iGEM 2025 — Team scau-guangzhou

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}This serves as a chronological record of your team's progress throughout the season. It documents your daily activities, experiments, discussions, and decisions.{% endblock %}
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![](https://static.igem.wiki/teams/5818/wiki-image/logo1.webp)

# Notebook

# Introduction

Below you can find the lab notes of all the performed experiments in chronological order:

1. Primers Designing
2. Amplification of the Target Genes and Promoters
3. Vector Construction
4. Soybean Genetic Transformation
5. Identification of Positive Plants
6. Folate Extraction and Detection

1. Primers Designing

*![](https://static.igem.wiki/teams/5818/wiki-image/down.webp)*

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2. Amplification of the Target Genes and Promoters

*![](https://static.igem.wiki/teams/5818/wiki-image/down.webp)*

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3. Vector Construction

*![](https://static.igem.wiki/teams/5818/wiki-image/down.webp)*

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4. Soybean Genetic Transformation

*![](https://static.igem.wiki/teams/5818/wiki-image/down.webp)*

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5. Identification of Positive Plants

*![](https://static.igem.wiki/teams/5818/wiki-image/down.webp)*

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6. Folate Extraction and Detection

*![](https://static.igem.wiki/teams/5818/wiki-image/down.webp)*

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{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
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![](https://static.igem.wiki/teams/5818/wiki-image/logo1.webp)

# Engineering

# Engineering

* [Overview](#section1)
* [Cycle1：Selection and Construction of Core Elements for High-Folate Soybean Biosynthesis](#section2)
* [Cycle2：Acquisition and Detection of Genetically Modified Soybeans](#section3)
* [Cycle3：Extraction of Folate and Detection of Its Active Components](#section4)

# Overview

![](https://static.igem.wiki/teams/5818/wiki-image/engineering1-1.webp)

Synthetic biology aims to design standardized biological systems. Drawing on engineering design principles, the "Design-Build-Test-Learn" (DBTL) cycle has gradually became the core strategy in synthetic biology. This cycle uses engineering predictability to control the construction of complex systems. The core of our project is to produce high-folate soybeans through biosynthetic methods.
During our engineering process, the DBTL cycle was applied multiple times. These applications included selecting and constructing core elements for folate biosynthesis in high-folate soybeans, obtaining and detecting transgenic soybeans, and extracting folate while testing its active components. Throughout the entire process, we kept learning, updating our approaches, and summarizing experiences to do better. In the end, we successfully confirmed that our biosynthetic elements are both operable and reproducible.

# Cycle1：Selection and Construction of Core Elements for High-Folate Soybean Biosynthesis

### Design

First, many enzymes are involved in folate biosynthesis, and we need to figure out which enzymes in soybeans can be selected to effectively increase folate levels. Second, since our goal is to boost folate content in soybean seeds, we need to decide on the right promoter to drive the expression of folate synthetase specifically in seeds. Finally, we will integrate the chosen promoter and folate synthetase gene into an expression vector, which will then be used for soybean genetic transformation.

Based on literature research, GCH1 (GTP Cyclohydrolase-1) gene and ADCS (Aminodeoxychorismate Synthase) gene encode the rate-limiting enzymes in folate synthesis. These enzymes catalyze the conversion of GTP to dihydroneopterin triphosphate (DHNTP) and chorismate to aminodeoxychorismate (ADC), respectively, thereby supplying the necessary precursor substances for folate production. These two enzymes have been widely used in bioengineering other plants like Arabidopsis and rice
(de La Garza et al., 2007; Liang et al., 2019; Zamberlan, 2024). For this reason, GCH1 and ADCS from soybeans were chosen as our primary candidate genes. Next, HPPK (Hydroxyphenylpyruvate Dioxygenase Kinase) and DHFR (Dihydrofolate Reductase) were also selected as candidate genes. These two enzymes are responsible for making dihydropteroate (a building block for dihydrofolate) and tetrahydrofolate, respectively (Waller et al., 2010). Since soybeans can naturally produce folate, these enzymes are all present at measurable levels in developing soybean seeds.

During soybean seed development, 4 genes that show high activity in the middle to late stages of seed ripening were screened. The promoters from these genes were then used to control the activity of the candidate enzymes involved in folate production. The four genes identified are Oleosin-1, Oleosin-2, LEA5, and LEA7, respectively.

![](https://static.igem.wiki/teams/5818/wiki-image/engineering2-2.webp)

![](https://static.igem.wiki/teams/5818/wiki-image/engineering3-3.webp)

![](https://static.igem.wiki/teams/5818/wiki-image/engineering4.webp)

### Build and Test

The screened promoters and candidate genes were constructed into the pTF-Flag-35S expression vector, and the following 4 vector constructions were obtained: GmOleosin-1p:GmGCH1, GmOleosin-2p:GmADCS, GmLEA5p:GmHPPK, GmLEA7:GmDHFR.

