iGEM 2024 — Team hubu-wuchang-china

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![](https://static.igem.wiki/teams/5167/headpic/engineering.png)

# Notebook

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# EXPERIMENT

| Year | Month | Wet-Lab Activity |
| --- | --- | --- |
| 2022 | Jan | Determine project |
| 2022 | Feb | Conducting induced polyploid experiment |
| 2022 | Mar | Screening and identification of tetraploid seedlings |
| 2022 | Apr | Planting the first generation of rice WT-4x |
| 2022 | May | Determine the use of CRISPR/Cas9 gene editing technology to mutate the OsGluB1 gene |
| 2022 | Jun | Constructing CRISPR/Cas9 gene editing vector targeting OsGluB1 |
| 2022 | Sept | Perform WT-4x fluorescence quantitative analysis |
| 2022 | Oct | Harvest WT-4x rice and observe the thickness of the endosperm layer and the structures of PSV and PB-II using scanning electron microscopy, Send for testing the total protein, gluten, soluble protein, globulin, and 17 types of amino acids in rice. |
| 2022 | Oct | Transfer the gene editing vector targeting OsGluB1 into E.coli DH5α for propagation |
| 2022 | Nov | Extract gene expression vectors and transform them into Agrobacterium. Cultivate WT-4x rice tissue and transform it during the callus stage |
| 2022 | Dec | Screening and identification of OsGluB1 mutation positive plants |
| 2023 | Apr | Planting second-generation OsGluB1-4x |
| 2023 | Sept | Perform OsGluB1-4x fluorescence quantitative analysis |
| 2023 | Nov | Harvest OsGluB1-4x rice and send it for testing to determine the total protein, gluten, soluble protein, globulin, and 17 types of amino acids in the rice. |
| 2023 | Dec | Constructing a dual target CRISPR/Cas9 gene editing vector targeting OsGluB1 and OsGlb |
| 2023 | Jan | Transfer gene editing vector into E.coli DH5α for propagation |
| 2023 | Feb | Extract gene expression vectors and transform them into Agrobacterium. Cultivate WT-4x rice tissue and transform it during the callus stage |
| 2023 | Mar | Screening and identification of OsGluB1 and OsGlb mutation positive plants |
| 2023 | Apr | Planting third-generation OsGluB1/OsGlb-4x |
| 2023 | Sept | Perform fluorescence quantitative analysis of OsGluB1/OsGlb-4x and harvest OsGluB1/OsGlb-4x rice, Send for testing the total protein, gluten, soluble protein, globulin, and 17 types of amino acids in rice. |
| 2024 | Jan | Transfer gene editing vector into E.coli DH5α for propagation |
| 2024 | Feb | Extract gene expression vectors and transform them into Agrobacterium. Cultivate WT-4x rice tissue and transform it during the callus stage |
| 2024 | Mar | Screening and identification of OsGluB1 and OsGlb mutation positive plants |
| 2024 | Apr | Planting third-generation OsGluB1/OsGlb-4x |
| 2024 | Sept | Perform fluorescence quantitative analysis of OsGluB1/OsGlb-4x and harvest OsGluB1/OsGlb-4x rice, Send for testing the total protein, gluten, soluble protein, globulin, and 17 types of amino acids in rice. |

| Year | Month | Dry-Lab Activity |
| --- | --- | --- |
| 2022 | Jan-Mar | Using the protein and amino acid content data of diploid and tetraploid rice studied by the research group, linear regression is performed to predict the extent of protein and amino acid content |
| 2022 | Apr-May | Using the research of other rice varieties mutants by the research group and the data of OsGluB1 mutants in the literature, the original linear regression was used as the [WT-2x, WT-4x] segment, and [WT-4x, OsGluB1-4x] was introduced |
| 2022 | Jun-Sept | Structural prediction of OsGluB1 mutant glutelin and wild-type gluten using designed target sequences |
| 2022 | Oct | Feedback protein and amino acid content data into a linear regression function for optimizing the [WT-2x, WT-4x] segment functions |
| 2022 | Sept-Nov | Comparative analysis of the structural characteristics of glutelin before and after mutation, and molecular docking simulation of the hydrolysis of gluten before and after mutation |
| 2023 | Nov | Feedback protein and amino acid content data into a linear regression function for optimizing the segment functions of [WT-4x,OsGluB1-4x] |
| 2023 | Dec | Structural prediction of OsGlb mutant globulin and wild-type globulin using designed target sequences |
| 2024 | Jan-Mar | Using the research conducted by our research group on mutants of other rice varieties and the data of OsGluB1 mutants in literature, we introduce the [OsGluB1-4x, OsGluB1/OsGlb-4x] segment |
| 2024 | Feb-Apr | Comparative analysis of the structural characteristics of globulin before and after mutation, and molecular docking simulation of allergenic changes in globulin |
| 2024 | Sept | Feedback protein and amino acid content data into a linear regression function for optimization of the [OsGluB1-4x,OsGluB1/OsGlb-4x] segment functions |

