iGEM 2024 — Team nnhs

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block page\_content %}

# Notebook

**March-April**

Project proposal: Brainstorming, project background research, consultation with relevant experts, proposal report.

**March-August**

Building Controllable Semi-Random Mutagenesis System

High Screening Selection over Human Selection

Development of a monitor to quantify the process mutagenesis

Improvement from GFP to metabolic system evolution using CBE-Mag-RNAP

Safety Consideration before Application on Advanced Life

Implementation of C. elegans

Construction of a Mutation Library Database Design

Potential Future Application in the medical field

**May-August**

Dry lab design: Plasmid Construction,Sequencing Analysis,Structural Prediction,Metabolic Pathways,Model

**June-August Wet lab**

-June

Laboratory safety training

experiment design

material preparation

hands-on training of nematodes and Escherichia coli

-July

Plasmid extraction, transformation, and amplification.

Nematode life cycle experiment.

Fluorescence intensity test of CEB-Mag-RNAP colonies.

Gel electrophoresis experiment and quality control of DNA.

Sequencing and sending samples of bacterial liquid.

-August

Flow cytometry sorting based on CEB-Mag-RNAP mutagenesis.

Large-scale extraction of plasmids.

PCR and DNA sequencing library construction.

High-throughput sequencing and analysis.

Modeling

**September**

Data analysis of experimental results.

Construction of mutation and function information database.

Protein structure prediction.

WIKI construction.

**October**

Prepare for Presentation

Prepare for Giant Jamboree

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block page\_content %}

# Engineering Success

## 1. Cycle 1-Building Controllable Semi-Random Mutagenesis System

### (1)Design

The development of random mutagenesis is primarily motivated by the goal of addressing large-scale challenges. Random mutagenesis can either enhance or reduce the expression of genetically coded products. By applying random mutagenesis, selecting beneficial mutations, and refining the final product, we can identify the most effective mutation for various applications, ultimately helping to solve a wide range of problems.

We designed a more controllable mutagenesis second generation. The new system consists of the Lac operon, CBE (cytidine deaminase), and a functional T7 RNA polymerase. This system is capable of mutating cytosine to thymine from the T7 promoter to the terminator. However, due to its uncontrollable nature-where CBE mutates all cytosines in its path to thymine-the first-generation system was not suitable as a universal solution for problem-solving.

### (2)Build

Seeing the shortcoming of the first-generation mutagenesis system, we seek to gain greater control over the entire random mutagenesis process. Building on previous experience, we have incorporated light-sensitive components-such as N-terminal RNAP, C-terminal RNAP, n-Mag, and p-Mag-into the existing T7 RNAP system. In this new, blue-light-sensitive T7 RNAP, exposure to blue light triggers the components to bind together, forming a functional RNAP that initiates transcription. By controlling the timing of transcription, we can more effectively select the gene that we hope to induce mutation on.

### (3)Test & Learn

We performed semi-random mutagenesis using Cytosine Base Editor- Mag-T7 RNA polymerase system (CBE-Mag-RNAP). After collecting the data, we fit it and compared it with the model. While adding IPTG to bacteria to start expressing CBE-nMag-nRNAP and pMag-cRNAP, we get the mutagenesis system ready to use. When performing the blue light, the RNAP can be assembled and start to work on the genes downstream of T7 promoter. We call it the black box system because this CBE-Mag-RNAP can generate a semi-random mutation on Cytosine in a region starting from T7 promoter to T7 terminator. In order to know how many mutations contributed to an optimized system, we next need to design a selection strategy.

![](https://static.igem.wiki/teams/5191/homepage/home-9-1.png)

Figure1. The Second Generation of controllable mutagenesis system

## 2. Cycle 2-High Throughput Screening Selection over Human Selection

### (1)Design

The system is encoded into a plasmid for transformation, and initially, human selection is used to identify the most successful host cell by observing fluorescence under a microscope and picking the most prominent monoclonal colony. However, this technique is inefficient for large-scale selection. To address this, we turned to high-throughput screening, which allows for the effective selection of large numbers of cells.

### (2)Build

We chose GFP as an indicator of successful mutation. In this approach, cells are placed in a harsh and selective environment to enhance plasmid production. For example, genetically modified *Estrecholia coli(E. coli)* can produce large amounts of plasmid in a petri dish containing over 20% glycerol and ampicillin. The host cells are then processed through the second-generation system and analysed using flow cytometry to select cells with the lowest fluorescence intensity. By Altering the GFP sequence entirely, it can indicate a positive mutagenesis of other enzyme coding parts in the plasmid. By high throughput screening techniques, we can select the most successful host cell in large quantities.