![](https://static.igem.wiki/teams/5818/wiki-image/engineering5.webp)

![](https://static.igem.wiki/teams/5818/wiki-image/engineering6-2.webp)

### Learn

Since soybeans can naturally produce folate and the key genes in their folate synthesis pathway show some level of activity during seed development, we didn't plan to use multi-gene stacking systems (which allow multiple genes to be expressed at once) when first designing the synthetic components. But when we work on improving the project later, we'll focus first on using these multi-gene systems. This should let us boost several enzyme reactions at the same time in a single step.
Next, when building the vectors, we'll look into adding components that can remove the vector backbone from transgenic plants. This would make the plants safer, help them pass regulatory reviews more easily down the line, and speed up the process of getting them into production.

# Cycle2：Acquisition and Detection of Genetically Modified Soybeans

### Design

The constructed expression vector was used for soybean genetic transformation, with the high-efficiency genetic transformation soybean variety HC6 selected as the recipient. This variety is also a widely promoted spring-planted cultivar in South China. Soybean genetic transformation was carried out using the half-cotyledon node method, which involves the following steps: explant preparation, infection and co-cultivation, induction and screening of clustered buds, stem elongation culture, and rooting culture.

### Build

We mastered and completed the entire soybean genetic transformation process, successfully obtaining transgenic soybean plants.

![](https://static.igem.wiki/teams/5818/wiki-image/engineering7.webp)

### Test

A total of 149 transgenic seedlings were obtained from 4 transformation events, with 115 positive seedlings confirmed by PCR. For each transformation event, 37 positive seedlings were produced by GmOleosin-1p:GmGCH1 transformation, 29 by GmOleosin-2p:GmADCS transformation, 21 by GmLEA5p:GmHPPK transformation, and 28 by GmLEA7:GmDHFR transformation.

![](https://static.igem.wiki/teams/5818/wiki-image/engineering8.webp)

![](https://static.igem.wiki/teams/5818/wiki-image/engineering9.webp)

### Learn

For the transgenic plants of the current generation obtained, the transgenic vector signal was only detected at the genomic DNA level. The high expression of the experimental target gene in soybean seeds by the overexpression vector at the transcriptional level was not detected. The expression level of the target gene will be detected in the next generation.

# Cycle3：Extraction of Folate and Detection of Its Active Components

### Design

This section involves two key tasks. First, a process for extracting folate from dry soybean seeds must be established to ensure the effective folate components in soybean seeds can be obtained. Second, the detection method for the active components of folate needs to be determined.

### Build

Based on previously published folate extraction steps (Sun et al., 2022), we optimized the process for extracting folate from dry soybean seeds, resulting in folate extracts with high purity.

![](https://static.igem.wiki/teams/5818/wiki-image/engineering10.webp)

Five folate standards were selected, including folic acid (FA), tetrahydrofolic acid (THF), 5-methyl-tetrahydrofolate (5M-THF), 5,10-methylene-tetrahydrofolic acid (5,10M-THF), and 10-formyl-folic acid (10F-THF). These standards were analyzed using HPLC-MS with an Agilent 1290UPLC-6470A triple quadruple mass spectrometer. All five folate components were found to be accurately identifiable.

![](https://static.igem.wiki/teams/5818/wiki-image/engineering11.webp)

### Test

Detection of the five folate components mentioned above in transgenic positive seedlings revealed that different candidate genes exert varying effects on the final folate content. Overexpression of the DHFR enzyme doubled the 5M-THF content in soybean seeds, rising from 410 μg/100g seeds in the control group to 867 μg/100g seeds in our transgenic plants. This marked increase was validated across multiple independent transgenic plants. Other folate components showed slight increases, though these changes were not statistically significant. Since 5M-THF represents the primary active form of folate, these findings indicate a significant advancement in folate biosynthesis research in soybeans.

![](https://static.igem.wiki/teams/5818/wiki-image/engineering12.webp)

### Learn

We selected 5 forms of folate for detection, and more forms of folate should be chosen for comprehensive detection in the future. Meanwhile, some forms of folate are extremely easy to be inactivated. For example, FA is extremely easy to be degraded upon exposure to light. We will enhance the management of sample storage and transportation next.

