Engineering

Designing and Building

1

In our initial design cycle, we aimed to develop a biological solution for treating parasite infestations in a sustainable, marketable way. After thorough discussions among the team members and instructors, we identified the human microbiome as an ideal platform for this approach, with Cutibacterium acnes emerging as our bacterial chassis. We chose C. acnes because it naturally resides in human skin, particularly in areas where scabies mites (Sarcoptes scabiei) may be found. This made it an optimal vector for delivering our treatment to the locations of infestations.

For the insecticidal component, we selected Cry 3Aa, a toxin from Bacillus thuringiensis. We chose Cry 3Aa after extensive research, which showed that this toxin specifically targets mites, including scabies mites, by binding to receptors in their gut, forming crystals, and ultimately disrupting the digestive cells. This specificity was key for our goal of minimizing off-target effects, such as harming beneficial organisms. Additionally, the large library of Cry proteins, some even in iGEM’s registry, offered us a well-documented resource with proven track records in various biological applications.

To ensure that our bacteria would produce the Cry 3Aa toxin efficiently, we chose the MG26 promoter. This promoter is known for driving strong gene expression in C. acnes, which was crucial for achieving high concentrations of Cry 3Aa and ensuring its effectiveness once inside the mite. Given the size and properties of the Cry3A protein, the MG26 promoter was expected to allow for robust production, leading to sufficient toxin levels that would be lethal to mites without harming the human host.

The expected outcome of this design was that the genetically modified C. acnes would colonize temporarily the human skin as a normal part of the microbiome and release Cry 3Aa when ingested by scabies mites. Once ingested, the Cry 3Aa toxin would create crystals in the mite's gut, effectively killing the parasite. This targeted killing mechanism aligned perfectly with our project goal: creating a safe, non-chemical treatment for scabies that can be applied as a lotion - the SkinBAIT product.

Overall, our choice of C. acnes, Cry 3Aa toxin, and MG26 promoter provided a tailored solution for scabies infestations, while laying the groundwork for a broader platform that could be adapted for other parasitic infestations, including in the veterinary field. This approach also positioned our product as a novel alternative to chemical treatments, addressing the growing need for safer, more sustainable parasitic treatments.

To achieve our project goals, we used various materials and software tools to guide our design and experimental processes. Below, you will find a list of the key resources we used, as well as an overview of the basic pipeline followed in our wet lab experiments.

Testing and Learning

Before diving directly into lab work, we recognized the importance of "testing" our concept beyond the experimental stage. Instead of rushing into the lab and potentially wasting valuable materials and resources, we first sought essential feedback to refine our pipeline. This phase was closely tied to our Human Practices work, where we consulted professionals and stakeholders—such as dermatologists, investors, and experts (see more on our Human Practicespage)—to assess whether our approach was promising or needed adjustments.

From these discussions and “testing”, we gathered several critical insights:

• Investors felt the project needed a more innovative and novel aspect to make it more attractive and marketable. The new approach, specifically highlighting the non-model chassis, was attractive, but it could be pushed further.
• Dermatologists were intrigued by the platform but raised concerns about potential dysbiosis, questioning the introduction of a genetically modified C. acnes strain on human skin.
• Our internal discussions led to doubts about whether the toxin would effectively reach the scabies mites if it remained enclosed in the bacteria.

These inputs highlighted the need to adjust our pipeline before moving forward, ensuring that our engineering work was efficient and resource-conscious. The following points were critical learning outcomes that would be considered in the following improvement of the project:

A safety mechanism was needed to ensure that the modified C. acnes would not disrupt the natural skin microbiome or cause unforeseen issues when applied through our SkinBAIT product. This would address the concerns raised by dermatologists and increase the safety of our platform.

A more efficient toxin delivery system was necessary to ensure that the toxin reached the parasites effectively, overcoming our doubts about whether the bacteria could deliver the treatment as intended.

Additional constructs should be explored to give us more options for comparison, allowing us to refine and optimize the best solution for our proof of concept of this new platform.

This part of the testing and learning process was essential in improving our design and addressing key safety and efficacy concerns. By integrating these learnings, we ensured that our project was robust, innovative, and capable of providing a unique edge—appealing not only to investors but also to the general public.

