The SkinBait project aims to create a platform for treating various skin-related diseases caused by external organisms. For this 2024 iGEM season, the focus of SkinBait is scabies, which is caused by the mite Sarcoptes scabiei. To demonstrate the project's effectiveness, we are using synthetic biology to engineer the skin microbiota to produce a protein that is harmless to humans but toxic to scabies mites. Concretely, the SkinBait project is composed of three initial modules: (1) chassis characterization, (2) actuators, and (3) safety and biocontention. Once we had defined these 3 modules we could start with the design of final 4th module which is defining the (4) key experiments.
For each module we have planned and executed specific engineering cycles [(D)esign, (B)uild, (T)est, and (L)earn]. Each cycle will be explained in our Engineering page. Here, we will describe how we designed each module of the Skinbait platform and our general workflow:
In this project, this team aims to study the skin microbiome to address issues caused by various organisms, such as scabies. Our main chassis for this work will be Cutibacterium acnes (formerly known as Propionibacterium acnes). However, we also utilized different strains of Escherichia coli throughout the project, as the transformation process required the use of E. coli prior to obtaining the final transformed version of C. acnes.
The first step in our design process was to do storage of our parts, to avoid having to request more of the same fragment to companies. During our design process we planned to use the NZY 5α E. coli strain, since it is very well documented and its relatively short period of incubation makes it suitable for this task. 1
The pipeline for the transformation of Cutibacterium acnes requires the usage of another E. coli strain which is the EC24; 1 this strain is engineered to contain different methylation mechanisms that will be applied into the plasmid with our insert of interest. The reason behind this methodology is because Cutibacterium acnes contain defense mechanism systems called RM (Restriction-Modification) that contains endonucleases that recognize non-methylated DNA to cut it.2,3
Finally, as mentioned in the introduction, our main focus is Cutibacterium acnes, more precisely the strain KPA171202, is a gram-positive bacterium naturally present on human skin. Its ability to detoxify oxygen and adapt to environmental changes makes it an ideal candidate for genetic modification. Since C. acnes is a commensal bacterium, it coexists well with the skin’s natural microbiota, causing minimal disruption to the skin's ecosystem. These unique traits ensure that C. acnes is particularly well-suited for our project. 4
In our project, the selection of appropriate backbones and promoters was critical for achieving efficient protein expression in our novel chassis, C. acnes. Due to the relatively recent exploration of C. acnes as a chassis in synthetic biology, there is a lack of well-characterized genetic tools for this organism, which introduced several challenges.
First, the backbones had to be compatible with the assembly methods we planned to use, such as Gibson assembly, while also ensuring the stability and expression of the genetic constructs in C. acnes.
Additionally, finding suitable promoters that could induce reliable expression in C. acnes was particularly challenging. Most promoters characterized for common bacteria like E. coli are not effective in C. acnes due to differences in regulatory elements and transcriptional machinery.
We prepared two different plasmid backbones: p-JET, which comes from the ThermoFisher cloning kit, and p-END, a backbone created by Javier Santos, Marc Güell, and Guillermo Nevot. Each backbone had different uses due to its unique characteristics, which suited them to specific tasks.
To complement the modifications made to the p-END plasmid for C. acnes, we also engineered all of our parts with specific prefixes and suffixes. These short DNA sequences, added to the 5' and 3' ends of each fragment, provide homology overlaps with neighboring fragments, facilitating efficient joining during Gibson assembly. By designing these homology regions, we ensured seamless integration of our parts, optimizing the overall assembly process and enhancing the stability and functionality of the plasmid within the C. acnes chassis.
During the design of our project, we had clear objectives. Our main goal was to express toxins that could kill S. scabiei and demonstrate their effectiveness. Additionally, we aimed to obtain preliminary results in E. coli to facilitate the crucial engineering process for project development.
To achieve this, we used two constitutive promoters, MG10 and MG26 while also considering implementing an inducible, leaky promoter at some stages despite this idea being discarded at the end of the design.
Both promoters are compatible with C. acnes, but only MG10 induces some gene expression in E. coli. Therefore, our protein expression results that were achieved in E. coli come from constructs that used the MG10 promoter. We chose not to rely on a single promoter, since we didn’t want our results to depend on only one promoter that could induce potential metabolic stress or cytotoxicity from overproduction of proteins. Thus, we included the MG26 promoter, which expresses in C. acnes but not in E. coli, to mitigate these risks.