# HUMAN PRACTICES

## 2022

**February:**
Carrying out transgenic public opinion analysis and abandon the strategy of directly reprogramming free lysine metabolic pathway through metabolic engineering.
**May:**
 Carry out a questionnaire survey on domestic and foreign social platforms on the necessity of increasing the lysine content of rice. Instead, adjust the storage protein components through CRISPR/Cas9 gene editing technology, and redefine the technology module to increase the lysine content in the experiment.

## 2023

**November:**
 Carry out statistical analysis of nutritional composition data of rice and rice paste products on major domestic e-commerce platforms and Sam's Club online flagship stores.
**December:**
Student leader Ziying Qiu and PI Professor He Yuchi attended the 2023 23rd Jingchu grain and oil boutique exhibition and trade fair.

## 2024

**February:**
The team carried out large scale offline market research and science popularization in Hubei, Hunan, Guangdong, Guizhou and Shandong.

---

**March 30:**
Yangyu He, the leader of the Dry-team, participated in the Hubei Rural Planning Research Association on behalf of the team.

---

**April 4:**
The team went to Wuhan University to participate in the iGEM exchange conference.
**April 27:**
 Organize and participate in the Hubei Synthetic Biology Open Conference with the other three iGEM teams of Hubei University.

---

**May:**
The team participated in two speech contests of "Reading at Lake University, Striving for a New Journey" and Baisheng Forum and won two bronzes.
**May 18:**
Laboratory Open Day

---

**June 1:**
Biological Art Exhibition
**June 4:**
 The team participated in the 2024 Wuhan Seed Industry Expo and held a synthetic biology science popularization month carnival.
**July 11-July 14:**
The team went to Suzhou to participate in the CCiC exchange conference at the beginning of the month.
**July 20-July 30:**
 The team went to Hubei Wuhu Rice Science and Technology Academy for support education

---

**August 8 - August 10:**
 The team went to Shenzhen to participate in the final of the 2024 Syn-Bio Challenges 3.0
**August 17:**
Shenzhen University, Tianjin University, University of Geosciences, Sichuan University online exchange HP and security

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![](https://static.igem.wiki/teams/5167/headpic/engineering.png)

# Engineering

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# • Menu

* [• 1st group models](#section1)
* [• 2nd group model](#section2)
* [• 3rd group model](#section3)
* [→ Primary structure prediction](#3.1)
* [→ Changes in Nutritional Value](#3.2)
* [→ Taste changes](#3.3)
* [→ Changes in food safety](#3.4)
* [→ Third level structure prediction](#3.5)
* [• 4th group of models](#section4)

# • 1st group models:

## a linear regression model with polyploidization and gene editing iterations

The first step of our experiment design is to induce genome duplication from diploid rice to tetraploid. We conducted linear regression analysis using the existing data on the difference in protein content between diploid and tetraploid rice in our research group to predict the potential of this experimental design.