References
*![](https://static.igem.wiki/teams/5818/wiki-image/down.webp)*

Garza R, Gregory J F, Hanson A D. Folate biofortification of tomato fruit[J]. Proceedings of the National Academy of Sciences of the United States of America, 2007,104(10):4218-4222.
Liang Q J, Wang K, Liu X N et al. Improved folate accumulation in genetically modified maize and wheat[J]. Journal of Experimental Botany, 2019,70(5):1539-1551.
Waller J C, Akhtar T A, Lara-Nunez A et al. Developmental and feedforward control of the expression of folate biosynthesis genes in tomato fruit[J]. Molecular Plant, 2010,3(1):66-77.
Zamberlan F. Metabolic engineering Forming folate-fortified rice[J]. Nature Chemical Biology, 2024,20(11):1388.
Agyenim-Boateng K G, Zhang S, Islam M S, et al. Profiling of naturally occurring folates in a diverse soybean germplasm by HPLC-MS/MS. Food Chem. 2022 Aug 1;384:132520

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your project. It is designed to provide sufficient information for other teams to replicate our work.{% endblock %}
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![](https://static.igem.wiki/teams/5818/wiki-image/logo1.webp)

# Experiments

# protocols

1. Design of Primers for Target Genes and Promoters Based on the Soybean Wm82 Reference Genome

*![](https://static.igem.wiki/teams/5818/wiki-image/down.webp)*

# Experimental Objectives

By using bioinformatics methods and reference genome databases such as SoyBase and NCBI, design specific PCR primers for target genes and promoters, which will be used in subsequent molecular biology experiments including gene cloning and expression analysis.

# Main Databases and Software

## Databases

NCBI (National Center for Biotechnology Information): Used for sequence alignment verification and retrieval of published gene sequences.

 SoyBase: A soybean genetic database.

## Primer Design Software

Primer-BLAST: An online primer design tool that can complete both specific primer design and genome-wide specificity verification in one step.

 OligoCalc: Used for online analysis of the physicochemical properties of primers.

 Snapgene: Used for visual pairing of primers and target sequences.

# Experimental Procedures

Obtain the sequences of target genes and promoters from the SoyBase database.

 Target genes: Primers for GmGCH1, GmDHFR, GmADCS, and GmHPPK.

 Promoters: Primers for GmLEA5, GmOleosin1, GmOleosin2, and GmLEA7.

 Use the Primer-BLAST online primer design tool in the NCBI database to design primers.

Use the BLAST tool in the SoyBase database to verify the specificity of the primers.

 Copy the screened primer sequences into OligoCalc, and reconfirm whether their Tm values, GC contents, and other parameters meet the requirements.

2.  RNA extraction

*![](https://static.igem.wiki/teams/5818/wiki-image/down.webp)*

# Reagents and Instruments Required for RNA Extraction

Reagents: Chloroform, isopropanol solution, RNase-free water, 75% ethanol

Instruments: Fume hood, vortex mixer, micropipette, refrigerated microcentrifuge

# Preparations

Pre-cool the centrifuge.

 Pre-cool the reagents (isopropanol, ethanol).

# Experimental Procedures

1．Grinding: Put approximately 2 cm³ of young leaves and 2 steel balls (sterilized by ethanol burning) into a 2 mL RNase-free centrifuge tube. Grind the mixture using a grinder at 60 Hz for 120 seconds.
2．Add 1 mL of TRIzol reagent, shake well, and let it stand at room temperature for 5 minutes.
3．Add 0.2 mL of chloroform, mix thoroughly, and let it stand at room temperature for 3 minutes.
4．Centrifuge the mixture at 12,000 rpm and 4°C for 15 minutes.
5．Transfer 500 μL of the supernatant to a 1.5 mL RNase-free centrifuge tube, add 500 μL of isopentanol, shake well, and incubate in a 4°C refrigerator for 10 minutes.
6．Centrifuge the mixture at 12,000 rpm and 4°C for 10 minutes.
7．Aspirate and discard the supernatant, retain the precipitate, add 1 mL of 75% ethanol, and shake for washing.
8．Centrifuge the mixture at 7,500 rpm and 4°C for 5 minutes.
9．Aspirate and discard the supernatant, and air-dry for 10 minutes to completely remove ethanol.
10．Add 50 μL of RNase-free water and mix thoroughly to dissolve the precipitate.
11．Use a Nanodrop spectrophotometer to determine whether the OD260/280 ratio and OD260/230 ratio are within the reasonable ranges.
12．Store the extracted RNA in a -20°C refrigerator for later use.

3.  Reverse Transcription

*![](https://static.igem.wiki/teams/5818/wiki-image/down.webp)*

# Experimental Procedure

1．Design reverse transcription primers in accordance with the basic principles of primer design.
2．Prepare a system similar to the PCR system for amplification:

| System Components(Total 20μL System) | Added Volume |
| --- | --- |
| Primer | 1μL |
| 2\*TS Bf | 10μL |
| Reverse Transcriptase | 1μL |
| gDNA remover | 1μL |
| RNA（Extracted in the Previous Step)） | 1μL |
| RNA-free H2O | 6μl（to make up the total volume to 20μL） |

3．Mix the above system, transfer it to an EP tube, and place the tube in a PCR instrument for amplification. The PCR program is as follows:

| Temperature | Time | Purpose Description |
| --- | --- | --- |
| 42℃ | 15min | Reverse transcribe RNA |
| 85℃ | 5s | Inactivate enzymes |

4．Determine concentration and OD value:
a.Determine the concentration to confirm whether reverse transcription is successful.
b.Judge whether the OD260/280 ratio is within the reasonable range.

| Nucleic Acid Type | OD260/280 |
| --- | --- |
| DNA | 1.8 ~ 1.9 |
| RNA | 1.9 ~ 2.0 |

In this experiment, if the measured ratio is higher than 1.9, it may indicate that RNA has not been completely converted; if the measured ratio is lower than 1.8, the sample may contain impurities such as phenol or protein.