### (3)Test & Learn

![](https://static.igem.wiki/teams/5191/imp/imp-2.jpg)

Figure2. Results for High Throughput screening selection for flourescent intensity

## 3. Cycle 3-Development of a monitor to quantify the process mutagenesis

### (1)Design

We used metabolic genes instead of GFP as a parameter. We first incorporate a GFP report system. This system can report how the base editor functions in the cells.

### (2)Build

By analysing the changes in fluorescence intensity of fluorescent proteins and sequencing results, we have verified the success of the second generation black box system and intend to apply it to three different objects (environmental, human, fungal).We use the system to increase the activity of several key enzymes in those areas, which we test in nematodes.

### (3)Test & Learn

We are surprised to find some mutation can make the GFP brighter after we a high-throughput screening process using fluorescence- activated cell sorting (FACS) combined with Next Generation Sequencing. Mutations that enhance GFP brightness, known as "Winner Mutations", are cataloged in our Dictionary System.

## 4. Cycle 4 - Improvement from GFP to metabolic system evolution using CBE-Mag-RNAP

### (1)Design

After confirming the second-generation of black box could successfully enhance the frequency of random mutations, we applied it to improve the pentose phosphate pathway (PPP), polyamine synthesis pathway and nicotine metabolic pathway in *E. coli*. We hypothesized that by randomly mutating and screening the key enzymes in these metabolic pathways, we could increase the production of the anti-aging compound spermidine and the antioxidant NADPH, and also can let it degrade more nicotine in *E. coli*. First, we need to combine the second-generation black box with the three plasmids we constructed that contain the key enzymes and transform them into BL21 for mutation. Through sequencing analysis, we can determine whether our second-generation black box system can function properly in these environments.

### (2)Build

We constructed plasmids for spermidine synthesis (spermidine synthase, S-adenosylmethionine decarboxylase, and agmatinase) and NADPH synthesis (6-phosphogluconolactonase, transaldolase, and transketolase), and co-transformed them with the second-generation black box plasmid into *E. coli* BL21. For each experiment, we established a control group (where the addition of water would not activate black box) and an experimental group (where the black box was activated by IPTG and exposed to blue light). After the second-generation black box mutated the synthesis plasmids, we performed sequencing analysis on the control and experimental groups.

### (3)Test & Learn

![](https://static.igem.wiki/teams/5191/engineering/wechatimg616.jpg)

Figure3. Testing results for Spermidine and NADPH synthesis Pathway

![](https://static.igem.wiki/teams/5191/engineering/wechatimg617.jpg)

Figure4. Testing results for Nicotinate Degradation Pathway

The results revealed that, compared to the control group, the experimental group exhibited a clear C-to-T substitution, indicating the successful mutation of the three plasmids by the second-generation black box, with the majority being C-to-T mutations.

## 5. Cycle 5 Safety Consideration before Application on Advanced Life

### (1)Design

To address the potential issue of gene leakage from host cells, we developed a suicide system to trigger cell death if the cells leak into the environment. We encoded an enhancer-containing plasmid that encodes essential enzymes for ATP synthesis. This plasmid is regulated by an arabinose repressor protein produced from an upstream sequence, which binds to the plasmid and prevents transcription of the downstream gene. If the cells leak into an environment containing arabinose, the arabinose will bind to the repressor protein, initiating the transcription process. Once RNA polymerase (RNAP) reaches the coding sequence, it will cleave the sequence responsible for producing the essential ATP-synthesizing enzymes, effectively disabling the cell’s energy production.

### (2)Build

![](https://static.igem.wiki/teams/5191/description/with.jpg)

Figure4. Regulatory sequence for safety (With Arabinose)

![](https://static.igem.wiki/teams/5191/description/without.jpg)

Figure5. Regulatory sequence for safety (Without Arabinose)

There are concerns about the potential leakage of genetically modified host cells into the environment. To address these, we designed a suicide system that triggers cell death upon environmental exposure. The system includes a plasmid enhancer that codes for essential enzymes involved in ATP synthesis. An arabinose repressor protein, produced from an upstream sequence, binds to the operator, preventing transcription. A Cas9 gene is placed downstream of the promoter to halt protein production if transcription is triggered. When the host cell is leaked into an environment containing arabinose, the arabinose binds to the repressor protein, initiating transcription. As RNA polymerase reaches the Cas9 sequence, the Cas9 protein is expressed and cuts the ATP-synthesis enzyme-coding sequence, effectively shutting down the cell's energy production and killing it. This positive control system ensures safety by preventing the survival of genetically modified cells outside their intended environment.