Improving

In the Improving phase of our first Engineering Cycle, we implemented several key enhancements based on the insights gained during the Testing and Learning stage. The primary concern areas —investor appeal, safety (dysbiosis), and toxin delivery— were addressed by incorporating new elements into our construct. We settled on a more sophisticated design by adding a couple of crucial parts to our plasmid:

Final Construct

The plasmid now contained a gene for constitutive production of the Cry protein and an RNA thermometer (sourced from the iGEM registry) that would activate at 37°C, the temperature of human skin. Once triggered, the RNA thermometer would induce the production of an endolysin tailored to break down the walls of C. acnes. This adjustment solved several critical issues:

• Investor appeal: The addition of the RNA thermometer and endolysin gave the project a more innovative and complex edge, making it more attractive as a novel solution.
• Safety: The risk of uncontrolled bacterial colonization on the skin was greatly diminished. The bacteria would begin to break down upon reaching body temperature, thereby mitigating the potential for dysbiosis.
• Toxin release: With the bacteria lysing, the Cry toxin would be released directly onto the skin, eliminating the need for the mites to ingest the bacteria, which provided a more reliable method of targeting the parasites.

These improvements resulted in a more complete and promising construct, which addressed the majority of the challenges identified in the initial testing phase. However, this also required changes to our experimental pipeline, with the inclusion of additional parts and tests (details on this can be found in the Second Engineering Cycle - Optimization).

In addition to enhancing the construct, we also decided to expand our testing scope by incorporating:

Multiple promoters: Relying solely on the MG26 promoter wasn't sufficient. We decided to include the MG10 promoter, which is also functional in E. coli. This allowed us to perform preliminary tests in E. coli —a faster and easier organism to transform than C. acnes —helping us gather early results without consuming excessive time and resources.

Additional Cry toxins: Testing just one Cry toxin was insufficient for a comprehensive evaluation of our system. We introduced the Cry 4Ba toxin to test alongside Cry 3Aa. While Cry 4Ba is typically used against Drosophila rather than mites, it was strategically chosen for safety and feasibility in the lab. This enabled us to test the platform's effectiveness in a more controlled environment, while also assessing whether the system could have broader applications in future experiments.

Through these improvements, we advanced our project significantly by addressing safety concerns, adding flexibility for testing, and refining the overall construct. These changes allowed us to develop a more robust and adaptable platform that aligns with both our technical goals and stakeholder expectations.

Designing and Building

In the second Engineering Cycle, we undertook a significant redesign and refinement of our experimental pipeline due to the introduction of additional components and increased complexity in our construct. The WetLab work was divided into three distinct Blocks, each focusing on a specific aspect of our project.

Block 1: Functionality of the Cry Proteins and Endolysin with Different Promoters on C. acnes

This block focused on assessing the functionality of various Cry proteins and our endolysin in C. acnes, utilizing different promoters to gauge their effectiveness. The goal was to determine which constructs produced the desired proteins effectively. The constructs tested included:

Where MG10 and MG26 were two different promoters. Successful transformation of C. acnes and characterization of these constructs were critical to understanding their expression profiles and functionality.

Block 2: Functionality of the RNA Thermometer

This block aimed to evaluate the performance of the RNA thermometer in C. acnes and its responsiveness at various temperatures. To test this, we constructed and analyzed the following:

These constructs were used to characterize the thermometer’s activity and its ability to regulate gene expression in response to temperature changes, ensuring that the final construct would function correctly under physiological conditions.

Block 3: Final Construct

Based on the results from Blocks 1 and 2, the final construct was assembled by integrating the most promising components. This involved combining the RNA thermometer, a promoter responsive to the thermometer, an endolysin gene, and a constitutively expressed Cry protein. The final design aimed to produce the endolysin when the temperature threshold was reached and to express the Cry protein continuously. This construct would be tested with both Cry proteins to evaluate its overall effectiveness.

To support these design and experimental processes, we used various materials and software tools. For detailed information on these resources and the basic pipeline followed in our wet lab work, please refer to the images provided below.

Testing and Learning

In the second engineering cycle, we began by focusing on the initial stages of Block 1 and Block 2, aiming to streamline the transformation process and ensure that the necessary parts were assembled correctly before moving to more complex experiments. This phase primarily involved the preparation of part stocks and the successful transformation of E. coli strains NZY 5α and EC24.

