iGEM 2024 — Team upf-barcelona


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### Engineering

* [Overview](#1)
* [Wetlab objectives](#2)
* [Initial Design](#3)
  + [Design and Build](#3.1)
  + [Test and Learn](#3.2)
  + [Improve](#3.3)
* [Optimization](#4)
  + [Design and Build](#4.1)
  + [Test and Learn](#4.2)
  + [Improve](#4.3)
* [Final Design](#5)
  + [Design and Build](#5.1)
  + [Test and Learn](#5.2)
  + [Improve](#5.3)

# Engineering

## iGEM is Design, Build, Test and Learn

### This section explains how we iterated through the Engineering Cycle.

## Project overview

Our project aims to create an innovative synthetic biology treatment for parasite infestations. We focus on genetically modifying bacteria to produce toxins that are lethal to parasites but safe for humans and animals. As a proof of concept, we are targeting scabies, a condition caused by the *Sarcoptes scabiei mite*, which has seen a resurgence globally and in local communities, including in Barcelona. Current treatments often have side effects, are unsuitable for certain vulnerable populations, and are not designed for preventive use.
By developing a novel treatment that specifically targets scabies mites, we address these shortcomings while also establishing a versatile platform that could be adapted to combat a wide range of other parasitic infections. This project not only offers an alternative for scabies management but also has the potential to transform how parasitic diseases are treated in the future.

## WetLab Objectives

The WetLab portion of our project aims to achieve a proof of concept for our innovative treatment approach. Our primary objectives include the successful genetic modification of *Escherichia coli* and later *Cutibacterium acnes* to express the Cry 3Aa and Cry 4Ba toxins, and the validation of these toxins' effectiveness in killing scabies mites. This involves the introduction of these genes into the bacteria, ensuring proper expression and functionality. Ultimately, the WetLab's role is to provide the foundational data and experimental results that will inform the development of a scabies treatment lotion, paving the way for future clinical applications.

To refine our solution, we executed three engineering cycles: Initial Design (1), Optimization (2), and Final Design (3), using the Design+Build - Test+Learn - Improve methodology.

![Blue disk representing cycle 1 rotating](https://static.igem.wiki/teams/5311/assets/engineering/cicle-1.png )

1

![Orange disk representing cycle 2 rotating](https://static.igem.wiki/teams/5311/assets/engineering/cicle-2.png)

2

![Gray disk representing cycle 3 rotating](https://static.igem.wiki/teams/5311/assets/engineering/cicle-3.png)

3

In each cycle, we first designed and planned the approach, then built and assembled prototypes. We tested these prototypes to evaluate their feasibility or performance, learned from the results to identify areas for improvement, and finally enhanced the design based on our findings. This iterative process enabled us to systematically advance our project and achieve an effective solution.

## Initial design

In the Initial Design phase, we laid the groundwork for our project by carefully choosing each component of the system. We selected ***Cutibacterium acnes*** as our chassis due to its **natural presence on human skin**, particularly in areas prone to scabies infestations. Our goal was to have ***C. acnes* produce Cry 3Aa toxin (Part:BBa\_K5311002)**, which would target the scabies mites without harming the human host.

* **Design:** We began by designing a biological system with *C. acnes* as the chassis, Cry 3Aa as the toxin, and the MG26 promoter to drive high toxin expression. **Our research into Cry proteins led to the selection of Cry 3Aa (Part: BBa\_K5311011) for its specificity in targeting mites.**
* **Build:** We assembled an in vitro model of the system using **Benchling software.**
* **Test:** Before lab testing, we **gathered feedback through our Human Practices work.** Dermatologists raised concerns about the potential for dysbiosis, and we questioned the efficiency of toxin delivery, sparking an internal discussion about whether the toxin could effectively reach the mites.
* **Learn:** From this initial testing phase, we learned that **additional safety measures** were needed to prevent dysbiosis, and we questioned the effectiveness of bacterial delivery of the toxin. This led us to **make significant adjustments** in the next phase of the engineering cycle.

This phase demonstrates how we followed the full engineering design cycle by **documenting the design, building the prototype, testing it through stakeholder feedback, and learning from those results** to inform our next steps.

### Designing and Building

![Blue disk representing cycle 1 rotating](https://static.igem.wiki/teams/5311/assets/engineering/cicle-1.png)

1

![Three students chatting and having ideas for the project](https://static.igem.wiki/teams/5311/assets/engineering/group-thinking.png)

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.

![A computer with the repository of different proteins open with CryA marked](https://static.igem.wiki/teams/5311/assets/engineering/17-fixed.png)

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](https://parts.igem.org/Main_Page), 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.

![](https://static.igem.wiki/teams/5311/assets/engineering/idea-skinbait.png)

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.

![](https://static.igem.wiki/teams/5311/assets/engineering/gros-1.png)

### 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 Practices](https://2024.igem.wiki/upf-barcelona/human-practices)page)—to assess whether our approach was promising or needed adjustments.

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

![](https://static.igem.wiki/teams/5311/assets/engineering/critical-insights-gray.png)

* [Investors](https://2024.igem.wiki/upf-barcelona/human-practices) 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:

![Left side image](https://static.igem.wiki/teams/5311/assets/engineering/1.png)

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.

![Right side image](https://static.igem.wiki/teams/5311/assets/engineering/dermatologist-blue.png)

![Left side image](https://static.igem.wiki/teams/5311/assets/engineering/box.png)

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.

![Right side image](https://static.igem.wiki/teams/5311/assets/engineering/2.png)

![Left side image](https://static.igem.wiki/teams/5311/assets/engineering/3.png)

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.

![Right side image](https://static.igem.wiki/teams/5311/assets/engineering/crane.png)

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](https://parts.igem.org/Main_Page)) 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:

![](https://static.igem.wiki/teams/5311/assets/engineering/critical-insights-blue.png)

* [Investor](https://2024.igem.wiki/upf-barcelona/human-practices) 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.

![Right side image](https://static.igem.wiki/teams/5311/assets/engineering/promoters.png)

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.

![Right side image](https://static.igem.wiki/teams/5311/assets/engineering/20-fixed-now.png)

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.

## Optimization

In the Optimization phase, we responded to the insights gained during the initial cycle by **refining our system to improve safety, performance, and toxin delivery.** The key change was the **addition of an RNA thermometer (Part:BBa\_K115002) and endolysin (Part:BBa\_K5311006)** to control toxin release and mitigate safety concerns, along with exploring a broader range of Cry toxins.