### (3)Test & Learn

As a result of our built-in safety mechanism, we did not see the growth of *E. coli* outside of the laboratory.

## 6. Cycle 6-Implementation of *Caenorhabditis elegans*

### (1)Design

Since we cannot directly experiment on humans, we chose an alternative organic organism for testing—a well-suited nematode. Given that spermidine has anti-aging effects, NADPH has antioxidant properties, and nicotine is toxic, all of these factors may directly or indirectly affect the lifespan of the nematode. Therefore, we can design a series of related experiments by feeding the nematodes with those winner mutants we have selected in the previous experiments to verify our hypothesis by observing and statistically analyzing the average lifespan of the nematodes.

### (2)Build

For the spermidine synthesis experiment, we set up a control group (fed nematodes with normal BL21) and an experimental group (fed nematodes with well-mutated BL21). We placed the nematodes on two LB plates containing the same amount of arginine. Since we predict that the nematodes fed with mutated BL21 will produce more spermidine, leading to a longer lifespan, the final data for the average lifespan of nematodes from the experimental group should be higher than that of the control group.

Similar to what we did in the previous experiment, for the NADPH synthesis experiment, we set up a control group (fed nematodes with normal BL21) and an experimental group (fed nematodes with well-mutated BL21). We placed the nematodes on two LB plates, both containing the same amount of H2O2. Since we predict that the nematodes fed with mutated BL21 will produce more antioxidants, and the extended lifespan of the nematodes would confirm this, the average lifespan of the experimental group should be higher than that of the control group.

In the nicotine degradation experiment, we also set up a control group (fed nematodes with normal BL21) and an experimental group (fed nematodes with well-mutated BL21). We placed the nematodes on two LB plates, both containing the same amount of nicotinate solution. Since we predict that the nematodes fed with mutated BL21 will degradate more nicotine, the average lifespan of the experimental group should be higher than that of the control group.

### (3)Test & Learn

We tested their lifespan and found that the nematodes that consumed the mutated bacteria had a significantly longer lifespan. This suggests that the mutations in the bacteria positively impacted the health and longevity of the nematodes.

![](https://static.igem.wiki/teams/5191/result/result-head.jpg)

Figure6. *C. Elegans* Observation

![](https://static.igem.wiki/teams/5191/engineering/wechatimg627.jpg)

Figure7. A）average lifespan of nematodes in spermidine and NADPH synthesis experiments B）average lifespan of nematodes in nicotine degradation experiment

## 7. Cycle 7-Construction of a Mutation Library Database Design

By leveraging this technology, we are able to do a semi-random mutation on genes from animals and humans. The next question is how to select those genes with "gain of function" mutation?

Here, since the fluorescence of GFP may change when mutagenesis is conducted on key genes, we utilized it as a real-time reporter and developed a high-throughput screening method based on fluorescence, employing fluorescence-activated cell sorting (FACS) and Next Generation Sequencing. However, while this process can help track the progress of mutagenesis, it doesn't provide information on the actual quality of the mutations.

For other metabolic genes we want to optimize, we use different selective conditions in LB plates to identify "gain-of-function" mutations. Colonies that are GFP-negative but survive selection are processed using our fluorescent-based high-throughput screening method, and specific sequencing results are identified as "gain-of-function" mutations. We further confirmed the functional mutations by feeding *Escherichia coli* with mutated proteins to nematodes who grew in environments intended to benefit nematodes with the desired “gain of function mutations.”

These beneficial mutations can be recorded in our Dictionary System. As we continue to accumulate knowledge on mutation-function relationships, this system will become increasingly useful for guiding gene therapy in the future!!

![](https://static.igem.wiki/teams/5191/homepage/home-7-1.png)

Figure8. Project Overview

## 8. Cycle 8-Potential Future Application in the medical field

### (1)Design

The use of probiotics has shown positive effects in improving the health of *C. elegans* (nematodes), leading to the question: could this be applied to humans as well? If the benefits observed in these organisms can be replicated in humans, it opens up the possibility of developing a product that specifically targets human health. We could design a probiotic-based solution tailored to enhance well-being by focusing on mechanisms that promote healthy gut flora or other beneficial biological processes in humans, building on the promising results seen in model organisms like nematodes.

### (2)Build

We introduce this mutated plasmid into Lactobacillus to create a Lactobacillus probiotic urachus.

