iGEM 2024 — Team botchanlab-tokyo


## What is Engineering Success

![Custom Description Banner](https://static.igem.wiki/teams/5349/botchanlab-wet/eef44460-b09c-46d4-8bd1-34fc0e2a272d.png)

**Engineering Success through the DBTL Cycle**

Engineering success is a crucial process in our activities, characterized by the Design-Build-Test-Learn (DBTL) cycle. This iterative approach involves designing unique systems, assembling them from various components, testing their functionality, and learning from the results. At the Bo-chan lab, we applied this method to create and refine colonics, continuously optimizing our designs through repeated cycles. By leveraging insights from both our experimental outcomes and existing literature, we enhance our systems incrementally, ensuring continuous improvement and effective problem-solving in our projects.

# Wet

## Wet Overview

We set the following three goals at wet. And we designed and executed an experiment to achieve them.

## Kill switch

As mentioned in Project Description, we introduce a suicide gene into the genetically modified *E. coli* strain Nissle 1917-**Gassle**.

Everyone knows the usefulness of implementing a kill switch in iGEM. However, We also recognize that the inclusion of this Kill switch system is indispensable in discussing how to implement this diagnostic system.
The herpes simplex virus thymidine kinase gene/ganciclovir (HSV-TK/GCV) system is currently being studied in cancer treatment[1]. And it is a system that specifically kills transformants (inhibits DNA replication) by transforming them to express HSV-TK.

## Bio sensor

We searched for a variety of biomarkers to detect IBD. We found a study that mentions an increase in nitric oxide levels in the intestines of patients during inflammation[2]. Furthermore, after exploring the iGEM Parts Registry page, we found a part”[BBa\_K K4387006](https://parts.igem.org/Part%3ABBa_K%20K4387006)” that aligns with our objectives, so we decided to adopt this expression system.
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## Production of a safe and strong-smelling substance (Propionic acid)

Our ultimate goal is to have the biosensor mentioned earlier produce an odorant.
The gene cluster necessary for *E. coli* to produce propionic acid is called the Sleeping Beauty Mutase Operon.
In wild-type *E. coli*, it is not expressed, but through genetic modification, we designed it downstream of a promoter, allowing the enzymes to be expressed for the first time. [3]
We identified the potential to detect diseases through the distinct smell of propionic acid and decided to incorporate this gene cluster into the vector.

# Preparation of Competent *Escherichia coli* Nissle 1917 (EcN) Cells for Transformation

Cycle 1 : Preparation of Competent EcN Cells for transformation

## Design and Build: Obtaining the desired strain and creating the protocol. Preparation of EcN competent cells.

First, we attempted to obtain *E. coli* strain Nissle 1917 (EcN), a bacterium capable of forming colonies in the colon.

Unexpectedly, EcN was very expensive to purchase from overseas through Japan's cell banks, and we ended up spending a considerable amount of time trying to obtain it. We found a team that had isolated the strain from a probiotic product called Mutaflor, but according to a professor at the National Institute of Infectious Diseases, sequencing of the strain isolated from that product revealed it was a completely different bacterium. (A word of caution to future iGEM teams.) While searching for someone to provide the strain, we were introduced to several professors, and eventually, Dr. Nakao kindly shared the strain with us.

From this day, our experiments began.

## Test - Verification of competent cell viability and comparison of transformation efficiency with DH5α (PI’s Lab stock) as a positive control.

To begin the genetic recombination experiments, we first started by preparing competent cells. However, despite creating the competent cells, we failed in our first transformation experiment. To identify the cause, we adjusted various conditions and retried the transformation.

We received *E. coli* DH5α as a positive control, as well as fresh SOC medium, from our PI's lab and conducted experiments to identify the cause.

## Learn - Consideration of the causes of the transformation failure.

We considered multiple possibilities and conducted various experiments, but fortunately, the transformation was successful under the condition where the SOC medium was freshly prepared for the first time. However, as a control, we also successfully transformed *E. coli* DH5α using the old SOC medium. This indicated that the EcN competent cells we had prepared had a significantly lower transformation efficiency. Since this could affect future experiments, we realized that we needed to explore various approaches to address this issue. On to Cycle 2.