- **Design:** Based on the feedback from the initial phase, we created new parts incorporating an **RNA thermometer (Part:BBa\_K5311016, Part:BBa\_K5311017, Part:BBa\_K5311018 and Part:BBa\_K5311019) and endolysin (Part:BBa\_K5311014 and Part:BBa\_K5311015)** to ensure that the *C. acnes* would lyse at body temperature, **releasing the Cry toxin** directly onto the skin. This addressed the potential issue of dysbiosis by ensuring the bacteria wouldn’t colonize the skin indefinitely. Additionally, we expanded the scope by **adding Cry 4Ba (Part: BBa\_K5311003) toxin as an alternative to Cry 3Aa** and tested both **MG26 (Part:BBa\_K5311000) and MG10 (Part:BBa\_J23119) promoters**.
- **Build:** We built new constructs that incorporated the RNA thermometer and endolysin, as well as constructs with Cry 4Ba and multiple promoters (MG10 and MG26) to compare their effectiveness.
- **Test:** Through lab testing, we faced **challenges related to the size of the Cry 4Ba construct**, which affected the **efficiency of the assembly** process and led to the need for modified protocols. Testing also revealed **metabolic strain in *E. coli*** with the MG10 promoter, requiring adjustments in growth conditions.
- **Learn:** From these tests, we learned that the construct **MG10+Cry 4Ba** did not showcase efficient transformation in *E. coli*, as it placed too much metabolic burden on the cells. This led us to **drop MG10+Cry 4Ba** from the pipeline, streamline our growth protocols, and focus on **optimizing the performance of Cry 3Aa, RNA, and Endolysin constructs with improved assembly and transformation protocols.**

This cycle demonstrates our **engineering success by iterating on the design, incorporating new components like the RNA thermometer, and improving our assembly process** based on experimental feedback.

![Image 1](https://static.igem.wiki/teams/5311/assets/engineering/cicle-2.png)

2

### 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:

![](https://static.igem.wiki/teams/5311/assets/engineering/block1-constructes.png)

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:

![Right side image](https://static.igem.wiki/teams/5311/assets/engineering/block-2.png)

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

![](https://static.igem.wiki/teams/5311/assets/engineering/block-3.png)

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.

![](https://static.igem.wiki/teams/5311/assets/engineering/gros-2.png)

![](https://static.igem.wiki/teams/5311/assets/engineering/gros-3.png)

![](https://static.igem.wiki/teams/5311/assets/engineering/gros-4.png)

### 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.

![](https://static.igem.wiki/teams/5311/assets/engineering/21-fixed-now.png)

![](https://static.igem.wiki/teams/5311/assets/engineering/adjusted-protocol.png)

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.

![](https://static.igem.wiki/teams/5311/assets/engineering/drop-cry4mg10.png)

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.

![](https://static.igem.wiki/teams/5311/assets/engineering/cry3-plates.jpeg)
![](https://static.igem.wiki/teams/5311/assets/engineering/endolisina-plates.jpeg)

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.

[![](https://static.igem.wiki/teams/5311/assets/engineering/results-termometer.png)](https://2024.igem.wiki/upf-barcelona/results)

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.

![](https://static.igem.wiki/teams/5311/assets/engineering/summary-improvements.png)

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.

![](https://static.igem.wiki/teams/5311/assets/engineering/time-up.png)

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.

![Image 1](https://static.igem.wiki/teams/5311/assets/engineering/cicle-3.png)

3

## Final Design

In the Final Design phase, we implemented the final round of improvements based on the optimization cycle and focused on **overcoming practical challenges** related to transformation efficiency and contamination. This phase culminated in the creation of a robust and functional system, ready for final testing and application.

* **Design:** We focused on **MG10+Cry 3Aa (Part:BBa\_K5311010), MG26+Cry 3Aa (Part:BBa\_K5311011), and MG26+Cry 4Ba (Part:BBa\_K5311013)** constructs during the later parts of our experimental pipeline. We further optimized the **RNA thermometer and endolysin systems**, ensuring efficient toxin release.
* **Build:** We **built these final constructs** using the insights gained from previous iterations. This included modifying the transformation protocol for *C. acnes*, improving freezing and sterilization steps, and **adjusting handling procedures to enhance transformation efficiency.**
* **Test:** During this phase, we encountered contamination issues, particularly from Staphylococcus species, which affected the transformation process. We also found that **Cry 3Aa and Endolysin were expressed intracellularly** but required extended growth times to achieve more detectable levels.
* **Learn:** We learned that contamination could be controlled with stricter sterilization protocols, and we **optimized our transformation** method by adjusting the freezing process and **improving colony screening techniques.** These improvements resulted in the successful transformation of *C. acnes* with the endolysin constructs. To test functionality, we also designed an **assay using Drosophila larvae as a model organism to assess the effectiveness of Cry proteins.**

This final iteration highlights our **engineering success by addressing issues related to transformation, optimizing protocols, and demonstrating the functionality of the system.** Each improvement was a direct result of the learning from previous cycles, showcasing a systematic approach to engineering design.

### 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:

* Block 1: Functional Testing of Cry Proteins and Endolysin.
* Block 2: RNA Thermometer Functionality.
* Block 3: Final Construct Assembly.

![](https://static.igem.wiki/teams/5311/assets/engineering/scheme-blocks.png)

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.

![](https://static.igem.wiki/teams/5311/assets/engineering/gros-5.png)

![](https://static.igem.wiki/teams/5311/assets/engineering/gros-6.png)

![](https://static.igem.wiki/teams/5311/assets/engineering/gros-7.png)

### 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.

![](https://static.igem.wiki/teams/5311/assets/engineering/28-fixed-now.png)

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.

![](https://static.igem.wiki/teams/5311/assets/engineering/contamination.png)

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.

![](https://static.igem.wiki/teams/5311/assets/engineering/preventive-mesures.png)

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.

![](https://static.igem.wiki/teams/5311/assets/engineering/petry-dishes-ok.png)

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](https://2024.igem.wiki/upf-barcelona/contributions)). 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.

![](https://static.igem.wiki/teams/5311/assets/engineering/c-acnes.png)

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.

![](https://static.igem.wiki/teams/5311/assets/engineering/liquid-culture.png)

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.