![](https://static.igem.wiki/teams/5167/engineer/engineer1.png)
![](https://static.igem.wiki/teams/5167/engineer/engineer2.png)

![](https://static.igem.wiki/teams/5167/engineer/engineer3.png)
![](https://static.igem.wiki/teams/5167/engineer/engineer4.png)

![](https://static.igem.wiki/teams/5167/engineer/engineer5.png)

Figure 1 Linear regression analysis of changes and trends in total protein, component proteins, and lysine content after polyploidization

The prediction results showed that total protein, glutelin and lysine increased significantly, gliadin increased significantly, and globulin did not increase significantly.Among them, the growth of total protein, lysine and gliadin is considerable, but glutelin is contrary to our expectations.Although there are flaws in the experimental design, overall it is considerable.
However, the problem that polyploidization does not selectively lead to the increase of glutelin content due to the increase of metabolite content remains to be solved. The second step of our experiment is to knock out the glutelin coding gene osglub1 in tetraploid rice using crispr/cas9 gene editing technology.(See  [Wet-Lab-Design](https://2024.igem.wiki/hubu-wuchang-china/design%20)  for details) We also conducted linear regression analysis on the existing data about the protein content of the GluB1 mutant rice in our research group to predict the potential of this experimental design.

![](https://static.igem.wiki/teams/5167/engineer/engineer6.png)
![](https://static.igem.wiki/teams/5167/engineer/engineer7.png)

![](https://static.igem.wiki/teams/5167/engineer/engineer8.png)
![](https://static.igem.wiki/teams/5167/engineer/engineer9.png)

![](https://static.igem.wiki/teams/5167/engineer/engineer10.png)

Figure 2 Linear regression analysis of changes and trends in total protein, component proteins, and lysine content after OsGluB1 gene mutation

Unfortunately, the model predicts that this experiment will lead to poor results. The increase in total protein and prolamine content was not significant, while the lysine content unexpectedly decreased. This reminds us to carefully analyze the failures of this experimental design and correct them. (Defect details can be found in  [Wet-Lab-Design](https://2024.igem.wiki/hubu-wuchang-china/design%20) )
Inspired by further literature review and suggestions from stakeholders (for details, see  [human practices - Integrated Human Practices - Market Research (offline & face to face)](https://2024.igem.wiki/hubu-wuchang-china/human-practices) ), we will pay attention to globin. The experimental design of this step is modified to use crispr/cas9 gene editing technology to knock out glutelin coding gene osglub1 and globin coding gene osglb in tetraploid rice.(See  [Wet-Lab-Design](https://2024.igem.wiki/hubu-wuchang-china/design%20)  for details) . We also conducted linear regression analysis on the protein content data of rice with OsGluB1 and OsGlb dual gene mutations that our research group already had, and predicted the potential of the improved experimental design in this step.

![](https://static.igem.wiki/teams/5167/change1.png)
![](https://static.igem.wiki/teams/5167/change2.png)

![](https://static.igem.wiki/teams/5167/change3.png)
![](https://static.igem.wiki/teams/5167/engineer/engineer15.png)

![](https://static.igem.wiki/teams/5167/change4.png)

Figure 3 Linear regression analysis of changes and trends. Total protein, component proteins, and lysine content after dual mutation of OsGluB1 and OsGlb genes

Excitingly, the model prediction results of the improved experimental design are highly consistent with our goals. The content of total protein, prolamine, and lysine significantly increased, while the content of glutelin and globulin significantly decreased.
Therefore, we have determined the experimental design:
STEP1. Inducing the genome doubling of diploid rice to polyploidize it into tetraploid
STEP2. Knockout OsGluB1 and OsGlb, using CRISPR/Cas9 gene editing technology

# • 2nd group model:

## Linear regression model for predicting and comparing the effects of different sequences of polyploidization and gene editing

We want to get rice with improved total protein, gliadin and lysine content, basically unchanged glutelin content, and almost removed globulin nutrition, taste quality and safety.（Defect details can be found in  [Project-Description](https://2024.igem.wiki/hubu-wuchang-china/description%20) ）We have preliminarily determined that the technical route of polyploidization followed by gene editing in the project, which is aimed at improving the nutritional quality of rice while reducing the adverse effects on its agronomic traits. However, when the project was established, some scholars questioned the difficulty of performing homozygous gene editing on tetraploids, wondering why we did not choose to first perform gene editing in a diploid background, screening and identifying the homozygotes, and then performing polyploidization lastly, which would greatly reduce the difficulty. We consulted our second PI, Dr. Lu Gan, who told us that as a higher eukaryotic organism, rice has a much higher complexity in genome structure and gene regulation than microorganisms. We cannot easily give up the advantage of increasing the biomass of tetraploid chassis when we are unsure whether carry out the step of polyploidization or gene editing first achieves the most significant protein nutrition improvement effect. In order to improve breeding efficiency, we organized and conducted linear regression analysis on the research data of rice polyploidization and CRISPR/Cas9 gene editing to improve protein nutritional quality in our research group. We predicted the effect of polyploidization followed by gene editing or gene editing followed by polyploidization, compared and analyzed which one better met our expectations, and evaluated the necessity of using the sequence of gene editing followed by polyploidization to simplify the experiment.