![](https://static.igem.wiki/teams/5191/description/wechatimg290.jpg)

Figure9. Potential Applications of Black Box System

### (3)Test & Learn

While we have not yet tested this capsule, it has already been synthesised in collaboration with our partner company. This capsule is designed to enable the gut microbiota to produce more spermidine. We expect to see an effect in humans, but further experiments are needed to confirm the results. Stay tuned for updates!

{% endblock %}

{% extends "layout.html" %}
{% block title %}Test{% endblock %}
{% block page\_content %}

# Experiments

During the experimental phase, we used 7 protocols to achieve our experimental goals.

1. [**Agarose Gel electrophoresis**](https://static.igem.wiki/teams/5191/dry-lab/kegg-metabolic-pathway.pdf)

2. [**Transformation**](https://static.igem.wiki/teams/5191/dry-lab/kegg-metabolic-pathway.pdf)

3. [**Monoclonal & Enlarge incubation**](https://static.igem.wiki/teams/5191/dry-lab/kegg-metabolic-pathway.pdf)

4. [**PCR**](https://static.igem.wiki/teams/5191/dry-lab/kegg-metabolic-pathway.pdf)

5. [**Sanger Sequencing**](https://static.igem.wiki/teams/5191/dry-lab/kegg-metabolic-pathway.pdf)

6. [**Flow Cytometry**](https://static.igem.wiki/teams/5191/dry-lab/kegg-metabolic-pathway.pdf)

7. [**Plasmid Extraction**](https://static.igem.wiki/teams/5191/dry-lab/kegg-metabolic-pathway.pdf)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block page\_content %}

# Results

![](https://static.igem.wiki/teams/5191/result/result-head.jpg)

## 1. The schematic design of semi-random mutagenesis using cytidine base editor-T7 RNA polymerase (CBE-RNAP)

For our second-generation black box, we first predicted the structure of the fusion proteins rAPOBEC1-n-Mag-N-terminal T7-RNA polymerase (RNA, 1-179) and p-Mag-C-terminal T7-RNA polymerase (RNA, 180+). As shown in Figure 1A, we predicted that the two fusion proteins will bind together to form a complex under blue light and bind to the T7 promoter, inducing random C to T mutations in the specific region from T7 promoter to T7 terminator. When the blue light illumination stops, the two fusion proteins will separate again in the dark environment, causing the mutation to cease. Therefore, we further predicted that compared to the unmutated sample, the Green Fluorescent Protein (GFP) gene in the second-generation black box plasmid will ultimately present two results after random mutation: negative mutations that reduce the GFP fluorescence and positive mutations that enhance the GFP fluorescence (as shown in Figure 1B).

![](https://static.igem.wiki/teams/5191/result/result1.jpg)

Figure1. Schematics of blue-light inducible base editor with split RNA polymerase and predicted GFP fluorescence results. A, Structure prediction of fusion proteins rAPOBEC1-n-Mag-N-terminal T7-RNA polymerase (RNA, 1-179) and p-Mag-C-terminal T7-RNA polymerase (RNA, 180+). B, Two predicted results of GFP fluorescence in mutated samples.

## 2. The validation in semi-random mutagenesis on GFP gene by CBE-RNAP

To verify the above theoretical predictions, we constructed the plasmid (as shown in Figure 2A) of the second-generation black box (CBE-Mag-RNAP). Subsequently, we used agarose gel electrophoresis to verify the successful construction of the plasmid. As shown in Figure 2B, the band positions shown in the electrophoresis results were consistent with the predicted restriction enzyme sites in the plasmid, indicating that the second-generation plasmid construction was successful. We then transformed the plasmid into *Escherichia coli* (*E. coli*) BL21 and set up a control group (where the addition of water will not activate the second-generation black box) and an experimental group (where the addition of IPTG can activate the second-generation black box), and placed them under the blue light. Fluorescence microscopy (as shown in Figure 2C) revealed that compared to the control group, the experimental group showed a significant increase in fluorescence intensity, indicating enhanced GFP expression in the experimental group. We further quantified the fluorescence intensity of the two groups, as shown in Figure 2D. Compared to the control group, where the fluorescence intensity was generally concentrated around 30, the experimental group had some points significantly higher than 30 and some significantly lower than 30, indicating that the second-generation black box successfully induced both positive and negative mutations in GFP. We further sequenced the plasmids from the control and experimental groups after the mutations, and the results (as shown in Figure 2E) revealed that the experimental group had randomly mutated the C in the control group to T, confirming the successful construction of the second-generation black box plasmid.