 5．Store the final sample in a -20℃ refrigerator.

4.  PCR amplification of target gene

*![](https://static.igem.wiki/teams/5818/wiki-image/down.webp)*

# Reagents and Instruments

Reagents: 2×Phanta™ Master Mix, ddH₂O (double-distilled water), template DNA, primers

Instrument: PCR instrument

# PCR Reaction System

| Total Volume | 50 μL |
| --- | --- |
| 2x PhantaTM Master Mix | 25 μL |
| Template DNA | 1 μL |
| Primer 1(10uM) | 2 μL |
| Primer 2(10uM) | 2 μL |
| ddH2O | 20 μL |

# PCR Amplification Conditions

![](https://static.igem.wiki/teams/5818/wiki-image/experiments1.webp)

5. Agarose gel electrophoresis

*![](https://static.igem.wiki/teams/5818/wiki-image/down.webp)*

# Reagents and Instruments

Reagents:Agarose, 1× TBE buffer, nucleic acid dye (GelRed)

Instruments: Microwave oven, Erlenmeyer flask, gel tray, sample comb

# Experimental Procedures

1．Weigh agarose:Weigh an appropriate amount of agarose according to the required concentration. For example, to prepare a 1% agarose gel, weigh 1 gram of agarose and place it in a 500-milliliter Erlenmeyer flask.
2．Add buffer: Add 100 milliliters of 1× TBE buffer to the Erlenmeyer flask, ensuring that the agarose is completely submerged in the liquid.
3．Heat to melt: Place the Erlenmeyer flask in a microwave oven and heat it on high power for 30 seconds. Take it out and shake gently to ensure that the agarose particles are fully dissolved. Repeat the heating and shaking process until the agarose is completely dissolved; usually, it needs to be heated to boiling.
4．Add dye: After heating until the agarose is completely dissolved, let it stand at room temperature to cool slightly. When the temperature drops to approximately 60℃, add 10 μL of the nucleic acid dye GelRed and shake thoroughly.
5．Pour the gel: Carefully pour the melted agarose solution into a pre-prepared gel tray, taking care to avoid generating air bubbles. When pouring the gel, the temperature should not be too low, otherwise the gel will solidify unevenly.
6．Insert the sample comb: Before the agarose gel is completely solidified, insert the sample comb, ensuring that there is an appropriate gap between the bottom of the comb and the bottom of the gel tray.
7．Cool and solidify: After the agarose gel is completely solidified, gently pull out the sample comb to form sample wells. Then add an appropriate amount of buffer to the tray, ensuring that the liquid level covers the surface of the gel.

6. Gel Purification

*![](https://static.igem.wiki/teams/5818/wiki-image/down.webp)*

# Reagents and Instruments

Reagent: HiPure DNA Mini Column Kit (Brand: Magen Biotechnology Co., Ltd., Model: D2111)

![](https://static.igem.wiki/teams/5818/wiki-image/experiments2.webp)

# Experimental Procedures

1．Prepare an agarose gel of appropriate concentration and perform electrophoresis to separate DNA fragments. After the DNA fragments are separated, place the gel under an ultraviolet lamp, quickly cut off the gel containing the target DNA fragment, and remove excess gel as much as possible.

2．Weigh the gel block and transfer it to a 2.0 mL centrifuge tube. Calculate based on the standard that 100 mg of gel block is equivalent to a volume of 100 μL, add 1.5 times the volume of Buffer GDP, and incubate in a 55℃ water bath for 15 minutes to completely dissolve the gel block. During the water bath, invert and mix 3 times to accelerate gel dissolution.

3．Perform a short centrifugation to collect the liquid droplets on the tube wall. Place the HiPure DNA Mini Column onto a collection tube. Transfer ≤700 μL of the gel-dissolved solution into the column, and centrifuge at 12,000×g for 30–60 seconds.

4．(Optional: If the gel-dissolved solution exceeds 700 μL) Discard the filtrate, place the column back onto the collection tube, transfer the remaining gel-dissolved solution into the column, and centrifuge at 12,000×g for 30–60 seconds.

5．Discard the filtrate, place the column back onto the collection tube, add 150 μL of Buffer GDP into the column, let it stand for 1 minute, and centrifuge at 12,000×g for 30–60 seconds.