### Figure 4: Changes in Total Protein Content (Prediction)

![](https://static.igem.wiki/teams/5167/engineer/engineer17.png)

![](https://static.igem.wiki/teams/5167/engineer/engineer18.png)

Polyploidization+gene editing Gene editing+polyploidization

As shown in the figure, the increase in total protein content of rice obtained by sequential "gene editing+polyploidization" is greater than that obtained by "gene editing+polyploidization".

### Figure 5 glutelin content change (prediction)

![](https://static.igem.wiki/teams/5167/engineer/engineer19.png)

![](https://static.igem.wiki/teams/5167/engineer/engineer20.png)

Polyploidization+gene editing Gene editing+polyploidization

As shown in the figure, the sequence "gene editing + polyploidization" restored the glutelin content to that of wild-type diploid rice (that is, traditional rice), while the sequence "gene editing + polyploidization" increased the glutelin content instead.

### Figure 6 Changes in prolamine content (predicted)

![](https://static.igem.wiki/teams/5167/engineer/engineer21.png)

![](https://static.igem.wiki/teams/5167/engineer/engineer22.png)

Polyploidization+gene editing Gene editing+polyploidization

As shown in the figure, the increase in prolamine content obtained by sequential "gene editing+polyploidization" is greater than that obtained by "gene editing+polyploidization".

### Figure 7 Changes in Globulin Content (Prediction)

![](https://static.igem.wiki/teams/5167/engineer/engineer23.png)

![](https://static.igem.wiki/teams/5167/engineer/engineer24.png)

Polyploidization+gene editing Gene editing+polyploidization

As shown in the figure, the sequence of "gene editing+polyploidization" successfully removed globulin, while "gene editing+polyploidization" was ultimately ineffective in removing globulin.

### Figure 8 Changes in Lysine Content (Prediction)

![](https://static.igem.wiki/teams/5167/engineer/engineer25.png)

![](https://static.igem.wiki/teams/5167/engineer/engineer26.png)

Polyploidization+gene editing Gene editing+polyploidization

As shown in the figure, the lysine content of rice obtained by the sequence of "gene editing+polyploidization" and "gene editing+polyploidization" is the same.
To sum up, the sequence "gene editing + polyploidization" is better than "gene editing + polyploidization" in improving the nutritional quality of rice (comprehensively considering the changes of  [total protein](#total) and  [lysine content](#total) ), eating quality (considering the [gliadin content](#prolamine)), food safety (comprehensively considering the changes of [glutelin conten](#glu) and  [globulin content](#glo) ).
Therefore, it is not advisable to perform gene editing before polyploidization in order to simplify the experiment, although it is indeed tempting in reducing technical difficulty.

# • 3rd group model:

## Protein structure (primary structure+tertiary structure) before and after mutation of glutelin and globulin

When we conducted mutation identification on OsGluB1 and OsGlb, we found that the CRSIPR/Cas9 gene editing caused frameshift mutations in them (see  [Wet-Lab-Result](https://2024.igem.wiki/hubu-wuchang-china/result) for details), which raised our concerns. Due to frameshift mutations, protein translation reading frames are altered, leading to codon rearrangements after the mutation site and significant changes in amino acid sequence and structure. Although globulin are hardly synthesized and the synthesis of glutelin is limited after mutation, in this case, on one hand we must take measures to predict the protein structure and function after mutation to ensure that it will not produce toxic proteins; on the other hand, we need to predict the function of the mutant protein is better than the wild-type one .Therefore, we predicted the primary and tertiary structures of glutelin and globulin before and after mutation, and analyzed their properties and functions.