![](https://static.igem.wiki/teams/5311/assets/engineering/fly-experiments.png)

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:

![](https://static.igem.wiki/teams/5311/assets/engineering/gros-8.png)

![](https://static.igem.wiki/teams/5311/assets/engineering/gros-9.png)

![](https://static.igem.wiki/teams/5311/assets/engineering/gros-10.png)

If you want to know more about other aspects of [SkinBAIT](https://2024.igem.wiki/upf-barcelona/) related to the WetLab Engineering work, you can check out these pages: [WETLAB DESIGN](https://2024.igem.wiki/upf-barcelona/design), [WETLAB PROTOCOLS](https://2024.igem.wiki/upf-barcelona/experiments), [WETLAB RESULTS](https://2024.igem.wiki/upf-barcelona/results) and [HUMAN PRACTICES](https://2024.igem.wiki/upf-barcelona/human-practices).

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# Experiments

## iGEM is Following a Pipeline

### This section explains the different laboratory protocols followed by the wetlab team.

On our Experiments page, you’ll find all the protocols used throughout our project. Each protocol outlines the step-by-step processes we followed to achieve our results. The accompanying image illustrates the sequence of steps taken during our experiments.

![](https://static.igem.wiki/teams/5311/assets/experiments/first-scheme.png)

Figure 1. C. acnes competent cells protocol. Scheme made with BioRender.

Plasmid digestion

For the plasmid digestion protocol add in an Eppendorf tube:

* 1 µg of DNA
* 5 µL of 10X rCutSmart Buffer
* 1 µL of SacI-HF
* Nuclease-free Water up to 50 µL

It must then be incubated at 37°C for 5–15 minutes. After this, the plasmid must go through a clean up by following the PCR purification protocol to obtain the digested plasmid.

Gibson assembly

The Gibson Assembly method is essential for creating our plasmid constructs. Whether it is for storage or the definitive backbone (pJet or pEnd, respectively), the exact same protocol was followed. To proceed with the Gibson Assembly, introduce the following components in a 0.2 ml Eppendorf tube, in the order listed:

* 50 ng of template (empty backbones).
* 3X molar ratio of the part being introduced, with respect to the template used in each case.
* The same amount of Gibson Mix as the sum of the last two.

The 0.2 ml Eppendorf tube will be then placed into the thermocycler for 1 hour, at 50 ºC.

The Gibson mix contains three key components: T5 exonuclease, DNA polymerase, and DNA ligase. T5 exonuclease creates 3′ overhangs by digesting 5′ ends of the DNA, DNA polymerase fills in any gaps between annealed fragments, and DNA ligase seals the nicks to form a continuous DNA molecule. These enzymes work together in an optimized buffer that maintains the proper conditions for the reaction. For its correct functioning, it is very important to work at low temperatures when manipulating this mix.

Note: It is important to note that this protocol is highly specific to the lab we worked in, as it relies on the prefix and suffix sequences flanking all of our parts, as well as the exact cleavage sites in the backbones.

DNA PCR / Colony PCR

To set up a DNA PCR or Colony PCR, use the following components in a 0.2 ml Eppendorf tube, in the order listed:

* 19 µL of H₂O if DNA PCR; 21 µL of H₂O if Colony PCR.
* 25 µL of 2x Phanta Master Mix.
* 2 µL of Primer 1.
* 2 µL of Primer 2.
* Template DNA (2 µL in case of DNA PCR).

Note: The primers used depend on the specific DNA fragment you want to amplify.

![](https://static.igem.wiki/teams/5311/assets/experiments/pcr.jpg)

Figure 2. Thermocycler machine used.

To perform the PCR, we must put the Eppendorf PCR tubes in the thermocycler. The specific settings depend on what you are performing the PCR on but here is a table with the most common settings:

| Step | Temperature | Time | Number of cycles |
| --- | --- | --- | --- |
| Initial Denaturation | 94 °C | 2 min | --- |
| Denaturation | 94 °C | 15 sec | 30 - 35 |
| Primer Annealing | 65 °C | 20 sec | 30 - 35 |
| Extension | 72 °C | 1 min/kb | 30 - 35 |
| Final Extension | 72 °C | 5 min | --- |
| Resting temperature | 4 °C | --- | --- |

After performing the PCR, you can analyze the results following the agarose gel protocol.

Agarose Gel

To prepare agarose gels, we used a tray that allowed the creation of four, twenty-well gels simultaneously. The measurements for one gel are as follows:

* 150 mL of TBE buffer.
* 1.5 g of agarose.
* 4.5 µL of Green Safe dye.

1. First, weigh out 1.5 g of agarose to make a 1% agarose gel. Add the agarose to an Erlenmeyer flask.
2. Add 150 mL of TBE buffer to the flask and gently mix the contents.
3. Heat the mixture in a microwave for 45-60 seconds, mixing it until the agarose is fully dissolved.
4. Allow the solution to cool slightly, then add the Green Safe dye and mix thoroughly.
5. Pour the solution into the gel tray, ensuring the well combs are in place.
6. Allow the gel to solidify for about 20 minutes.

![](https://static.igem.wiki/teams/5311/assets/experiments/agarose-gel.jpg)

Figure 3. Agarose Gel machinery used.

PCR purification

PCR purification is typically done after PCR amplification to prepare the product for sequencing. Follow these steps:

1. Load 25 µL of the PCR mixture into a PCR purification column.
2. Add 125 µL of PB buffer and centrifuge at 13,000 rpm for 1 minute.
3. Discard the flow-through.
4. Add 750 µL of PE buffer and centrifuge again at 13,000 rpm for 1 minute.
5. Discard the flow-through.
6. Centrifuge at 13,000 rpm for 2 minutes to remove any extra buffer.
7. Transfer the PCR purification column into a new Eppendorf tube.
8. Add 20 µL of water, wait for 1 minute, then centrifuge at 13,000 rpm for 1 minute.

The purified PCR product will be in the Eppendorf tube.

Sequencing

For the sequencing:

* In case of sequencing a PCR product, 50-250 ng. If it is a MiniPrep, 150-500 ng.
* 2.5 µL of desired primer.
* 2.5 µL of H₂O.

![](https://static.igem.wiki/teams/5311/assets/experiments/sequencing.jpg)

Figure 4. Sequencing samples.