Cycle 2 : Optimization of the protocol

## Design and Build -Transformation using various protocols.

In order to compensate for the low transformation efficiency of the competent cells, we conducted a literature review. Transformation protocols are often unique to each lab. Our PI pointed out that, while the NEBuilder Vector Assembly we adopted for cloning is effective for short fragments of 1-2 pieces, the success rate is very low for long fragments with more than 3 pieces. Therefore, we decided that it was necessary to at least optimize the transformation protocol and proceeded to do so.

We modified the following protocols:

・Changed the heat shock duration at 42°C from 60 seconds to 45 seconds [4].

We incorporated protocols used by other teams that performed transformations with *E. coli* Nissle 1917.

・Added a step to pellet the cells after recovery culture, resuspend them, and concentrate them before plating [5].

## Test -Transformation using the optimized protocol.

The success of vector assembly using NEBuilder is determined by whether the transformation was successful. Although we were concerned about this step due to the low transformation efficiency of our competent cells, we successfully completed both the vector assembly and transformation, likely thanks to the optimized protocol.

## Learn and conclusion

Through the optimization of the transformation protocol, we learned that even small adjustments, such as reducing the heat shock duration and introducing additional steps like pelleting and concentrating the cells after recovery, can significantly improve the transformation efficiency of *E. coli* Nissle 1917 competent cells. This was particularly important for our work with NEBuilder, as the low transformation efficiency had previously posed a major bottleneck, especially for complex assemblies with longer DNA fragments.

The success of the optimized protocol allowed us to proceed with our vector assembly confidently, demonstrating that protocol adjustments can effectively compensate for inherent challenges such as low competence of the cells. Moving forward, this optimized protocol will be invaluable for further experiments and will serve as a foundation for future iGEM teams working with similar strains and assembly methods.

# Kill switch

Cycle 1 : kill switch (Check HSV-TK/GCV system)

## Design - Design of the vector.

Since we would be incorporating genetically modified *E. coli* into the body, it was necessary to control the recombinant *E. coli* appropriately in consideration of our relationship with the world. After hearing about the HSV-TK/GCV system from Dr. Nishikawa, Dr. Kusamori, and Dr. Kamakura, we decided to incorporate the UL23 gene, which expresses herpes simplex virus thymidine kinase (HSV-TK), into the vector.

## Build - Vector Assemble, Transformation

We aimed to find the optimal solution by utilizing the free DNA synthesis service available through iGEM. After exploring various cloning methods, reflecting on our experimental techniques, and learning from numerous failures, we arrived at one conclusion: seamless cloning using NEBuilder.

This method, as long as overlap sequences are designed, is an exceptionally efficient technique with fewer operational errors, allowing for quicker results. It is becoming more mainstream than traditional cloning methods that use restriction enzymes. iGEM participants can fully benefit from NEBuilder.

First, since the DNA synthesis service is available, it's very easy to create overlap sequences at both ends. By using IDT's gBlocks, which do not have adapter sequences, the fragments can be used immediately for vector assembly without needing to go through PCR. Additionally, if point mutations need to be introduced, it is surprisingly simple and quick to do so with NEBuilder by designing primers with approximately 15 bp of complementary sequence during inverse PCR.

For these reasons, we chose NEBuilder for our assembly

## Test - HSV-TK/GCV Assay 1

We conducted an assay to demonstrate the growth inhibitory effect by observing the turbidity progression of *E. coli* during the logarithmic growth phase.

However, although there was a slight delay in the rate of turbidity increase, no significant difference was observed.

## Learn - Consideration of the causes for the failure of growth control to work.

After considering several factors that could have contributed to the lack of growth inhibition observed in the previous HSV-TK/GCV assay, we identified potential reasons for the failure and have developed a plan for the next steps. The primary hypothesis is that the **wild-type** HSV-TK expressed by the UL23 gene does not exhibit sufficient sensitivity to GCV, as previous studies used HSV-TK variants, such as **SR39**, with increased GCV sensitivity. As a result, the current system may not be functioning as an effective kill switch.

To address this, we decided to **introduce mutations** to the UL23 gene to replicate these more sensitive variants, specifically focusing on introducing the **SR39 mutation**. This mutation has been reported to increase the GCV susceptibility of *E. coli* 4000-fold [6]

## Next Steps: Mutation Introduction by Inverse PCR and DNA Assembly

→Cycle 2

Cycle 2 : Mutation Introduction by Inverse PCR

## Design - Inverse PCR and PCR primer Design

For the next assay, we plan to use *E. coli* expressing **mutant UL23** (SR39 variant) to increase the system's efficiency. To introduce the mutation, we determined that the most efficient method would be **inverse PCR**, followed by **linear DNA assembly using NEBuilder® HiFi DNA Assembly**. This approach ensures precise mutation insertion without affecting other regions of the gene.