## Primary structure prediction

The primary structure prediction was completed on the  [ProtParam platform.](http://web.expasy.org/protparam%20) The wild-type gene information glutelin and globulin are all from  [NCBI database.](https://www.ncbi.nlm.nih.gov/)

|  | GlutelinB1 | | | | | Globulin | |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  | WT | WT | Mutant-1(50%) | Mutant-2(50%) | Mutant-2(50%) | WT | Mutant |
| Number of amino acids | 499 | 499 | 583 | 583 | 583 | 186 | 185 |
| Molecular weight | 56550.58 | 56550.58 | 67178.78 | 67136.70 | 67136.70 | 21054.74 | 20346.4 |
| PI | 9.26 | 9.26 | 9.66 | 9.66 | 9.66 | 7.48 | 10.22 |
| Total number of negatively charged residues | 39 | 39 | 38 | 38 | 38 | 21 | 20 |
| Total number of positively charged residues | 50 | 50 | 70 | 70 | 70 | 22 | 29 |
| Instability index | 52.11 | 52.11 | 58.90 | 58.42 | 58.42 | 69.24 | 72.62 |
| Aliphatic index | 76.19 | 76.19 | 85.95 | 85.45 | 85.45 | 52.53 | 75.46 |
| Grand average of hydropathicity | -0.495 | -0.495 | -0.226 | -0.234 | -0.234 | -0.629 | -0.317 |
| Amino acids composition | | | | | | | |
|  | Glutelin-WT | Glutelin-Mutant1 | Glutelin-Mutant2 | Globulin-WT | Globulin-Mutant |
|  | ![](https://static.igem.wiki/teams/5167/engineer/engineer27.png) | ![](https://static.igem.wiki/teams/5167/engineer/engineer28.png) | ![](https://static.igem.wiki/teams/5167/engineer/engineer29.png) | ![](https://static.igem.wiki/teams/5167/engineer/engineer30.png) | ![](https://static.igem.wiki/teams/5167/engineer/engineer31.png) |  |  |

| Rice | GlutelinB1 - Mutation | Globulin - Mutation |
| --- | --- | --- |
| Biological Value(BV) | ↑ | ↓ |
| taste | Unable to determine | Unable to determine |
| digestibility | Unable to determine | Unable to determine |
| allergenicity | Unable to determine | Unable to determine |

## Changes in Nutritional Value

1、The biological value(BV) of rice is an indicator of its nutritional value in nutritional science, which reflects the efficiency of amino acids in rice being absorbed and utilized by the human body. WHO/FAO has proposed recommended amino acid ratios based on human nutritional needs to facilitate the measurement of biological value, with foods closer to the recommended ratio having higher biological value. Because amino acids are more balanced, digestion and absorption rates, metabolic assimilation rates, and nutrient utilization rates are higher. Due to the low content of globulin in the total protein of rice endosperm, especially after mutation, its amino acid ratio deviates from the WHO recommendation and will not have a significant impact on the biological value of rice.
2、After the mutation of glutelin and globulin, the hydrophilicity of the primary structure (peptide chain) becomes worse, while the interior of the active center pocket of the corresponding digestive enzymes (trypsin and chymotrypsin) is hydrophobic, which is conducive to their binding with the enzyme.Trypsin recognizes and cleaves Lys-Arg, Phe-Tyr, and Trp-Tyr.
3、The protein molecules are larger after mutations of glutelin and globulin.Pepsin, trypsin and chymotrypsin, the three key digestive enzymes of glutelin and globulin, have positive charges in the active center pocket.The Pi of glutelinB1 before and after mutation is greater than the pH value of gastric juice and small intestinal juice, resulting in its positive charge in the stomach and small intestine, especially in the stomach.The wild-type Pi of globulin fluctuates within the pH range of small intestinal fluid, but is higher than the pH value of gastric acid. After mutation, the Pi is higher than the pH values of gastric acid and small intestinal fluid. The efficiency of digestive enzymes in decomposing these two proteins decreases, which may be unfriendly to whom has digestive system diseases, especially gastric disease patients. Due to the fact that the protein with the highest lysine content and the most balanced amino acid ratio in rice protein is not one of these two types, the loss of digestive performance and nutritional value caused by their mutation can be compensated for by other proteins.