![](https://static.igem.wiki/teams/5191/result/result2.jpg)

Figure2. Verification of the second-generation black box’s success. A, Plasmid of the second-generation black box (CBE-Mag-RNAP). B, Agarose gel electrophoresis result of the plasmid of the second-generation black box (CBE-Mag-RNAP). C, Fluorescence microscopy results of the experimental group and the control group. D, The GFP fluorescence intensity of the experimental group and the control group. E, Sequencing results of the experimental group and the control group.

## 3. High throughput screen for "gain-of-function" mutations on GFP by CBE-RNAP

At the same time, in order to compare the differences in mutation frequencies caused by the second-generation black box (CBE-Mag-RNAP) and the first-generation black box (CBE-RNAP), we transformed the two plasmids separately into *E. coli* BL21, and added continuously expressed mCherry as an internal control (as shown in Figure 3A). We set up a control group (where the addition of water prevented the black box from activating mutations), a second-generation black box group (where IPTG was added and placed under blue light to activate mutations), and a first-generation black box group (where IPTG was added to activate mutations). We then performed fluorescence-activated cell sorting (FACS) on the three groups based on the fluorescence intensities of GFP and mCherry, and sorted out the cell populations with increased and decreased fluorescence intensities (as shown in Figure 3B). The results (as shown in Figure 3C) revealed that the proportion of cells with increased fluorescence intensity in the second-generation black box group (14.78%) was significantly higher than in the first-generation black box group (3.42%) and the control group (11.95%). This indicates that the second-generation black box significantly enhanced the mutation frequency compared to the first-generation black box, and the majority of the mutations were C-to-T substitutions (as shown in Figure 3D). We inferred that the significant mutations caused by the second-generation black box may have substantially altered the aggregation ability of the GFP, leading to the changes in fluorescence intensity (as shown in Figure 3E). Here, we concluded that GFP with mutations T10I, S29F, A155V are the top ranked which may accelerate the combination and assembly between GFP proteins.

![](https://static.igem.wiki/teams/5191/result/result3-1.jpg)

Figure3. Differences in mutation frequencies caused by the second-generation black box (CBE-Mag-RNAP) and the first-generation black box (CBE-RNAP). A, Plasmids of the second-generation black box and the first-generation black box. B, Fluorescence-activated cell sorting (FACS) based on the fluorescence intensities of GFP and mCherry. C, Proportion of cells with increased and decreased fluorescence intensity in the three groups. D, Statistical analysis of the proportions of different types of random mutations. E, Schematics of the mutated GFP T10I, S29F, A155V . Gray part refers to the wild type while green part refers to the mutant.

## 4. High throughput screen for "gain-of-function" mutations on metabolic enzymes in *E. coli* by CBE-RNAP

After confirming that the second-generation black box could successfully enhance the frequency of random mutations, we applied the second-generation black box to improve the pentose phosphate pathway (PPP) and polyamine synthesis pathway in *E. coli*. By randomly mutating and screening the key enzymes in these metabolic pathways (as shown in Figure 4A), we hypothesized that we could increase the production of the anti-aging compound spermidine and the production of antioxidant NADPH in *E. coli*. Therefore, we constructed plasmids for spermidine synthesis (spermidine synthase - S-adenosylmethionine decarboxylase - agmatinase) and NADPH synthesis (6-phosphogluconolactonase - transaldolase - transketolase), and co-transformed them with the second-generation black box plasmid into *E. coli* BL21 (as shown in Figure 4B). We set up a control group (where the addition of water will not activate the black box) and a second-generation black box group (where the black box was activated by IPTG and placed under blue light). After the second-generation black box randomly mutated the spermidine synthesis plasmid and NADPH synthesis plasmid, we performed sequencing analysis on the plasmids from the control and experimental groups to verify the success of the mutations. The results (as shown in Figure 4C) revealed that, compared to the control group, the experimental group exhibited a clear C-to-T substitution, indicating the successful mutation of the two plasmids by the second-generation black box, with the majority being C-to-T mutations (as shown in Figure 4D). We then screened the mutated *E. coli* and tested them on *Caenorhabditis elegans* (*C. elegans*) in LB culture medium. Since spermidine and can delay aging and NADPH can scavenge reactive oxygen species (ROS), we recorded the survival times of the *C. elegans*, which were cultured on the LB culture mediums with the mutated *E. coli* (experimental group) and the unmutated *E. coli* (control group). The results (as shown in Figure 4E) demonstrated that the average lifespan of the *C. elegans*. in the experimental group was significantly longer than the control group, indicating that the second-generation black box successfully enhanced the production of spermidine and NADPH in *E. coli*, ultimately leading to the extended lifespan of *C. elegans*.