6．Discard the filtrate, place the column back onto the collection tube, add 600 μL of Buffer DW2 (diluted with absolute ethanol) into the column, and centrifuge at 12,000×g for 30–60 seconds. Note: Before using Buffer DW2, dilute it with absolute ethanol according to the instructions on the bottle label.

7．Discard the filtrate, place the column back onto the collection tube, add 300 μL of Buffer DW2 (diluted with absolute ethanol) into the column, and centrifuge at 12,000×g for 2 minutes. When taking out the column, do not let the bottom of the column come into contact with the liquid; if it does, discard the liquid and centrifuge for another 1 minute. Open the lid of the column and air-dry for 5–10 minutes to completely remove ethanol.

8．Place the column onto a 1.5 mL centrifuge tube, add 15–30 μL of Elution Buffer to the center of the column membrane, let it stand for 2 minutes, and centrifuge at 12,000×g for 1 minute. Discard the column and store the DNA at -20℃.

7. Plasmid Extraction

*![](https://static.igem.wiki/teams/5818/wiki-image/down.webp)*

# Reagents and Instruments for Plasmid Extraction

Reagent: HiPure Plasmid Micro Kit (Brand: Magen Biotechnology Co., Ltd., Model: P1001)

![](https://static.igem.wiki/teams/5818/wiki-image/experiments3.webp)

# Experimental Procedures

1．Isolate a single colony from a freshly streaked selective plate and inoculate it into 1–5 mL of LB medium containing the appropriate selective antibiotic. Incubate at 37°C for 12–16 hours with vigorous shaking. Centrifuge at 13,000 rpm for 1 minute at room temperature. Pour off or aspirate and discard the medium. It is strongly recommended to use endA-negative strains of E. coli for routine plasmid isolation. Examples of such strains include DH5α® and JM109®. The HiPure Mini protocol in this manual can be used for the preparation of low-copy-number plasmid DNA or cosmids. Use 1–10 mL of overnight cultured E. coli grown in LB medium. When a 10 mL culture volume is used, it is recommended to double the volumes of Buffer P1, P2, and P3.

2．Resuspend the pelleted bacterial cells in 250 μL of Buffer P1 and transfer to a microcentrifuge tube. Ensure that RNase A has been added to Buffer P1. No cell clumps should remain after this step. For resuspension of the pellet: Bacteria should be completely resuspended by vortexing. Mix by repeated pipetting until no cell clumps are left.

3．Add 250 μL of Buffer P2 and mix thoroughly by inverting the tube 8–10 times; gently invert to mix the liquid in the tube. Do not use a vortex mixer, as this will cause shearing of genomic DNA and contamination of the plasmid. Continue inverting the tube until the solution becomes viscous and slightly translucent. Do not allow the lysis reaction to proceed for more than 5 minutes.

4．Add 350 μL of Buffer P3. Mix immediately and thoroughly by inverting the tube 8–10 times. To avoid local precipitation, the solution should be mixed thoroughly immediately after adding Buffer P3. For large-volume cultures (e.g., ≥5 mL), inversion may need to be done 10 times. The solution should become turbid.

5．Centrifuge at 13,000 rpm for 10 minutes in a table-top microcentrifuge.

6．Insert a HiPure DNA Mini Column II into a 2.0 mL Collection Tube (provided). Pipette 800 μL of the supernatant from Step 5 onto the column. Centrifuge for 30–60 seconds. Discard the flow-through.

7．Recommended: Wash the column by adding 0.5 mL of Buffer PW1 and centrifuge for 30–60 seconds. Discard the flow-through. This step is necessary when using endA+ strains (e.g., JM series, HB101 and its derivatives) or any wild-type strains that have high levels of nuclease activity or high carbohydrate content. Host strains such as XL-1 Blue and DH5α do not require this additional washing step.

8．Wash the column by adding 0.75 mL of Buffer PW2 and centrifuge for 30–60 seconds.

9．Discard the flow-through and centrifuge at full speed for an additional 1 minute to remove residual wash buffer. Residual wash buffer will not be completely removed unless the waste flow is discarded before performing this additional centrifugation step. Residual ethanol in Buffer PW2 may inhibit subsequent enzymatic reactions.

10．Place the column in a clean 1.5 mL microcentrifuge tube. To elute the DNA, add 50–100 μL of Elution Buffer or water to the center of each column, let stand for 1 minute, and centrifuge for 1 minute. When the plasmid DNA or cosmids are larger than 10 kb, preheat the Elution Buffer (or water) to 70°C before use to elute the DNA from the HiPure membrane.