Transformation of *E. coli*

For *E.coli* transformation:

1. Gently mix the thawed competent cells by tapping the tube. Do not vortex.
2. Add 1-5 µL of plasmid DNA (10-100 ng) to the competent cells.
3. Gently flick the tube to mix the DNA with the cells. Do not vortex.
4. Incubate the cells with the DNA on ice for 20-30 minutes. This allows the DNA to associate with the cell membrane.
5. Transfer the tubes to a 45°C Thermoblock for 45-60 seconds.
6. Quickly return the tubes to ice and incubate for another 2-5 minutes.
7. Add 250-500 µL of LB or SOC medium to the cells.
8. Incubate the cells at 37°C for an hour with shaking (~200 rpm) to allow the bacteria to recover and express the antibiotic resistance gene.
9. After the recovery period, plate 50-200 µL of the transformation mix onto LB agar plates with the appropriate antibiotic.
10. Spread the cells with an L-loop.
11. Incubate the plates overnight at 37°C.

The day after, there should be colonies on the plates.

![](https://static.igem.wiki/teams/5311/assets/experiments/tranformationecoli.jpg)

Figure 5. E. coli transformation plates.

Liquid cultures for *E. coli*

To grow liquid cultures:

1. Add 5 mL of LB to a culture tube.
2. Add 5 µL of the appropriate antibiotic, based on the resistance gene present in the plasmid.
3. Using a pipette tip, pick a single bacterial colony from a plate and inoculate the LB.

Incubate the culture under the appropriate conditions, shaking at 37ºC, during 24 hours.

Plasmid DNA purification from *Escherichia coli* cells (MiniPrep)

To purify plasmid DNA using the MiniPrep method, you must start with a grown liquid culture.

1. Pellet the bacterial cells by centrifuging the liquid culture.
2. Resuspend the cell pellet in 250 µL of Buffer A1.
3. Add 250 µL of Buffer A2, then gently mix by inverting the tube 6-8 times. Incubate at room temperature for no more than 4 minutes (do not vortex).
4. Add 300 µL of Buffer A3 and gently invert the tube 6-8 times to mix (do not vortex).
5. Centrifuge the mixture for 5-10 minutes at room temperature.
6. Place a spin column in a 2 mL collection tube and load the supernatant from the previous step into the column.
7. Centrifuge for 1 minute at 11,000 xg and discard the flow-through.
8. Add 500 µL of Buffer AY, centrifuge for 1 minute, and discard the flow-through.
9. Add 600 µL of Buffer A4, centrifuge for 1 minute, and discard the flow-through.
10. Centrifuge again for 2 minutes to ensure all liquid is removed.
11. Transfer the column to a new 1.5 mL Eppendorf tube, add 50 µL of Buffer AE, and incubate for 1 minute.
12. Centrifuge for 1 minute to elute the plasmid DNA.

Note: The exact protocol may vary depending on the specific MiniPrep kit. This protocol is based on the NZYTech MiniPrep kit.

![](https://static.igem.wiki/teams/5311/assets/experiments/miniprep.jpg)

Figure 6. Centrifuge needed for the plasmid DNA purification.

Preparing competent *C. acnes* cells

First, you must grow a liquid culture of the desired C. acnes strain in liquid medium.

1. Spin 50 mL of liquid culture at 4200 rpm for 10 minutes at 4ºC.
2. Decant the supernatant carefully to remove it.
3. Resuspend the cell pellet in 2 x 40 mL of ice-cold sucrose buffer.
4. Centrifuge again at 4200 rpm for 10 minutes at 4ºC.
5. Decant the supernatant.
6. Resuspend the pellet in 1 mL of ice-cold sucrose buffer.
7. Spin at 4200 rpm for 10 minutes at 4ºC.
8. Decant the supernatant.
9. Resuspend the pellet in 1 mL of ice-cold sucrose buffer and centrifuge at 10,000 rpm for 1 minute at 4ºC. Repeat this resuspension and centrifugation step three more times.
10. After the final spin, resuspend the pellet in 100 µL of ice-cold sucrose buffer per 50 µL of competent cell suspension.

If you need to store the competent cells for future use you can split the competent cells made into smaller volumes. Freeze these immediately using liquid nitrogen and then store them at -80ºC.

Transformation of *C. acnes*

Transforming C. acnes requires two days and the use of pre-prepared competent cells. Always work in a laminar flow hood to prevent contamination.

1. In an Eppendorf tube, add the following in order:

* 30 µL of sucrose buffer
* 500-1000 ng of plasmid DNA
* 10 µL of C. acnes competent cells

2. Transfer the contents to a 1 mm cuvette and electroporate (1.5 kV, 25 µF, 400 ohm).
3. Quickly add 100 µL of warm BHI medium and transfer the mixture back to an Eppendorf tube.
4. Plate the mixture onto non-selective Brucella plates.
5. Incubate the plates anaerobically at 37ºC for 24 hours.

The next day:

1. Prepare Eppendorf tubes with 1 mL of BHI.
2. Using a sterile swab, collect the entire contents from the plate and transfer them into the BHI.
3. Centrifuge at 4,200 rpm for 5 minutes and discard the supernatant.
4. Resuspend the pellet in 200 µL of BHI.
5. Plate 100 µL on a Brucella + Erythromycin plate.
6. Incubate the plates anaerobically at 37ºC for 6 days.

![](https://static.igem.wiki/teams/5311/assets/experiments/cacnes-transformation.jpg)

Figure 7. Our wet lab team doing the C. acnes transformation.

Liquid cultures for *C. acnes*

To grow liquid cultures for *C. acnes:*

1. Resuspend the grown C. acnes plate into 10 ml of Brain Heart Infusion medium.
2. Do a 1/10 dilution of the resuspension in more BHI medium, and then measure the OD 600. Values will go from 0.1 to 0.2, which means the original resuspension is around 1 to 2.
3. The liquid culture will need to be at 0.1 OD 600 for correct growth. In a culture flask, add the right amount of the original resuspension and fresh BHI to achieve the desired volume at 0.1 OD 600.

The culture flask is going to be grown under anaerobic conditions, at 37ºC and shaking.

Glycerol stocks

In an Eppendorf tube add:

* 800 µL of grown culture.
* 400 µL of glycerol at 50%

Freeze at -80ºC until needed for use.

![](https://static.igem.wiki/teams/5311/assets/experiments/glycerolstocks.jpg)

Figure 8. Freezer where we kept our glycerol stocks.

Western blot

Preparation of cell cultures

To perform a Western blot, you must first grow 30 mL of liquid culture of the cells you want to analyze. In our case, we worked with two different bacteria: *E. coli* and *C. acnes*.

