we chose to use engineered bacteria. The E. coli expression system is the earliest expression system to be applied, and it has been studied more intensively, and among all the methods of genetically engineered bacteria for the production of antimicrobial peptides the E. coli expression system is the most common. It has the advantages of clear genetic background, easy genetic engineering operation, rapid reproduction, low growth requirements, cheap, good safety, wide sources, and various modifications are mature.
2. Build and Test
Through literature review, we found that Propionibacterium acnes secretes substances such as porphyrins, CAMP factors, extracellular enzymes and other toxin substances, and inflammatory factors such as IL1-β and IL-6. Or they may produce group sensing and form a biofilm to invade the strain. However, we failed to find a substance that is secreted only by Propionibacterium acnes. We plan to be able to study some Propionibacterium acnes in subsequent experiments, and then achieve a more precise targeted treatment by the same manoeuvre.
However, we found a positive correlation between the severity of inflammation and the amount of NO secreted, and we wanted to find a promoter for our antimicrobial peptide to be produced when the amount of NO secreted reaches a certain level of inflammation severity.
Click to view the secretion survey of Propionibacterium acnes
3. Learn
Through consulting with professors and communication within the group, we explored that the causative agent of acne is also Staphylococcus aureus. In previous years' team study, we knew that only S. aureus secretes V8 protease, an extracellular protease.V8 protease is a single-chained globular protein with a molecular weight of about 29 kDa and consists of 275 amino acid residues.The active centre of the V8 protease consists of one serine residue (Ser221), one histidine residue (His64) and one aspartic acid residue (Asp32), which form a catalytic triangle.
NO secretion was not able to find a suitable standard line to qualify when it should be initiated through a literature search, and the reason for this was analysed because there were too many individual factors that led to varying results in the literature.
Click to view nitric oxide secretion statistics table
Cycle stage C
1. Design
Based on the nature of S. aureus, we intend to use V8 protease as a specific link.The cleavage mechanism of V8 protease is by hydrolysis of the carboxy side peptide bond of glutamic acid or aspartic acid residues on the polypeptide chain. Positively charged antimicrobial peptides are easy to act on negatively charged bacterial cell walls and can bind to negatively charged biological membranes under electrostatic forces, penetrating and destroying the membrane structure to cause cell death. Therefore, we designed a negatively charged antimicrobial peptide and used the mechanism of V8 protease to make the antimicrobial peptide active only when the pathogenic bacteria, and make the broad-spectrum antimicrobial peptide with specificity.
For the NO sensor, we found that the NO sensor initiates an increase in NO secretion as it increases, so we changed our thinking to design a dynamic regulatory mechanism in response to inflammatory changes.
2. Build and Test
We re-screened the antimicrobial peptides and selected six of them for further screening and comparison with respect to antimicrobial peptide source, mechanism of action, strengths and weaknesses, MIC against Propionibacterium acnes, Staphylococcus aureus and engineered bacteria, haemolysis, and toxicity to cortical cells. At the same time, the antimicrobial peptides were optimised and modified, point mutations were performed, and three antimicrobial peptides were modified for comparative experiments, with the intention of screening the most suitable antimicrobial peptides.
3. Learn
During our discussion on the experimental design, we found out that E. coli is able to express antimicrobial peptides but is unable to secrete them, which interferes with the NO sensor we want to use. Also direct contact of our engineered bacteria to the skin could possibly cause skin flora disturbance.
Cycle stage D
1. Design
To address the secretion issue, we intend to use the E. coli haemolysin secretion system, which can achieve extracellular secretion to obtain active proteins, while reducing the toxicity of the producing cells and eliminating the need for cell lysis for downstream processes. To address the safety of engineered bacteria, we envision having a carrier wrapped around the bacteria that allows the antimicrobial peptide to pass through, but the bacteria cannot.
2. Build and Test
We enquired about a number of methods and finally decided to use a hydrogel, with reference to a double-layer dressing based on Pluronic F127 hydrogel, using the same approach to encapsulate our bacteria inside. The internal functional layer consists of engineered bacteria embedded in a hydrogel of a specific composition (DA X), and the external inhibitory layer consists of hydrogels of different compositions (DA 100). This ensures that the engineered bacteria do not escape the hydrogel matrix, but also that they are able to exchange substances with the external environment, receive NO signalling molecules and release antimicrobial peptides out of the hydrogel.
Click to view the application design summary
3. Learn
We intend to design a dressing of our own with reference to the above hydrogel dressing. Through inquiring a lot of information, we conclude that we can optimise the hydrogel by increasing the drug release function of the hydrogel, improving the hydrogel mechanical properties and anti-swelling ability, improving the cooling and analgesic function of the hydrogel, increasing the anti-bacterial and anti-inflammatory effect of the hydrogel, and constructing a self-healing hydrogel.
Cycle stage E
1. Design
We reported back to our teacher for guidance and designed a dressing with a modified alginate base, which will be designed with the idea of a three-layer structure, which consists of an internal nutrient layer, a central overlayer and an external inhibitory layer.
2. Build and Test
Because high molecular weight sodium alginate has better amphiphilicity and more branched chains, we choose high molecular weight sodium alginate among polymer, monomolecular and oligosaccharide sodium alginate. Then we modified sodium alginate by adding phenylborate, formaldehyde and isonitrile to make it amphiphilic. Through stirring, filtration, dialysis, lyophilisation and other steps followed by the addition of PVA and calcium chloride solution, the ratio was constantly adjusted to determine the optimal ratio for the preparation of hydrogel.