## Taste changes

1、According to the primary structure prediction, both glutelin and globulin hide hydrophobic residues in the interior of the protein and expose hydrophilic residues on the surface of the protein through peptide chain folding. The following tertiary structure prediction will verify this.During the cooking process, they gradually denature from hydrophilic proteins to hydrophobic peptide chains.
2、The solubility and emulsifying properties of rice protein during the cooking stage have a comprehensive impact on the taste of rice. During the cooking process of rice, in the first few minutes of low-temperature soaking, the protein has good hydrophilicity and fully absorbs water, transitioning from a dry state to a wet state; In the middle heating stage, the rice is nearly cooked, the protein structure becomes loose, hydrophobic residues are gradually exposed, and protein emulsification begins to occur, and protein and fat form a stable lotion; In the later stage of rice water harvesting, the hydrophobic residues of protein are almost completely exposed, and the emulsification of rice protein reaches the peak to form a stable lotion system. Adequate protein absorption and emulsification are necessary to make the taste of rice softer and more delicate. Therefore, from a structural perspective, it is necessary to meet the requirement of having a high content of hydrophobic residues in the primary structure and hiding them as much as possible inside the protein in the tertiary structure, in order to ensure sufficient emulsification without interfering with the early water absorption process. The primary structure of glutelin and globulin has met the above conditions, but whether the tertiary structure meets the requirements needs further prediction through modeling.

## Changes in food safety

The allergenicity of rice containing glutelin and globulin is related to the primary and tertiary structure of protein.Antigen epitopes include linear epitopes and conformational dependent epitopes, and protein folding and denaturation can affect antigen epitopes. Therefore, the allergenic changes of glutelin and globulin can be judged by comparative analysis of the primary and tertiary structure epitopes of wild-type and mutant glutenin.The expected result is a reduction in the number of mutant antigen epitopes, with the proportion of linear epitopes remaining as high as possible and the proportion of conformational dependent epitopes as small as possible. This can reduce the difficulty of preventing and controlling allergy risks and improve food safety from the perspectives of detection and regulation.

## Third level structure prediction

This round of modeling is to supplement the analysis of the predicted results of the primary structure and answer the following questions.
1、Is the hydrophobic residue of glutelin B1 and globulin more hidden inside the protein after mutation (proportion)?
Using Alphafold3 to predict protein tertiary structure and perform confidence scoring

![](https://static.igem.wiki/teams/5167/engineer/engineer32.png)

Figure 9 prediction of tertiary structure of glutelin B1 mutant (high confidence)

![](https://static.igem.wiki/teams/5167/engineer/engineer33.png)

Figure 10 Prediction of Tertiary Structure of Globulin Mutations (High Confidence)

We used the protein tertiary structure visualization software Pymol to process the structures of wild-type and two mutant types of gluten B1. For the convenience of differentiation, we unified the hydrophilic residues as pink and stained the hydrophobic residues Ala, Val, Leu, Ile, Phe with Cyan series colors. And open the sticks of hydrophobic residues in surface display mode.

![](https://static.igem.wiki/teams/5167/engineer/engineer34.png)

Figure 11 hydrophilic / hydrophobic changes of glutelinB1 before and after mutation

![](https://static.igem.wiki/teams/5167/engineer/engineer35.png)

Figure 12 Changes in hydrophilicity/hydrophobicity before and after globulin mutation

As shown in the figure, hydrophobic residues in their respective mutants (Figure 11B, C, figure 12b) are significantly more clustered in the interior of the protein than glutelin B1 and globulin wild-type (Figure 11, 12a). Therefore, it verifies our speculation on the improvement of food quality after gluten B1 mutation in the primary structure analysis.

# • 4th group of models:

## molecular docking of glutelin, globulin wild-type and mutant with human immunoglobulin E

This round of modeling is to supplement the analysis of the predicted results of the primary structure and answer the following questions. (See the table below)
1、Changes in the number of antigenic epitopes after mutations in glutelin B1 and globulin?
2、Does the proportion of linear epitopes increase after mutations in glutelin B1 and globulin?