![](https://static.igem.wiki/teams/5191/result/result4.jpg)

Figure4. Application of the second-generation black box on the pentose phosphate pathway (PPP) and polyamine synthesis pathway in *E. coli*. A, Pentose phosphate pathway (PPP) and polyamine synthesis pathway *E. coli*. B, Plasmids for spermidine synthesis (spermidine synthase - S-adenosylmethionine decarboxylase - agmatinase) and NADPH synthesis (6-phosphogluconolactonase - transaldolase - transketolase). C, Sequencing results of plasmids from the control and experimental groups. D, Statistical analysis of the proportions of different types of random mutations. E, Statistical analysis of lifespans of the control and experimental groups.

## 5. Protein structure prediction for "gain-of-function" mutations on metabolic enzymes in *E. coli* by CBE-RNAP

To further explain the reasons for mutations that can provide evidence for the production of more NADPH and spermidine, we performed protein structure analysis on each mutation detected through next-generation sequencing. As shown in Figure 5A, the mutations A66T and A26T in spermidine synthase may enhance the efficiency of converting putrescine to spermidine. Considering systems biology, the A66T and A26T mutations in spermidine synthase, along with the G12D and T60I mutations in S-adenosylmethionine decarboxylase, can collectively support the entire metabolic system in generating more spermidine. More importantly, the R244C mutation in 6-phosphogluconolactonase, along with the A31V, A159V, and A314V mutations in transaldolase, as well as the E85K and H47Y mutations in transketolase, represent ideal mutations that could work synergistically to improve the metabolic system's capacity to generate more NADPH (as shown in Figures 5B-E). Here, we aim to propose a systematic hypothesis linking bio-brick mutations to final functions. Much like playing the piano, where not every key needs to be struck forcefully, the collaboration of these mutations may result in more harmonious and efficient outcomes.

![](https://static.igem.wiki/teams/5191/result/result5.jpg)

Figure5. Structure based analysis of mutations in improving NADPH and Spermine synthesis.

## 6. High throughput screen for "gain-of-function" mutations on degradation of Nicotine in *E. coli* by CBE-RNAP

Considering the toxicity of nicotine and the fact that nicotinate is the substrate for generating nicotine, we constructed a bio-brick containing three enzymes: nicotinate dehydrogenase, 6-hydroxynicotinate reductase, and enamidase (Figure 6A). We applied the second-generation black box approach to enhance the degradation of nicotinate in *E. coli*. By randomly mutating and screening key enzymes in these metabolic pathways (as shown in Figure 6B), we hypothesized that we could increase the degradation of nicotinate on an LB plate. We then performed Sanger sequencing on nicotinate dehydrogenase, resulting in a successful mutation, as shown in Figure 6C. However, Sanger sequencing did not reveal mutations with low frequencies. We subsequently conducted next-generation sequencing on bacteria with multiple mutations. As shown in Figure 6D, the next-generation sequencing results indicated a significant increase in C to T mutations in the nicotinate dehydrogenase gene. To further confirm that these mutations in *E. coli* can protect *C. elegans* from high concentrations of nicotinate, we performed a lifespan assay, demonstrating that the mutant *E. coli* significantly protected *C. elegans* (Figure 6E).

![](https://static.igem.wiki/teams/5191/result/result6-1.jpg)

Figure6. Application of the second-generation black box on the nicotinate degradation pathway in *E. coli*. A, Nicotinate degradation pathway in *E. coli*. B, Schematic processing. C, Sequencing results of plasmids from the control and experimental groups. D, Statistical analysis of the proportions of different types of random mutations. E, Statistical analysis of lifespans of the control and experimental groups.

## 7. High throughput screen for "gain-of-function" mutations on degradation of Nicotine in *E. coli* by CBE-RNAP

To further explain the top mutations that improve nicotinate degradation, we performed protein structure analysis using AlphaFold3. As shown in Figure 7, the P122S mutation in nicotinate dehydrogenase, along with the A32V mutation in hydroxynicotinate reductase, are beneficial compared to the non-mutated group, indicating functional mutations that enhance the system's ability to degrade nicotinate.

![](https://static.igem.wiki/teams/5191/result/result7.jpg)

Figure7. Structure based analysis of mutations in improving Nicotine degradation.

{% endblock %}