8. Enzyme Digestion Treatment

*![](https://static.igem.wiki/teams/5818/wiki-image/down.webp)*

# Experimental Reagents

Restriction enzymes: HindIII and XbaI (Brand: Yugong Biotechnology Co., Ltd.)
Corresponding buffer: 10×CutOne Color Buffer
PTF101 empty vector

# Experimental Procedures

① Prepare the reaction system on ice according to the recommended sample addition order as follows:

| Component | Plasmid DNA | PCR Product | Genomic DNA |
| --- | --- | --- | --- |
| ddH₂O | 15 μL | 30 μL | 16 μL |
| 10xCutOne Buffer or10xCutOne Color Buffer | 2 μL | 5 μL | 3 μL |
| Substrate DNA | 10 μL (~0.2 μg) | 10 μL (5 μg) | 2 μL (up to 1 μg) |
| LightNing HindIII | 1 μL | 1 μL | 5 μL |
| Total Volume | 20 μL | 30 μL | 50 μL |

② Mix by gently pipetting up and down or flicking the tube wall, then perform a brief centrifugation to collect the liquid droplets adhering to the tube wall.
③ Incubate at 37℃ for 15 minutes.
④ Incubate at 80℃ for 20 minutes to inactivate the enzyme.

9. Electrophoresis Detection + Gel Extraction

*![](https://static.igem.wiki/teams/5818/wiki-image/down.webp)*

# Instruments for Electrophoresis Detection

Electrophoresis apparatus, developer
Settings for electrophoresis apparatus: Voltage = 220 V, Time = 10 min

# Reagents and Instruments for Gel Extraction

Reagent: HiPure DNA Mini Column Kit (Brand: Magen Biotechnology Co., Ltd., Model: D211)

![](https://static.igem.wiki/teams/5818/wiki-image/experiments4.webp)

# Experimental Procedures

1．Prepare an agarose gel of appropriate concentration and perform electrophoresis to separate DNA fragments. After the DNA fragments are separated, place the gel under an ultraviolet lamp, quickly cut off the gel containing the target DNA fragment, and remove excess gel as much as possible.

2．Weigh the gel block and transfer it to a 2.0 mL centrifuge tube. Calculate based on the standard that 100 mg of gel block is equivalent to a volume of 100 μL, add 1.5 times the volume of Buffer GDP, and incubate in a 55℃ water bath for 15 minutes to completely dissolve the gel block. During the water bath, invert and mix 3 times to accelerate gel dissolution.

3．Perform a short centrifugation to collect the liquid droplets on the tube wall. Place the HiPure DNA Mini Column onto a collection tube. Transfer ≤700 μL of the gel-dissolved solution into the column, and centrifuge at 12,000×g for 30–60 seconds.

4．(Optional: If the gel-dissolved solution exceeds 700 μL) Discard the filtrate, place the column back onto the collection tube, transfer the remaining gel-dissolved solution into the column, and centrifuge at 12,000×g for 30–60 seconds.

5．Discard the filtrate, place the column back onto the collection tube, add 150 μL of Buffer GDP into the column, let it stand for 1 minute, and centrifuge at 12,000×g for 30–60 seconds.

6．Discard the filtrate, place the column back onto the collection tube, add 600 μL of Buffer DW2 (diluted with absolute ethanol) into the column, and centrifuge at 12,000×g for 30–60 seconds. Note: Before using Buffer DW2, dilute it with absolute ethanol according to the instructions on the bottle label.

7．Discard the filtrate, place the column back onto the collection tube, add 300 μL of Buffer DW2 (diluted with absolute ethanol) into the column, and centrifuge at 12,000×g for 2 minutes. When taking out the column, do not let the bottom of the column come into contact with the liquid; if it does, discard the liquid and centrifuge for another 1 minute. Open the lid of the column and air-dry for 5–10 minutes to completely remove ethanol.

8．Place the column onto a 1.5 mL centrifuge tube, add 15–30 μL of Elution Buffer to the center of the column membrane, let it stand for 2 minutes, and centrifuge at 12,000×g for 1 minute. Discard the column and store the DNA at -20℃.

10. Ligation of Target Gene and Vector

*![](https://static.igem.wiki/teams/5818/wiki-image/down.webp)*

# Experimental Reagents

ClonExpress Ultra One Step Cloning Kit V2 from Thermo Fisher Scientific Inc.

![](https://static.igem.wiki/teams/5818/wiki-image/experiments5.webp)

# Amount of Fragments

Optimal amount of cloning vector = [0.02 × number of base pairs of cloning vector] ng (0.03 pmol)Optimal amount of insert fragment = [0.04 × number of base pairs of insert fragment] ng (0.06 pmol)

# Experimental System

| Reagent | Volume |
| --- | --- |
| 2×Clone Express mix | 5 μl |
| Vector | 40 ng |
| Vector | 200 ng |
| ddH₂O | Up to 10μl |

Gently pipette up and down to mix (do not vortex), then centrifuge briefly to collect the reaction solution at the bottom of the tube.Subsequently, incubate at 50°C for 5 minutes; cool down to 4°C or place on ice immediately for cooling.