・**Inverse PCR**: This method will allow us to amplify the circular plasmid containing UL23 while introducing the desired mutation at specific locations.

・**NEBuilder® Assembly**: Once the PCR produces linear DNA fragments with the mutation, we will assemble these fragments using NEBuilder, recreating the circular plasmid with the SR39 mutation.

After careful consideration of various methods, we have determined that inverse PCR is the optimal approach for introducing the UL39 mutation. I proceeded with primer design to enable this next step in the process.

![Custom Description Banner](https://static.igem.wiki/teams/5349/botchanlab-wet/inverse-pcr.webp)

*Fig 1. Design of inverse primers using SnapGene*

![Custom Description Banner](https://static.igem.wiki/teams/5349/botchanlab-wet/vectormap-inv.webp)

*Fig 2. Vector map for inverse PCR.*

・Inverse PCR, NEBuider Assembly and Transformation

![Custom Description Banner](https://static.igem.wiki/teams/5349/botchanlab-wet/trans-sr39.jpg)

*Fig3. Transformation of E. coli using the vector with introduced mutations.*

## Vector Assembly and Successful Transformation

After successfully assembling the vector and transforming it into competent cells, the transformed bacterial colonies were cultured. The plasmid DNA was extracted using a miniprep procedure and subsequently sent for Sanger sequencing to confirm the accuracy of the assembly and the presence of the desired genetic elements.

## Test - HSV-TK/GCV Assay 2, 3, 4

In these assays, we aimed to test the growth control effects of the HSV-TK/GCV system by measuring the bacterial growth rates in the presence of ganciclovir. By monitoring the turbidity of the bacterial cultures, we sought to observe any delay or inhibition in growth as an indicator of the system's efficacy. Unfortunately, while slight delays were observed, there was no statistically significant difference between the test and control groups. This suggested that further optimization of the system or mutations may be necessary to achieve stronger growth inhibition.

## Learn

We learned that while the SR39 variant of UL23 increased ganciclovir sensitivity, the overall inhibitory effect on bacterial growth was still insufficient for strong control. This points to the need for either additional mutations or improvements in the expression and delivery of the kill switch system. We also realized that the transformation efficiency and subsequent assay conditions play a critical role in the success of the system, and further refinements are needed for reliable results.

Cycle 3 : Evaluation Method Adjustment

## Design

After observing limited success in previous assays, we decided to modify the evaluation methods to more accurately measure the effects of the HSV-TK/GCV system. This included adjusting the concentration of ganciclovir, extending the incubation period, and using more sensitive detection methods to capture subtle differences in growth inhibition.

## Build

We implemented the necessary changes to the assay protocol, including using more precise spectrophotometry for measuring turbidity, altering the timing of measurements, and testing a wider range of ganciclovir concentrations to determine the system's optimal sensitivity.

## Test -HSV-TK/GCV Assay 5, 6

These assays incorporated the adjusted evaluation methods, and preliminary results indicated a more noticeable delay in bacterial growth when treated with ganciclovir. However, while this improvement was promising, the results still fell short of the robust growth control we aimed to achieve. Further testing is required to refine the system.

## Learn

From these tests, we learned that minor adjustments in assay conditions can significantly impact the observed results. While the modifications led to improved growth inhibition, the system's overall efficacy remains suboptimal. This suggests that future efforts should focus not only on assay conditions but also on potential structural improvements to the vector or the mutation itself to achieve stronger kill switch functionality.

# Bio sensor

Cycle 1 : Verification of nitric oxide responsiveness (pNorV-norR Plate reader Assay)

## Design and Build: Vector assembly using NEBuilder.

We performed the assembly in parallel with the kill switch. Since the number of fragments was the same, we were able to conduct a good comparative experiment. The fact that one assembly worked while the other did not, despite following the same procedure, suggested that the issue was not with experimental technique but with the inherently low success rate.

## Test

We exposed the transformed EcN strains to solutions of different concentrations of NO and assayed how many GFP the transformed EcN strains produced, reacting NO concentrations with measuring the fluorescence intensity.