For this second module we chose to use different Cry Toxins as actuators against the skin parasites that we want to treat. During the design of the project we decided to choose two different Cry proteins which were the Cry3Aa and the Cry4Ba and thanks to this decision we could demonstrate some proof of concept while having a Cry toxin potentially useful against S. scabiei.
For the Cry toxin, in order to act they need to be ingested by insect larvae and is activated in their alkaline gut. After that, It binds to receptors in the intestinal cells, creating pores that disrupt cell function, causing cell lysis. This leads to digestive failure and the death of the insect 6, 7.
Most Cry proteins, including those selected, share a similar structure consisting of three domains: Domain I, responsible for pore formation in insect gut cell membranes; Domain II, which binds to specific receptors in the insect midgut; and Domain III, which stabilizes the toxin-receptor interaction 7
To evaluate the efficiency of our actuators, we selected two different Cry proteins to address distinct proof-of-concept objectives for our project. We chose Cry3Aa, which has potential to target S. scabiei, and Cry4Ba, which allows us to demonstrate our mechanism in Drosophila. This dual approach enabled us to progress with both a relevant toxin for scabies treatment and a model organism for validating our concept.
We believe Cry3Aa has potential to target S. scabiei due to its demonstrated effectiveness against similar organisms. For instance, in previous iGEM projects like iGEM Wageningen 2016,8 Cry3Aa showed promise beyond beetles9, notably in combating Varroa destructor, a mite that parasitizes bees. Since V. destructor is an arachnid like S. scabiei, we aim to explore its efficacy against this parasite in future experiments.
On the other hand, Cry4Ba has been proven highly effective against mosquito larvae, particularly Aedes and Anopheles species 10, and has also been tested on Drosophila melanogaster to evaluate its broader insecticidal potential. These specific targets make Cry4Ba an ideal choice for our project, providing numerous opportunities to demonstrate our proof of concept.
Both of the sequences that we used to develop our project were designed using Benchling 11, and codon optimization was performed through the ATGme.org database 12 as well as a custom Python script to ensure compatibility with C. acnes.
An additional challenge was encountered with Cry4Ba, which was too large to be ordered as a single part. To address this, the gene was divided into two segments, including a 30 bp homology sequence, enabling alignment and assembly of the complete sequence through Gibson assembly.
After designing the second module, we received valuable feedback from investors and healthcare professionals suggesting the need to enhance our design by incorporating containment mechanisms ensuring greater patient safety when using our lotion.
Motivated by this new feedback, our team conducted further research and identified an innovative mechanism to enhance control over our engineered bacteria. The next step in our containment design was to incorporate an RNA thermometer, which triggers the expression of a gene inducing cell death at a specific temperature. With this concept in mind, we initiated a new engineering cycle to design and optimize the updated construct, ensuring both functionality and safety.
We selected the FourU RNA thermometer from the iGEM registry 13 for its ability to regulate gene expression at temperatures close to that of human skin (36-37°C). This RNA thermometer functions like a "zipper", forming a stable secondary structure at lower temperatures that conceals the ribosome-binding site (RBS) 14, preventing translation. As the temperature rises, the hydrogen bonds destabilize, causing the RNA to "unzip" and expose the RBS. This allows the ribosome to bind and initiate protein synthesis once the appropriate temperature is reached.
Once we have defined which RNA thermometer to use, we aim to use this structure to regulate the expression of endolysin 15 which will allow us to regulate the growth of our engineered bacteria as a containment measurement. The sequence chosen codes for the CAP 10-3 endolysin which is an enzyme derived from a bacteriophage that specifically targets and lyses C. acnes. Its inclusion in our project is essential, as it allows us to regulate the growth of C. acnes, ensuring a balanced interaction between the patient’s original skin microbiota and the newly introduced microbiota from our product, thereby preventing dysbiosis.
The primary function of this enzyme is to induce cell death in the transformed C. acnes. It achieves this by degrading the peptidoglycan links in the bacterial cell wall, creating pores that destabilize the cell membrane. Ultimately, the exposed C. acnes cells succumb to osmotic pressure, leading to their death. 15
To effectively demonstrate the efficiency of this combined gene circuit, we aimed to characterize the behavior of the FourU RNA thermometer in C. acnes, assessing its viability in this novel chassis. For this characterization, we designed an experiment coupling the RNA thermometer with different fluorescent proteins, allowing us to quantify and visualize the regulatory mechanism's performance.