* *E. coli* liquid cultures need to be grown for 24 hours.
* *C. acnes* liquid cultures must be grown for 3 days.

You also need to determine whether you're interested in analyzing protein expression in the bacterial pellet or the supernatant:

* For *E. coli*, we focused only on the pellet.
* For *C. acnes*, both the supernatant and pellet were of interest.

Protein Precipitation

Supernatant:

1. Add 100 µL of 100% TCA (Trichloroacetic acid) per mL of supernatant.
2. Precipitate the protein on ice for 2 hours.
3. Centrifuge at 10,000 xg for 20 minutes at 4°C.
4. Carefully aspirate the supernatant.
5. Wash the pellet with ice-cold 100% acetone, then centrifuge at 10,000 xg for 5 minutes at 4°C.
6. Repeat the washing step twice.
7. Air-dry the pellet and resuspend it in 100 µL of PBS.

Pellet:

1. Add 2 mL of PBS, resuspend the pellet, and centrifuge at 10,000 xg for 5 minutes at 4°C. Repeat this 2 more times.
2. Resuspend the pellet in 1 mL of PBS.
3. Transfer the resuspended pellet to a Precellys tube (for mechanical lysis using a homogenizer).
4. Centrifuge the Precellys tubes at 10,000 xg for 10 minutes at 4°C.
5. Wash the pellet with ice-cold 100% acetone, then centrifuge at 10,000 xg for 5 minutes at 4°C.
6. Repeat the washing step twice.
7. Transfer as much supernatant as possible to a clean Eppendorf tube.

![Western Blot centrifuge used.](https://static.igem.wiki/teams/5311/assets/experiments/westernblot1.jpg)

Figure 9. Homogenizer for mechanical lysis being set up.

Western Blot Procedure

Protein quantification:

* The BCA Protein Assay is used to quantify the total protein concentration in the sample. The kit consists of Reagent A and Reagent B, which are mixed in a 50:1 ratio. This is our working solution.
* A standard curve of known protein concentrations is needed to calculate the concentration (ng) of our samples. BSA protein is prepared from 10 ng/uL to 0 ng/uL by dividing the concentration in half each time.
* Measure the absorbance using a multiwell plate reader with a measuring wavelength of 562 nm, loading 10 µL of each sample along with 190 µL of working solution.
* Use the standard curve equation to determine the total protein concentration of our samples.

SDS-Page

Sample preparation:

1. Prepare the sample mix: 15 µL of sample + 5 µL of sample buffer (Final volume = 20 µL per well). It is important to add a positive control to our gel.
2. Sample buffer preparation: LDS sample buffer (4x, 190 µL) + β-mercaptoethanol (10 µL).
3. Heat the samples at 95°C for 5 minutes using a Thermoblock to denature the proteins.

Running buffer:

1. Prepare the running buffer: 15 mL of MES buffer + 285 mL of distilled water.
2. Pour the running buffer into the Western blot chamber.

Loading the gel:

1. Load 10 µL of protein ladder (SeeBlue) into the first well and 20 µL of each sample into subsequent wells.
2. Run the gel at 120 V for 1 hour and 20 minutes.

![Loading the gel.](https://static.igem.wiki/teams/5311/assets/experiments/westernblot2.jpg)

Figure 10. Loading the gel.

Wet Transfer

Transfer Buffer Preparation:

Mix the following:

* 50 mL of 20X transfer buffer.
* 850 mL of deionized water.
* 100 mL of methanol (MeOH).

Prepare for the wet transfer:

1. Cut 4 filter papers and 1 PVDF membrane to the size of the gel.
2. Soak the sponges in the transfer buffer.
3. Activate the membrane by soaking it in methanol for 30 seconds, rinse with water, and then place it in transfer buffer.
4. Assemble the "sandwich" in the MiniBlot module, from negative to positive: sponge, filter paper, gel, membrane, filter paper, sponge.
5. Place the "sandwich" into the MiniBlot transfer tank, add transfer buffer, and run at 20 V for 1 hour.

Extract the membrane, never touching it with our hands, and place it in a small container, in this container:

1. Prepare the blocking solution by mixing 2 g of milk powder with 40 mL of TBS-T.
2. Block the membrane with 4% non-fat milk in TBS-T (TBS + 0.1% Tween 20) for 1 hour.
3. Add the primary antibody (mouse anti-histidine tag) at a 1:800 dilution in 4% non-fat milk.
4. Incubate overnight at 4°C.
5. Wash the membrane 3 times with TBS-T for 10 minutes each.
6. Add the secondary antibody at a 1:1000 dilution in 4% non-fat milk and incubate for 1 hour at room temperature.
7. Wash the membrane 3 times with TBS-T for 10 minutes each.

Chemiluminescence Detection:

1. Mix the luminol/enhancer solution and peroxide solution in a 1:1 ratio.
2. Apply the solution above to the membrane and use a Biorad Imaging System to visualize the WB membrane with the corresponding bands.

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←

Open Sidebar

### Results

* [Introduction](#1)
* [*E. coli*](#2)
* [*C. acnes*](#3)
* [Conclusions](#4)
* [Future work](#5)
* [Annex](#6)

# Results

## iGEM is extracting Results and Reaching to Conclusions.

### This section provides an overview of the diverse results and achievements our team has made.

## Introduction

On this page, you will find all the results and accomplishments our team has achieved throughout our participation in iGEM. These range from successfully developing an efficient storage system for biological parts to demonstrating the proof of concept of some module with the application of the key experiments.

As outlined in the [project design](https://2024.igem.wiki/upf-barcelona/design), we divided our workflow into different modules. This modular approach allowed us to meet several objectives and produce significant results across various aspects of the project. However, certain processes, such as part storage, were more efficient to manage collectively. This is why some of our results are specific to individual modules, while others are more comprehensive and apply to the project as a whole.

We split the work between two chassis: *E. coli* for initial proof-of-concept experiments and *C. acnes* for testing the constructs in a more relevant biological context. This strategy allowed us to optimize our system in *E. coli* before addressing the more challenging transformations in *C. acnes*, improving overall efficiency.

Below you can find the documentation of the outcomes of our experiments. The key experiments appear with a ★ sign at the beginning.

## *Escherichia coli*

During the first weeks at the lab one of the main objectives was to transform the *Escherichia coli* NZY 5α strain into having the parts that we requested to IDT.