11. Transformation of Competent Cells (E. coli)

*![](https://static.igem.wiki/teams/5818/wiki-image/down.webp)*

# Materials

Trelief 5α Chemically Competent Cells (from Tsc-col Qingke Biotechnology Co., Ltd.) ,Luria-Bertani (LB) Liquid Medium without Antibiotic Resistance

# Experimental Procedure

1．Thaw the competent cells on ice.
2．Add 1 μL of recombinant plasmid to 20 μL of competent cells and mix gently.
3．Place on ice for 30 minutes, then incubate in a 42°C water bath for 45 seconds, and then take it out and place it on ice for 2 minutes.
4．Add 500 μL of LB medium without antibiotic resistance, place it in a 37°C shaker for 1 hour, and then perform plate spreading for screening.

12. Colony PCR Protocol

*![](https://static.igem.wiki/teams/5818/wiki-image/down.webp)*

# Experimental Materials and Reagents

## Instrumentation and Equipment

PCR Thermocycler, Electrophoresis Power Supply and Gel Tank, Agarose Gel Imaging System, Vortex Mixer, Microcentrifuge
Pipettes and Tips (10μL, 200μL, 1000μL), PCR Tubes (0.2mL)

## Reagents

2×Taq Master Mix (PCR Premix): Contains Taq DNA Polymerase, dNTPs, Mg²⁺, and reaction buffer. Using the premix simplifies operations and reduces contamination.
Sterile Ultra-Pure Water
Primers: Target primers designed for the experiment, diluted to working concentration with sterile ultra-pure water.
Positive Control: Plasmid DNA or colonies known to contain the target fragment.
Negative Control: PCR system without template DNA (replaced with water).
LB Agar Plates: Agar plates with single colonies grown after transformation.
Reagents for Agarose Gel Electrophoresis: Agarose, Indicator, DNA Molecular Weight Standard (DNA Marker).

# Experimental Procedures

## Step 1: Pre-Experiment Preparation

1．Calculate and prepare the PCR Master Mix according to the number of required reactions. It is recommended to prepare 1-2 extra reactions to compensate for pipetting losses.

| Reaction Component | Volume per Reaction |
| --- | --- |
| 2×Taq Master | Mix 10μL |
| Forward | Primer 0.5μL |
| Reverse | Primer 0.5μL |
| ddH₂O | 9μL |
| Total | Volume 2μL |

2．Vortex to mix the Master Mix thoroughly, centrifuge briefly, and aliquot 20μL into each PCR tube.

## Step 2: Addition of Template (Colony)

1．Gently touch a single colony on the transformation plate with a sterile 10μL pipette tip. Stir the tip with the colony gently in the PCR tube containing the aliquoted Master Mix to fully suspend the bacterial cells in the liquid. Replace the pipette tip and repeat the above operation to pick the next colony.

2．Do not add any template to the negative control tube, or add an untransformed colony as the negative control.

3．Tighten the PCR tube caps and centrifuge briefly to concentrate the liquid at the bottom of the tubes.

## Step 3: PCR Amplification

Place the PCR tubes into the PCR thermocycler and set the appropriate reaction program.

| Step | Temperature | Time | Number of Cycles |
| --- | --- | --- | --- |
| 1. Pre-denaturation | 95°C | 5-10 min | 1 |
| 2. Denaturation | 95°C | 30 sec |  |
| 3. Annealing | Tm-5°C | 30 sec | 30-35 cycles |
| 4. Extension | 72°C | 1 min/kb |  |
| 5. Final Extension | 72°C | 5-10 min | 1 |
| 6. Hold | 4-16°C | ∞ | 1 |

## Step 4: Detection of PCR Products

1．Prepare an agarose gel of appropriate concentration.
2．Load the mixture into the gel wells. Meanwhile, load the DNA Molecular Weight Standard into an adjacent well.
3．Perform electrophoresis at an appropriate voltage until the bromophenol blue indicator migrates to 2/3 of the gel length.
4．Observe and record the results using the gel imaging system.

## Step 5: Result Analysis

Positive Clones: A specific amplified band appears at the expected DNA fragment size, consistent with the size of the positive control band.
Negative Clones:No bands appear, or only faint bands appear at the position of primer dimers (below approximately 100bp).
Negative Control: No bands should appear. If bands appear, it indicates contamination in the reaction system, and the experimental results are unreliable.