![](https://static.igem.wiki/teams/5167/engineer/engineer36.png)

Figure 13 modeling results for molecular docking between wild-type and mutant glutelin B1 and Fc region of human immunoglobulin E

![](https://static.igem.wiki/teams/5167/engineer/engineer37.png)

Figure 14 Molecular docking modeling results of wild-type and mutant immunoglobulin E with human immunoglobulin E in the fc region

|  | GlutelinB1-WT | GlutelinB1-M1 | GlutelinB1-M2 | Globulin-WT | Globulin-M |
| --- | --- | --- | --- | --- | --- |
| Number of ionic bonds | 9 | 7 | 7 | 7 | 0 |
| Number of hydrogen bonds | 10 | 11 | 11 | 20 | 9 |
| Number of identification bits | 8 | 8 | 8 | 14 | 9 |
| Total number of recognition site residues | 8 | 9 | 9 | 16 | 9 |
| The adjacent residues make up the recognition site frequency | 25% | 62.5% | 62.5% | 25% | 0 |
| analysis | The binding of glutelin B1 to IgE decreased, but not significantly | | | The binding of globulin to IgE decreased significantly | |
| Speculation | The allergenicity of glutelin B1 decreases but is not obvious, but the proportion of linear epitopes will increase, making it easier to prevent and control. | | | The allergenicity of globulin decreased significantly, and the proportion of linear epitopes would decrease, but it was relatively insignificant. | |

Table 2 Analysis of Molecular Docking Results for Glutinous Protein B1 and Globulin Allergy

As the cases of glutelin intolerance or allergy are not as common as globulin, we can improve food safety by strengthening prevention and control.However, cases of globulin intolerance and even allergy are very common in children with high protein food intolerance. Therefore, directly removing allergens and reducing rice protein allergy is more direct and effective than strengthening prevention and control. In conclusion, we predict that mutations in glutelin B1 and globulin genes can effectively improve the nutritional quality, food safety and eating quality of rice, but may be unfriendly to patients with digestive system diseases. In the future, they need to carefully consider whether they are suitable for eating our products under the advice of doctors.

### Reference

[[1] Kynurenine formamidase: determination of primary structure and modeling-based prediction of tertiary structure and catalytic triad - ScienceDirect](https://www.sciencedirect.com/science/article/pii/S0167483802002327)
[[2] Kynurenine formamidase: determination of primary structure and modeling-based prediction of tertiary structure and catalytic triad - ScienceDirect](https://www.sciencedirect.com/science/article/pii/S0167483802002327)
[[3] Interaction between rice protein and soybean 11S globulin: Effect on the characteristics of rice dough - ScienceDirect](https://www.sciencedirect.com/science/article/pii/S0733521024001309)
[[4] Comparative DNA-methylome and transcriptome analysis reveals heterosis- and polyploidy-associated epigenetic changes in rice - ScienceDirect](https://www.sciencedirect.com/science/article/pii/S2214514122001593)
[[5] Efficient breeding of low glutelin content rice germplasm by simultaneous editing multiple glutelin genes via CRISPR/Cas9 - ScienceDirect](https://www.sciencedirect.com/science/article/pii/S0168945222002746)
[[6] Efficient breeding of low glutelin content rice germplasm by simultaneous editing multiple glutelin genes via CRISPR/Cas9 - ScienceDirect](https://www.sciencedirect.com/science/article/pii/S0168945222002746)

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![](https://static.igem.wiki/teams/5167/paddy2.png)

# Design

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# • Menu

* [→ Improving nutritional quality through rice polyploidization](#section1)
* [→ Choose to edit gluten to increase lysine content in Rice](#section2)
* [→ Knockout of globulin to reduce rice allergenicity](#section3)
* [→ Gene editing technology and vector construction](#section4)

## • Improving nutritional quality through rice polyploidization

We told our PI Professor Yuchi He about our idea of improving the endogenous nutritional quality of rice, and she affirmed our idea. Based on her research on polyploid rice breeding over the years, she told us that, like other polyploid crops that have been widely used, such as triploid watermelon, polyploid rice has the characteristics of generally improved nutrients, which can meet our expectations, and its shape in biomass, stress resistance and other aspects is better than that of conventional ploidy, that is, diploid rice. She suggested that we first double the genome of diploid rice to tetraploid, and then targeted to improve the nutrients we urgently want to improve, namely lysine. Because as a project widely beneficial to people, it is hoped that its product is not only a tool for the production of target products, but also can help the development of food nutrition balance and seed industry.