Storage of the requested parts in *Escherichia coli* NZY 5α

With this procedure we were able to have an unlimited supply of our genes and parts of interest without needing to order more.

In order to achieve these transformations we digested the plasmids with restriction enzymes and introduced our parts with Gibson Assembly. To confirm that the transformations were properly achieved we did agarose gels for electrophoresis to identify the band that corresponds to our gene of interest. For this case we also confirmed our positive results with Sanger sequencing and performed the alignments of our sequenced results with our designed parts on Benchling. Since for most of the parts its length was much higher than 1000 bp we used reverse and forward priming to obtain the maximum possible information on our alignment results.

After we obtained the results we did liquid cultures of our samples and saved glycerol stocks and minipreps of them. We also measured the DNA concentration results of the colony PCR purified products and the backbone we wanted to use (p-JET) with the Nanodrop, obtaining the following results:

|  | DNA Concentration [ng/μL] |
| --- | --- |
| Cry 3Aa Sample 1 | 25.7 |
| Cry 3Aa Sample 2 | 109.6 |
| Cry 3Aa Sample 3 | 144.3 |
| Cry 4Ba | 224.4 |
| Endolysin Sample 1 | 60.8 |
| Endolysin Sample 2 | 155.9 |
| p-JET | 69 |
| sfGFP | 138.6 |
| mCherry | 144 |

Table 1. Concentrations of the purified inserts of interest after amplifying with a PCR.

Once we had the parts of interest, our main focus for both modules was to create the constructs we wanted to insert into *Cutibacterium acnes.*

Construction of the parts of interest in *Escherichia coli* NZY 5α

To achieve this, we began with the Gibson assembly to introduce our parts along with our [two promoters](https://2024.igem.wiki/upf-barcelona/parts) inside p-END.

After obtaining promising results with the Gibson Assembly and transforming NZY5α *E. coli* cells, we incubated them at 37°C. However, during this first cycle, we discovered that the transformation containing the [MG10 promoter](https://2024.igem.wiki/upf-barcelona/parts) and [Cry4Ba](https://2024.igem.wiki/upf-barcelona/parts) toxin constructs did not yield any colonies on any of our plates.

With the grown colonies we performed a PCR Colony and an agarose gel for electrophoresis, it confirmed that some of the constructs had been integrated properly, since the results for the Module 3.1 required more time we sent to sequence the PCR purified products for the parts corresponding to the Module 2 and the Module 3.2 and analyze its results with Benchling.

![](https://static.igem.wiki/teams/5311/assets/results/good-primer-gel-nzyalpha.png)

Figure 1. PCR colony of NZY 5α cells that had been previously transformed with all our constructs.

Before transforming *C. acnes*, our constructs needed to undergo one last step for correct incorporation.

Cloning of our constructs in EC24

This consisted in transforming yet again another *E. coli* strain, that has the particularity of having the same methyltransferase as *C. acnes*. The goal is to obtain the constructs with a pattern of methylation that ressemblance that of *C. acnes*.

To start off, we performed minipreps from the liquid cultures of the NZY 5α confirmed colonies, obtaining the following concentrations:

|  | DNA Concentration [ng/μL] |
| --- | --- |
| Cry3Aa+MG10 | 80.4 |
| Cry3Aa+MG26 | 137.4 |
| Cry4Ba+MG26 | 255.6 |
| Endolysin+MG10 | 54.8 |
| Endolysin+MG26 | 151.3 |
| sfGFP+MG10 | 106.5 |
| sfGFP+MG26 | 109.5 |
| mCherry+MG10 | 149.9 |
| mCherry+MG26 | 101.4 |

Table 2. Concentrations of the purified minipreps for each of the constructs of interest.

After transforming the *E. coli* EC24 strain with the minipreps and culture overnight, once again we performed PCR colonies, in order to send the purified products to sequence. The colonies were again confirmed through alignment, the analysis of these sequencing can be seen on the document attached below.

![](https://static.igem.wiki/teams/5311/assets/results/plates.png)

Figure 2. Cry 3 EC24, Cry 4 EC24 and Endolysin EC24 plates, respectively.

We wanted to conduct a first proof of concept to evaluate the functionality of the RNA thermometer in *E. coli* by monitoring temperature-dependent gene expression.

★ Evaluation of RNA thermometer functionality in *E. coli* via comparative analysis of fluorescent intensities

For this experiment, the EC24 strain of *E. coli* was transformed with plasmids encoding two fluorescent proteins. The transformed *E. coli* cells were grown in liquid culture at different temperatures (room temperature, 30ºC, and 37ºC) to assess the effect of temperature on protein production. Fluorescence intensity was measured two days after for 30ºC and 37ºC, and we waited two more days to measure the room temperature sample, to assure correct growth. By doing so, we quantified the expression levels of the fluorescent proteins. We anticipated that higher temperatures would lead to increased fluorescence, reflecting the activation of transcription via the RNA thermometer.

Just by looking at the liquid culture growth, it was apparent that temperatures close to 37ºC had significantly more protein production, in comparison to those that had grown at room temperature and 30ºC.

![](https://static.igem.wiki/teams/5311/assets/results/fluorescence.png)

Figure 3. Liquid cultures of EC24 containing the RNA thermometer coupled with GFP and mCherry, grown at different temperatures.

Nevertheless, we also performed a quantitative analysis of the amount of fluorescence in all the samples. The protocol was straightforward: after incubating the transformed *E. coli* EC24 cells at different temperatures, we transferred aliquots of each culture into a 24-well plate and measured fluorescence using a plate reader. For GFP, the excitation and emission were measured at 485 nm, while for mCherry, it was measured at 575 nm. The absorbance was also measured in all samples at 660 nm.

To ensure accuracy, all fluorescence readings were normalized by applying a simple formula:

*Sample reading (sfGFP/mCherry) - LB reading (sfGFP/mCherry)*
*Sample ABS 660 - LB ABS 660*

The LB served as the blank and this normalization allowed us to account for the background fluorescence and ensured that the fluorescence values correspond only to the expression of the different proteins.

![](https://static.igem.wiki/teams/5311/assets/results/sfgfp.png)

Figure 4. sfGFP: Graphical representation of the normalized values of the fluorescence emitted by the sfGFP samples at different temperatures. (To check the raw data, see [annex](#annex) at the end of the results section).