The assembly of all constructs in Block 1 and Block 2 was largely successful on the first attempt, except for those containing Cry 4Ba. We quickly identified the issue: the larger size of the Cry 4Ba construct required adjustments in our Gibson Assembly and PCR protocols. Running an agarose gel to verify the constructs' size and subsequent sequencing confirmed that the correct parts were assembled after implementing a modified protocol. This underscored the need for precise optimization in the assembly of larger parts, which we successfully achieved.

In the transformation step, we encountered a challenge with larger plasmids in E. coli NZY 5α. Smaller plasmids integrated well, as confirmed by colony PCR and sequencing, but larger constructs required adjustments to our thermal shock protocol. We extended the cooling phase while maintaining the same heat shock conditions, followed by a slightly longer post-shock incubation. These modifications worked for all constructs except MG10 + Cry 4Ba, suggesting that Cry 4Ba posed too high a metabolic burden on E. coli, leading to toxicity. Consequently, this construct had to be dropped from Block 1.

Figure 1.

Figure 1. Transformed E.coli colonies in petri dishes.

Further testing showed that constructs containing MG10 (which drives expression in both C. acnes and E. coli) affected colony growth, with MG10+Cry3 and MG10+Endolysin growing slower than MG26 constructs. This indicated that the expression of our proteins placed a metabolic burden on the cells. To address this, we extended the growth period for these constructs, ensuring sufficient colony development for further testing.

Additionally, we observed that the expression of endolysin in E. coli did not kill the cells, aligning with our expectations that endolysin would specifically target C. acnes. However, this emphasized the importance of performing a viability test on C. acnes in the later stages of Block 1 to confirm endolysin's targeted effect.

For Block 2, we successfully tested the RNA thermometer in E. coli by measuring the fluorescence of GFP and RFP constructs at different temperatures. These tests confirmed that the thermometer was functional and responsive, and we determined that testing at three specific temperatures would suffice in future experiments with C. acnes.

These early stages of testing were essential for refining our experimental protocol and addressing potential issues before progressing to more complex steps. By engineering each process to work efficiently at the outset, we set ourselves up for smoother execution in the later stages of both Block 1 and Block 2.

Improving

Based on the insights gained from the first stages of Block 1 and Block 2, we implemented several key improvements to enhance the performance of our experimental processes.

The most significant challenge was the difficulty with Cry 4Ba constructs, which led us to modify our assembly and transformation protocols for larger parts. By adjusting the timing of cooling and incubation steps, we ensured more reliable integration of plasmids into E. coli, though ultimately, the MG10 + Cry4Ba construct had to be dropped from Block 1 due to its toxicity. This decision allowed us to focus resources on more viable constructs, optimizing the performance of the remaining parts in the pipeline.

We also improved the growth conditions for E. coli transformed with MG10 constructs. Recognizing that protein expression imposed a metabolic strain on the cells, we extended their growth time in petri dishes to ensure larger and more stable colonies. This adjustment improved the overall reliability of our results and ensured that we could proceed with testing these constructs.

Moreover, the successful functionality test of the RNA thermometer in E. coli allowed us to fine-tune our protocol for Block 2. We confirmed that testing at three temperatures would be sufficient, streamlining future experiments with C. acnes and avoiding unnecessary resource expenditure.

These improvements not only ensured the success of our initial testing but also laid the groundwork for Block 3, where the final construct would be assembled. By addressing issues early in Blocks 1 and 2, we ensured that the final assembly and testing would be based on the most efficient constructs and protocols, allowing us to achieve optimal performance in the final stage of our second engineering cycle. This structured approach provided a strong foundation for further optimization in the next iteration.

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Designing and Building

In the final design cycle, we built upon the improvements and optimizations made in the second iteration, refining our experimental protocols. The design process was largely consistent with the previous cycle, with few key updates and optimizations to ensure a streamlined workflow and successful outcomes.

The main update in this cycle was the decision to drop the MG10 + Cry 4Ba construct from Block 1. After multiple testing attempts, it became clear that the Cry 4Ba protein caused excessive metabolic strain on E. coli, rendering the construct non-viable. With this part removed, our focus shifted to maximizing the performance of the remaining constructs and ensuring the successful assembly and transformation of C. acnes.