In terms of how to realize the technology of targeted improvement of lysine content, our initial consideration was to choose the way of adjusting the lysine metabolism pathway of rice, which is also a common method to improve the lysine content of rice through genetic engineering and metabolic engineering, but its strategy and technical means are contrary to our project (add a hyperlink: see iHP and plant synthetic biology for details, you can click to jump to these two pages respectively). We hope to edit the endogenous genes of rice in the project, rather than the exogenous genes introduced from other species. We sought the help of our PI, who suggested that we might start with gluten, the protein with the highest content of rice endosperm protein, and recommend Dr. Lu Gan, who is engaged in the research of rice storage protein, to us as our second pi to provide us with specific guidance in experiments.

## Choose to edit gluten to increase lysine content in Rice

Glutenin is the most abundant protein in rice endosperm, accounting for about 80% of the storage protein in rice endosperm. It is encoded by four gene families, GluA, GluB, GluC and GluD. First, it synthesizes protein precursors in the rough endoplasmic reticulum, transports them into the lumen of the endoplasmic reticulum, and then carries them through the processing of the Golgi apparatus to the protein storage vacuole (PSV). The processed mature storage proteins form protein bodies (PB-Ⅱ) in the vacuole and accumulate. On the other hand, in theory, polyploidization will increase the content of gluten, which will also be verified in our experimental results.

![](https://static.igem.wiki/teams/5167/experiment1.png)

How can the increase of gluten be associated with the increase of lysine? Our PIs suggests that there is often a dose compensation effect at the expression level among the coding genes of proteins encoded by multiple families in rice, that is, if one of the genes encoding less lysine content of glutamine is selected for site directed mutagenesis, then other synthetic genes in this subfamily can be expressed compensably, and correspondingly, the overall increase in lysine content is finally achieved. By constructing the evolutionary tree, we found that the GluB subfamily is the largest, with a total of eight synthetic genes (GluB1 includes two coding genes, GluB1-a and GluB1-b), and whether GluB1 is in the GluB subfamily or in four subfamilies, it the number of lysine residues is the least. It is advantageous to select it as the target gene of site directed mutagenesis to improve the overall lysine content of gluten by using the dose compensation effect.

## • Knockout of globulin to reduce rice allergenicity

In addition, during the investigation, we found that for some susceptible people, allergenic proteins in high protein foods have a greater risk of sensitization, which is one of the sources of greater uncertainty in food safety. (add a hyperlink: see iHP for details) through consulting the literature, we found that α- globulin is the main allergen in rice, accounting for about 2-8%. It is a 26kDa globulin, and its precursor is 19kDa globulin, which is encoded by a single gene Glb located on chromosome 5. Therefore, we also selected OsGlb as the target gene for site directed mutagenesis.

## • Gene editing technology and vector construction

In terms of gene editing technology, we chose CRISPR/Cas9 technology with relatively mature research and high specificity to construct PYLCRISPR/Cas9Pubi-H multi-target binary vector.
1. first, pCAMBIA-1300 was selected as the vector backbone, and the Cas9 protein expression cassette was integrated into the binary vector. The multi cloning site Bsa I used to load multiple sgRNA expression cassettes was located close to the binary vector RB. The sgRNA expression cassette elements were set on the intermediate plasmid vector, assembled by enzymatic ligation and PCR methods, and then assembled on the binary vector by Golden Gate or Gibson Assembly cloning methods.
2. using self-made isothermal in vitro recombination reaction mixture, multiple fragments of plasmid vector can be cloned and deleted simultaneously.
4. on the basis of constructing the vector of the first group of target (OsGluB1) sgRNA expression cassettes, add the second group of other target (OsGlb) sgRNA expression cassettes, change the Mlu I site in the primer Pgs-GGR by 1 base, so that the Mlu I upstream of LacZ becomes the only site. Mlu I was used to cut the vector into linear shape, and Gibson Assembly was used to clone the second group of target sgRNA expression cassettes.
6. after connecting the linker, amplify the sgRNA expression cassette fragment and directly use Golden Gate position specific primers for a round of PCR amplification.
7. the target OsGluB1 sequence is GGTAAGTCGCAGGGCAACC, and the target OsGlb sequence is TCGTCGTCGGAGTACTACGG. The molar ratio of vector to each insert is 1:4-6. Enzyme digestion and ligation were carried out with variable temperature cycles for about 10-15 cycles, and the unused ligation products were frozen for further amplification if necessary.

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