![](https://static.igem.wiki/teams/5311/assets/results/mcherry.png)

Figure 5. mCherry: Graphical representation of the normalized values of the fluorescence emitted by the sfGFP samples at different temperatures. (To check the raw data, see [annex](#annex) at the end of the results section)

In both cases, there is a significant difference between higher and lower temperatures. When comparing 30ºC and 37ºC, there is around a two-fold difference in each case, proving the functioning and ideal working temperature of the FourU RNA thermometer (at least in *E. coli*).

Note: we measured the fluorescence of each liquid culture three times in order to have replicates.

## *Cutibacterium acnes*

Following the successful transformation of *E. coli* as a proof-of-concept, we proceeded to transform *C. acnes* with all of our constructs. However, this phase of the project presented significant challenges. *C. acnes* is intrinsically more difficult to transform due to its slow growth and sensitivity to environmental conditions. Additionally, the process was complicated by frequent contamination with *Staphylococcus* species, forcing us to do multiple repetitions of the transformation protocol. Despite the tedious and time-consuming nature of these setbacks, we continued to optimize our methods to achieve the desired transformation efficiency. Here, we present the results of these efforts and the progress made in overcoming the technical difficulties.

![](https://static.igem.wiki/teams/5311/assets/results/c-acnes-failure.png)

Figure 6. First C. acnes transformation failure. The plates were left to grow for 5 days, only to find zero growth.

Despite the challenges, we ultimately succeeded in transforming *C. acnes* with all of our constructs. However, we were only able to conduct experiments with the *C. acnes* cells that had correctly incorporated the construct containing the endolysin gene. These transformed cells allowed us to move forward, and we were able to assess the activity of the endolysin, as well as to detect protein production through a Western Blot.

Although further experiments with the remaining constructs were not completed within the time frame of the iGEM project, the successful transformation provides a solid foundation for future work.

First *C. acnes* successfully transformed

As mentioned, the first couple of transformations were a failure due to both inexperience and the intrinsic difficulty of correctly transforming *C. acnes*. After these two first attempts, we finally succeeded in introducing our first construct into *C. acnes*: the CAP 10-3 Endolysin.

![](https://static.igem.wiki/teams/5311/assets/results/good-gel-acnes-endolysin.png)

Figure 7. PCR colony of C. acnes transformation, analyzing both our region of integration, and the 16S region.

As can be seen in the gel, apparently all of the constructs had been correctly integrated. However, the latter 16S region analysis through alignment (see [*C. acnes* alignments](#annex) pdf), would show that *C. acnes* was only present in the endolysin samples. The other “positive” samples were *Staphylococcus* species that had been able to incorporate our construct.

Just before the wiki freeze, we were finally able to obtain *C. acnes* cells that had successfully integrated all our constructs.

Final and definitive *C. acnes* transformation

Successfully obtaining the correct transformations in *C. acnes* was a crucial step for our project. After many setbacks, this achievement opens up the possibility for further experiments that couldn't be finished before the iGEM wiki freeze. Moving forward we can focus on testing the remaining constructs and exploring additional applications. This progress gives a strong foundation for future work and ultimately, the development of our final product.

![](https://static.igem.wiki/teams/5311/assets/results/good-gel-definitiu-acnes.png)

Figure 8. PCR colony of C. acnes transformation, analyzing both our region of integration, and the 16S region.

We conducted a Western blot analysis to evaluate protein expression across various samples.

★ Performing a Western blot to evaluate protein concentration

The samples analyzed included:

* *E. coli* NZY 5α with Cry3Aa+MG10 (pellet)
* *E. coli* NZY 5α with endolysin+MG10 (pellet)
* *C. acnes* with endolysin+MG10 (pellet)
* *C. acnes* with endolysin+MG26 (pellet)
* *C. acnes* with Cry4Ba+MG26 (pellet)
* *C. acnes* with Endolysin+MG10 (supernatant)
* *C. acnes* with Endolysin+MG26 (supernatant)
* *C. acnes* with Cry4Ba+MG26 (supernatant)

For *E. coli*, proteins are primarily intracellular, requiring cell lysis to isolate the proteins from the pellet. For *C. acnes*, we anticipated protein expression both within the cell and in the supernatant. Thus, we evaluated both pellet and supernatant samples to capture the full protein profile.

To ensure consistency, we loaded 20 ng of total protein per well, adjusting sample volumes based on protein concentration. Before loading in the samples, we evaluated the concentration of protein found in each one to know how much was needed to have the 20 ng.

| Sample | DNA Concentration [ng/μL] |
| --- | --- |
| *E. coli* Cry3A+MG10 (pellet) | 1.179 |
| *E. coli* Endolysin+MG10 (pellet) | 1.499 |
| *C. acnes* Endolysin+MG10 (pellet) | 0.512 |
| *C. acnes* Endolysin+MG26 (pellet) | 0.692 |
| *C. acnes* Cry4Ba+MG26 (pellet) | 0.745 |
| *C. acnes* Endolysin+MG10 (SN) | 0.686 |
| *C. acnes* Endolysin+MG26 (SN) | 1.072 |
| *C. acnes* Cry4Ba+MG26 (SN) | 1.018 |
| Control sample | 1.32 |

Table 3. Concentrations of the proteins of interest evaluated previous to the Western Blot.

Once we have obtained the total protein concentrations, we can proceed with the results of the gel:

![](https://static.igem.wiki/teams/5311/assets/results/good-wb-endolysin.png)

Figure 9 & 10. SeeBlue Pre-Stained Standard Western blot ladder and Western blot membrane revealed to study the presence of desired proteins.

We have also attached an image of the ladder used for reference. For reference, the expected kDa were the following:

* Cry3Aa: 76.8 kDa
* Cry4Ba: 130.6 kDa
* Endolysin: 31 kDa

### Results

After revealing the Western blot, we noted the following:

* Well 1: *E. coli* Cry3Aa+MG10 (pellet) showed no significant protein bands, indicating no detectable protein expression.
* Well 2: *E. coli* Endolysin+MG10 (pellet) showed two clear bands, one around 76 kDa and another around 31 kDa. This suggests that Cry3Aa protein contaminated this well, leading us to conclude that both Cry3Aa and Endolysin+MG10 are expressed.
* Well 3: *C. acnes* Endolysin+MG10 (pellet) showed a faint band at 31 kDa, suggesting some expression of Endolysin+MG10.
* Well 4: *C. acnes* Endolysin+MG26 (pellet) displayed a strong band at 31 kDa, confirming successful protein expression of Endolysin+MG26.
* Well 5-8: No detectable protein bands were observed. Specifically, for the *C. acnes* supernatants (wells 6–8), the lack of protein expression may be due to insufficient time for the protein to be secreted into the supernatant since we only left the liquid cultures growing for 48 hours.