## Learn

From the experimental results, we obtained graphs similar to those from teams that had conducted similar measurements in the past, confirming the reproducibility of our experiment. However, since our ultimate goal is to detect inflammation in the intestines, we planned a more in-depth assay to take the next step. On to Cycle 2.

Cycle 2 : Verification of responsiveness to hydrogen peroxide and other reactive oxygen species

## **Design and Build** - Verification of the response of pNorV to hydrogen peroxide and other reactive oxygen species.

The purpose of this experiment is to validate the responsiveness of the pNorV system to hydrogen peroxide and other reactive oxygen species (ROS). The pNorV promoter is known to be activated in response to nitric oxide, but since reactive oxygen species such as hydrogen peroxide (H2O2) also play a significant role in inflammation and oxidative stress in the intestines, it is crucial to assess the sensitivity of pNorV to these compounds.

Previous experiments have focused on detecting nitric oxide as a biomarker for inflammation, and the results showed that our biosensor system, which employs the pNorV promoter, successfully responded to NO. However, because intestinal inflammation involves a complex interplay of various oxidative species, we are expanding our focus to include other ROS, such as H2O2, to improve the robustness and sensitivity of our biosensor system.

By exposing *E. coli* carrying the pNorV system to controlled concentrations of hydrogen peroxide and other ROS, we aim to measure the fluorescence intensity as an indicator of the system’s activation. This will allow us to determine whether the pNorV promoter can also be used to detect oxidative stress beyond nitric oxide, thus moving us closer to our ultimate goal of detecting intestinal inflammation more comprehensively.

## Test

We exposed the transformed EcN strains to solutions of different concentrations of H2O2 and assayed how many GFP the transformed EcN strains produced, reacting H2O2 concentrations with measuring the fluorescence intensity.

## Learn and Conclusion

From this experiment, we learned that the pNorV promoter demonstrated a measurable response to hydrogen peroxide (H2O2) and other reactive oxygen species (ROS), similar to its response to nitric oxide (NO). The fluorescence intensity increased in a dose-dependent manner, confirming that pNorV is sensitive not only to NO but also to oxidative stress signals induced by ROS.

However, the magnitude of the response to H2O2 and other ROS was lower than that observed with NO, suggesting that while pNorV can detect multiple forms of oxidative stress, it may exhibit a stronger specificity for nitric oxide. This insight is important as it suggests that while pNorV could serve as a broader oxidative stress sensor, its application may be more suited for NO detection in inflammatory conditions.

Moving forward, we conclude that the pNorV promoter can be a valuable component of a biosensor designed to detect inflammation-related oxidative stress in the intestines. However, to achieve our ultimate goal of detecting intestinal inflammation with high specificity and sensitivity, further optimization of the sensor system or the incorporation of additional ROS-specific promoters may be necessary. This would allow us to improve the overall robustness and reliability of our detection system in complex biological environments such as the gut.

# References

[1] Black ME, Kokoris MS, Sabo P. Herpes simplex virus-1 thymidine kinase mutants created by semi-random sequence mutagenesis improve prodrug-mediated tumor cell killing. Cancer Res. 2001 Apr 1;61(7):3022-6. PMID: 11306482.

[2] Lundberg JO, Hellström PM, Lundberg JM, Alving K. Greatly increased luminal nitric oxide in ulcerative colitis. Lancet. 1994 Dec 17;344(8938):1673-4. doi: 10.1016/s0140-6736(94)90460-x. PMID: 7996962.

[3] Akawi L, Srirangan K, Liu X, Moo-Young M, Perry Chou C. Engineering Escherichia coli for high-level production of propionate. J Ind Microbiol Biotechnol. 2015 Jul;42(7):1057-72. doi: 10.1007/s10295-015-1627-4. Epub 2015 May 7. PMID: 25948049.

[4] University of Zurich. 2022 iGEM Team. Retrieved July 1, 2024.

[5] (Japanese) Thermo Fisher Scientific. Bacterial Transformation Workflow. Retrieved August 21, 2024

[6] Kokoris MS, Black ME. Characterization of herpes simplex virus type 1 thymidine kinase mutants engineered for improved ganciclovir or acyclovir activity. Protein Sci. 2002 Sep;11(9):2267-72. doi: 10.1110/ps.2460102. PMID: 12192082; PMCID: PMC2373606.