The fluorescent genes that were designed for this experiment, were sfGFP which is a variant of the Green Fluorescent Protein (GFP) engineered for enhanced folding efficiency 16 and the mCherry which is a red fluorescent protein derived from the coral Discosoma 17.
To optimize our budget, we ordered oligonucleotides containing the RNA thermometer, along with a prefix and a 23 bp aligning region, from IDT, as we already had fluorescent genes optimized for C. acnes in our laboratory. We then employed PCR to align these oligonucleotides with existing fluorescent protein parts (MG140 for sfGFP and MG111 for mCherry), enabling us to create the final constructs without needing to order them in full. It’s worth noting that these parts were also optimized for C. acnes by following the same pipeline used for the Cry proteins. We began by designing the parts using Benchling11, and then optimized their codons using the ATGme.org database 12 and a custom Python script.
After completing the design of the previous modules, we began considering the next steps once the team successfully transformed C. acnes to produce the target toxins. This led to the development of the final module, where we meticulously modeled three key experiments to demonstrate that the objectives of both the actuator and biosafety modules have been achieved through effective design and chassis characterization. This is the designed point that represents the convergence of all modules, which will be culminating in the production of our results.
To demonstrate that the engineered organism produces sufficient Cry toxins to effectively treat scabies, the SkinBait team designed an experiment comparing the toxin’s lethality to a non-hazardous model organism, Drosophila melanogaster. In this experiment, survival rates of different D. melanogaster populations are compared: one group is exposed to C. acnes engineered to produce the Cry4Ba toxin, while the other group is exposed to C. acnes that does not constitutively produce this toxin. This experiment is designed to be conducted with Cry3Aa allowing us to determine the specificity of our toxin of interest against different organisms.
The SkinBait team designed experiments to evaluate the effectiveness and optimal working temperature of the FourU RNA thermometer for C. acnes. To assess this, the RNA thermometer was first going to be coupled with two different fluorescent proteins, sfGFP and mCherry. The transformed C. acnes cells would then be placed to grow at different temperatures (room temperature, 30ºC and 37ºC), and the transcription of the fluorescent proteins would be quantified over the following days. We anticipated that the fluorescence intensity would be greater at higher temperatures.
The next step would be the coupling of the FourU RNA thermometer with the endolysin, which would allow for targeted disruption of C. acnes cells at specific temperature conditions. By linking the endolysin expression to the RNA thermometer activity, we aimed to create a temperature-responsive system that only activates endolysin expression when the temperature is optimal for bacterial lysis.
This experiment aims to assess the effectiveness of endolysin in inducing cell death in C. acnes. To quantify its efficiency, the team developed an experiment comparing survival rates of C. acnes across different conditions. One group contains C. acnes with an empty plasmid, another contains C. acnes expressing a GFP plasmid to increase cellular stress, and a third group is engineered to express the endolysin. By analyzing these populations, the experiment seeks to characterize the endolysin's apoptotic capabilities.
In this design phase, the SkinBait team has conceptualized various gene circuits utilizing the different promoters discussed earlier. Each circuit is designed to pair with a specific Cry toxin, along with an RNA thermometer and endolysin, aiming to ensure safe and effective skin treatments for patients after a proper combination of the modules.
The design process involved developing each component individually to enhance cell viability and allow for proper characterization. Prior to integrating the RNA thermometer with the endolysin, the team planned to test its functionality by coupling it with genes expressing fluorescent proteins, such as sfGFP and mCherry, to measure outputs and validate the RNA thermometer's performance in C. acnes.
Looking ahead, the designed circuits are categorized into two types: those with a single promoter and one part, and those combining multiple parts. This structured approach sets the stage for future experimental cycles aimed at achieving our project objectives.
In the figures below we show the different circuits that are planned to be developed during the experimental phase, this are the proposed circuits:
| Module 2: Genetic circuits | |
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| MG10 + Cry3Aa | MG26 + Cry3Aa |
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| MG10 + Cry4Ba | MG26 + Cry4Ba |
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| Module 3.1: Characterization of the RNA Thermometer | |
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| Low temperature (~20 °C): | |
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| Higher temperature (~30 °C): | |
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| Highest temperature (~37 °C): | |
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| Module 3.2: Characterization of the endolysin | |
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| MG10 + Endolysin | MG26 + Endolysin |
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| Final construct | |
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| Low temperature (~20 °C): | |
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| Higher temperature (~30 °C): | |
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| Highest temperature (~37 °C): | |
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