In conclusion, protein expression was confirmed for *E. coli* Endolysin+MG10 and *C. acnes* Endolysin+MG26. Protein expression is concluded to also apply for *E. coli* Cry3Aa+MG10. We would have liked to perform another Western Blot with newly transformed *C. acnes* cells, however, we did not have enough time to do this.

We wanted to evaluate the effectiveness of the CAP 10-3 endolysin in reducing the viability of *Cutibacterium acnes.*

★ Evaluation of endolysin activity in transformed *Cutibacterium acnes* cells

To determine the effectiveness of the CAP 10-3 endolysin in reducing *C. acnes* viability, we compared the growth of three different strains: one expressing an endolysin-coding plasmid, one with a plasmid providing only antibiotic resistance (WT), and another one with a plasmid with constitutive GFP expression. Growth was monitored over the four following days by measuring the OD600. The OD600 of the blanks was subtracted from all the experimental readings to account for background absorbance.

![](https://static.igem.wiki/teams/5311/assets/results/endolysinmg10corrected.png)

![](https://static.igem.wiki/teams/5311/assets/results/endolysinmg26corrected.png)

Figure 11 & 12. Growth comparison of C. acnes strains expressing endolysin with MG 10 and MG 26 promoters, WT, and GFP constructs (to check the raw data, see [annex](#annex)).

This indicates that the expression of endolysin, regardless of the promoter used, is effective in impairing *C. acnes* viability. The consistent reduction in growth across strains with different promoters suggests that endolysin has a significant impact on its overall fitness.

The endolysin-expressing strain exhibited a lag phase that was noticeably longer than that of both the WT and GFP strains. This extended lag phase suggests that the presence of endolysin may require additional time for the cells to acclimate before starting to grow. Once the lag phase concluded, the growth rate of the endolysin strain appeared slightly slower than that of the WT and GFP strains. The OD600 measurements indicated that, although the endolysin strain eventually increased in cell density, it did so at a reduced rate, further supporting the fact that endolysin affects cellular viability.

These findings are important as they support the idea that endolysin may assist in the delivery of the Cry proteins. Future research should focus on investigating the specific role of endolysin in facilitating protein release, and evaluate whether this mechanism enhances the therapeutic effectiveness against the targeted skin infestations

The endolysin has been essential to our project, playing a key role in both ensuring the safe use of our GMO and facilitating the release of the Cry protein. We are particularly proud of the endolysin DNA part, which we are presenting for the Best New Part special prize due to its innovation and importance in our design. You can find more details about the part in the iGEM registry [here](https://parts.igem.org/Part%3ABBa_K5311006).

## Conclusions

In conclusion, we successfully stored our parts of interest and used them to construct the desired assemblies. We successfully combined the promoters with the corresponding proteins, resulting in the transformation of *E. coli* NZY 5α with the following constructs: Cry3Aa+MG10, Cry3Aa+MG26, Cry4Ba+MG26, Endolysin+MG10, Endolysin+MG26, sfGFP+MG10, sfGFP+MG26, mCherry+MG10, and mCherry+MG26. We then repeated these transformations with *E. coli* EC24, which was also successful. We observed a difference in expression between higher and lower temperatures, confirming that the [FourU RNA thermometer](https://2024.igem.wiki/upf-barcelona/parts) functions as expected at 37ºC. For *C. acnes*, we successfully extracted the plasmids from the *E. coli* EC24 transformations and inserted them into *C. acnes*, achieving successful transformations with all the constructs mentioned. Finally, our Western blot confirmed protein expression for *E. coli* NZY 5α Endolysin+MG10, *C. acnes* Endolysin+MG26, and *E. coli* NZY 5α Cry3Aa+MG10.

Overall, the wet lab experience during the last 4 months of the iGEM competition has been a very challenging yet rewarding process, providing us with valuable insights into synthetic biology and hands-on lab work. From the initial transformations in *E. coli* to the more complex work with *C. acnes*, each step has required readjustments, repetition, and adaptation. Working with *C. acnes* brought its unique challenges, from transformation efficiency issues to contamination with other bacteria like Staphylococcus. Initially, this seemed like a major setback, as this kind of contamination was a recurrent issue. However, we turned this situation into an opportunity to help future iGEM teams that are willing to work with *C. acnes*, to better distinguish both types of bacteria with our own [poster](https://2024.igem.wiki/upf-barcelona/contribution), contributing to the broader iGEM community.

This experience highlighted the importance of planning, iteration, and collaboration in the lab work. It has also taught us how to refine experimental protocols to achieve reliable results. Looking ahead, all the work has also established a very solid basis to continue working with the project.

## Future work

As we look towards immediate future work after the wiki freeze, we have several experiments designed to advance our iGEM project further. Our first objective is to test the antiparasitic activity of the Cry proteins produced by *C. acnes* against *Drosophila* as a proof-of-concept. This will help us demonstrate the efficacy of the Cry proteins in combating parasitic infestations, specifically those related to the skin, like scabies infections.

Additionally, we plan to evaluate the effectiveness of the RNA thermometer in *C. acnes*, which is crucial for our strategy of controlling the expression of endolysin. The expression of endolysin will be regulated by the RNA thermometer, allowing us to address the safety concerns associated with having a genetically modified organism (GMO) continuously present on the skin. This regulation also ensures that the endolysin is exclusively expressed at body temperature (around 37ºC), which aligns with our goal of releasing the Cry proteins when entering contact with the skin.

Ultimately, we aim to join these elements to achieve constitutive expression of the Cry proteins, while simultaneously controlling endolysin expression through the RNA thermometer. This integrated approach will not only enhance the therapeutic potential of our engineered strain but also mitigate safety concerns by ensuring precise control over protein expression in response to environmental conditions.

## Annex

In this annex section, you will find Document 1, which contains the different alignments we have performed to demonstrate the success of the transformations in *E. coli*; Document 2, which details the alignments we have conducted to show the success of the transformations in *C. acnes*; and Document 3, which includes the raw data generated from the experiments mentioned in the results section.

E. coli
C. acnes
Raw data

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