As in the second cycle, the design was divided into three separate blocks:

With our optimized protocols and streamlined design process, this final design cycle aimed to bring together all the previous insights and learnings into a robust and functional construct. The key modifications allowed us to refine our work for maximum effectiveness and move closer to achieving our goals with the SkinBAIT platform.

For detailed information on these resources and the basic pipeline followed in our wet lab work, please refer to the images provided below.

Testing and Learning

In this final cycle, we concentrated on the later stages of both Block 1 and Block 2, which involved transforming C. acnes and overcoming the challenges we encountered with contamination, transformation efficiency, and expression analysis. Throughout this phase, we iterated several steps of the process, constantly optimizing protocols based on the insights gained from our earlier experiments.

Our first major challenge was achieving successful C. acnes transformation. Initially, our attempts failed due to improper competence of the cells. After reviewing our methods, we realized that we needed to ensure that some reagents, like the sucrose buffer, were strictly ice-cold, and we optimized our handling of the cells by improving freezing methods and timings with liquid nitrogen. This adjustment improved transformation efficiency but introduced a new challenge: contamination.

We consistently faced contamination issues, particularly with Staphylococcus species (another species of bacteria that resides in our skin). This was especially problematic during the transformation process since C. acnes takes five days to form visible colonies, leaving plates vulnerable to contamination. Initially, contamination wasn't immediately apparent since C. acnes and Staphylococcus show the same result in colony PCR, and we only identified the contamination after sequencing.

To address this, we implemented stricter sterilization protocols: increasing UV light exposure in the hood, thoroughly cleaning all materials, and adopting designated lab coats for work with C. acnes (which was periodically washed by professional services of the lab). By the third round of iterations, we finally achieved correct transformation of two constructs: MG10 + Endolysin and MG26 + Endolysin.

To further streamline the transformation process, we developed a quick visual guide for distinguishing Staphylococcus from C. acnes colonies by size, color, and texture (you can check it out in our Contributions page). This allowed us to perform early colony screening after some hands-on training, minimizing the need to wait for sequencing to identify contamination. The combination of all the measures described above made possible the successful transformation of all C. acnes constructs from both Block 1 and Block 2.

Additionally, we optimized our stock storage method for C. acnes. Instead of continuously replating colonies, which was time-consuming, more prone to contamination, and inefficient (it takes between 3 to 5 days to be of use), we developed a new method: using half of one plate for glycerol stocks and the other half to continue generating fresh plates. This ensured faster and more reliable colony growth for future experiments.

Another key area of improvement was in the testing of Cry protein expression (Block 1). We conducted a western blot to check for both intracellular and extracellular expression of the Cry protein, but found that only intracellular expression was detectable. Given that the liquid cultures of C. acnes didn’t appear turbulent enough, we concluded that the standard 3-day growth period was insufficient, prompting us to extend the culture time for better results.

The insights gathered during our Testing and Learning phase were then applied to improve our WetLab design and obtain optimal results and efficiency.

Improving

As we iterated through the testing phases and improved our methodology and protocols, we also identified crucial improvements to fine-tune our wet lab pipeline and ensure optimal results. One major realization was the need for a functionality assay to test the effectiveness of our Cry proteins. After addressing transformation challenges and successfully expressing the proteins, we still needed a clear method to determine whether they were effective in eliminating mites, our target organisms.

To resolve this, we designed a new experiment to test our constructs in Drosophila larvae. By analyzing the effect of both the transformed bacteria and the lysate of our product on Drosophila, we could assess and compare the effectiveness of the Cry3 and Cry4 proteins and their performance when paired with different promoters. This functionality test would also allow us to evaluate specificity and offer deeper insights into whether our platform could target other organisms beyond mites.

These testing and learning processes, combined with the improvements we implemented, brought us closer to a fully optimized final design and a robust wet lab pipeline for the C. acnes transformation, Cry protein expression, and efficacy testing of all constructs.

Below you can find our Final Design of the WetLab pipeline:

If you want to know more about other aspects of SkinBAIT related to the WetLab Engineering work, you can check out these pages: WETLAB DESIGN, WETLAB PROTOCOLS, WETLAB RESULTS and HUMAN PRACTICES.