3. Learn
Safety is very important, so we also carried out a more detailed experimental design in terms of safety so as not to cause safety problems.
Cycle stage F
1. Design
As for safety, we take into account the safety of the human body as well as the safety of the environment. In both aspects we carry out double security design to achieve double security.
2. Build and Test
In terms of the human body, in addition to the design of hydrogel three-layer design to achieve the prevention of bacterial leakage, we also intend to use nitrocellulose membrane to prevent bacterial leakage again. In terms of the environment, we will choose the nutritional defective strains of bacteria, and after the source, toxicity, price, effectiveness and other aspects of the study selected can be artificially processed to join the titanium oxide doped silver to kill the engineering bacteria.
Click to view the list of killing substances
So we constructed it on pET-32a(+) vector and expressed it with B834(DE3)pLysS. B834(DE3)pLysS is a derivative of B834(DE3), which is a methionine-deficient strain that expresses both T7 RNA polymerase and Escherichia coli RNA polymerase, and it is suitable for protein expression of the pET series of plasmids.
3. Learn
The increase in safety performance also means a decrease in sensitivity, which prompted us to conceptualise how to solve the sensitivity problem.
Cycle stage G
1. Design
We decided to incorporate amplifiers to improve the sensitivity of the NO sensor by taking advantage of the properties of the T7 strong promoter and T7 RNA polymerase. We also used CRISPR technology to modify the pre-existing engineered bacteria to create the best fit for our design.
2. Build and Test
Upon addition of the T7 promoter and T7 RNA polymerase, it was clear that the signal was amplified.
Click to view the amplifier screening documentation
3. Learn
We hoped that in addition to eliminating the causative organisms for acne, we could also reduce the inflammatory factors or improve our improvement results. Through our studies, we found that we could adjust the ratio of herbs or chemicals to combine to improve acne skin problems.
Cycle stage H
1. Design
We found that hyaluronic acid, a chemical that helps us to improve the skin environment, that high molecular weight hyaluronic acid effectively interferes with advanced glycosylation products (AGE) induced NF-κB transduction, and that both high and low molecular weight HA reduces IL-6 and IL-8 expression at the transcriptional level in a cellular model, with good anti-inflammatory effects. We are ready to use hyaluronic acid to combine with our compresses for treatment, but the exact fit is not clear. And we continue to look for other substances, such as Chinese herbs, to improve the therapeutic effect.
We also intend to use a combination of light therapy and related equipment, LED red and blue light irradiation to accelerate the growth of fibroblasts in the acne area and to kill bacteria to effectively treat acne.
2. Build and Test
We searched for light therapy and summarised the methods, aiming to pave the way for the subsequent invention of related instruments.
3. Learn
During our discussion, we thought that although we designed our design to use ice packs and ice cubes during transport so that the bacteria would not die in large quantities in a dormant state, the lack of nutrients would prevent the bacteria from presenting the best condition and the nutritional deficiencies of the design would lead to their death more easily, so we needed to add the appropriate nutrients.
Cycle stage I
1. Design
We plan to provide nutrients to enable the bacteria to secrete antimicrobial peptides properly in addition to the essential amino acids. This can be explored in a couple of ways, one is just adding media like in the lab and the other is using biomaterials.
2. Build and Test
By checking the literature, there is literature that after encapsulating the cultured strain in hydrogel, the strain grows and secretes normally and the hydrogel can also provide nutrients to the strain. Also by checking the literature, there are studies that added sucrose as a nutrient, which can ensure that the strain can survive on the skin for more than tens of days. After discussion and continued searching for information, we found that a fully synthetic medium of the defective strain could be selected to ensure that the strain functioned properly.
3. Learn
At the same time, we want our dressings to be up to standard in terms of functionality, but we also want them to be in line with what the public likes. And the drug odour of both the culture medium and the dressing may cause the user not to want to use it. Our odour treatment is mainly considered from two major aspects, one is the medium itself in terms of composition and principle, and the other is masked by external conditions.
Cycle stage J
1. Design
We plan to use a variety of fragrances to mask odours, even if odourless, to enhance the user's sense of use.
2. Build and Test
We compared and chose several kinds of substances containing special flavour, and plan to carry out self-production and self-use in Hainan University, and start experimenting on a small scale and eventually promote it to a large scale, so as to drive the development of agricultural economy.
Click to view the fragrance arrangement
3. Learn
But we realised that hydrogels are inherently sticky, which can also contribute to a poor user experience.
Cycle stage K
1. Design
We were reminded of the band-aid and decided to copy its construction for our design.
2. Build and Test
By comparison, we decided to use a carrier debridement hydrogel, which facilitates the adhesion and fixation of the dressing to the wound, is non-toxic and non-irritating, and has good breathability.
3. Learn
We never stop learning and hope to build a product that meets the market and standards.
Future prospects
We hope to further optimise our designs and constructs, e.g. to increase the stability and selectivity of the antimicrobial peptides, to reduce their impact on normal flora, to improve their expression and secretion efficiency, etc. In addition, we hope that our project can really bring benefits to acne patients and expand to other fields, such as using different kinds of antimicrobial peptides or other biomolecules to treat other types of skin inflammation or infectious diseases.