13. Agrobacterium tumefaciens transformation

*![](https://static.igem.wiki/teams/5818/wiki-image/down.webp)*

# Materials

Competent Cells: K599 (pSoup) Chemically Competent Cells (from AC1082, Weidi Biotechnology Co., Ltd.)
Medium: Leagene TY Agar Medium (TY Medium); Antibiotic-free TY Liquid Medium (for post-heat shock culture)

# Experimental Steps

1．Thaw the Agrobacterium competent cells on ice.
2．Add 1 μg of plasmid to every 100 μL of competent cells, and mix quickly by pipetting.
3．Incubate on ice for 5 minutes → place in liquid nitrogen for 5 minutes → incubate in a 37°C water bath for 5 minutes → incubate on ice again for 5 minutes.
4．Add 700 μL of antibiotic-free TY liquid medium, culture in a 28°C shaker for 2 hours, then perform plate spreading for screening.

14. Soybean Genetic Transformation Method

*![](https://static.igem.wiki/teams/5818/wiki-image/down.webp)*

# Reagents and Instruments Required for RNA Extraction

1．Seed Sterilization: Take out the screened and stored Huachun 6 seeds from the -20°C refrigerator, soak them in 75% ethanol for 1 minute, then wipe dry. Arrange the seeds in a single layer in a petri dish and place them in a desiccator in a fume hood. Put a 250 mL beaker in the desiccator, add 5 mL of concentrated hydrochloric acid to 100 mL of sodium hypochlorite, cover the desiccator for sterilization for 13.5 hours. Take out the seeds, blow them in a ultra-clean workbench, and store them in a 4°C refrigerator for later use.

2．Seed Soaking: Soak the soybeans in sterilized ddH₂O, and place them in a dark environment at 24°C for 10-16 hours.

3．Bacterial Solution Preparation:Take out the EHA105 bacterial solution transformed with the target plasmid, and add it to YEP liquid medium containing 100 mg/L Spe and 30 mg/L Rif. Set the constant temperature shaker at 28°C and 240 rpm for overnight culture. The next day, measure the absorbance value of the bacterial solution (OD600 = 0.8-1.0). Pour the bacterial solution into a sterile 50 mL centrifuge tube, centrifuge at 5000 rpm for 10 minutes. Pour off the supernatant, resuspend the bacterial cells with CCM liquid medium to make OD600 = 0.6-0.8, and store at 4°C for the next experiment.

4．Explant Preparation and Transformation:In the ultra-clean workbench, use tweezers and a scalpel to cut the hypocotyl of the soaked seeds, cut the seeds vertically along the cotyledons, and remove the young buds, epicotyls and seed coats on the cotyledons.

5． Inoculation and Co-Cultivation:Soak the explants in the prepared CCM resuspension, treat with ultrasound for 3 minutes, vacuum for 10 minutes, then shake at 120-150 rpm and 28°C for 40 minutes. After inoculation, pour out the bacterial solution, spread the explants on the CCM solid medium covered with filter paper using tweezers. Seal with medical tape, place in the tissue culture room, set the temperature at 22°C, culture in the dark for 48 hours, then culture in light for 3-5 days until the cotyledons turn completely green.

6．Shoot Induction:Use a scalpel to cut off the overgrown hypocotyls, insert the explants obliquely into the shoot induction medium, seal with medical tape, and place in the tissue culture room. Set the temperature at 22°C and the light time at 16/8 hours, and culture for 14 days. Transfer the explants to a new shoot induction medium in a sterile plate, discard the explants without clustered shoots, place 6-7 explants in each medium, seal with medical tape, and place in the tissue culture room for induction for 14 days.

7．Shoot Growth:Four weeks after shoot induction, discard the undifferentiated materials, cut off the cotyledons of the explants with clustered shoots, transfer them to the shoot growth medium, and make the incisions fit the medium. Place 6-7 explants in each petri dish, place in the tissue culture room, and replace the medium every 14 days.

8．Rooting:Use a scalpel to cut off the shoots with a length of more than 3 cm from the root of the tissue, soak them in auxin for 1 minute, then transfer them to the rooting medium, and place them in the tissue culture room to continue growing. When 5-6 roots grow and there are also lateral roots, gently take out the plants from the rooting medium, and carefully rinse off the medium on the roots with tap water. Transplant the seedlings into nutrient soil (vermiculite: substrate soil = 1:2) sterilized by high temperature and high pressure, place them in a heat-preserving and moisture-preserving seedling tray, and place the seedling tray in an incubator. Set the temperature at 26°C and the light time at 12/12 hours. Water with nutrient solution for cultivation until pod formation.

15. DNA Extraction and PCR

*![](https://static.igem.wiki/teams/5818/wiki-image/down.webp)*

# Reagents and Instruments for DNA Extraction

## Reagents

CTAB (Hexadecyl Trimethyl Ammonium Bromide), Tris-HCl, EDTA, NaCl, β-Mercaptoethanol, Chloroform, Anhydrous Ethanol, ddH₂O

## Instruments

Grinder (equipped with zirconia steel beads), 65°C Oven, Centrifuge, EP Tubes

# Steps for DNA Extraction

1．Put 2 cm³ of young leaves into a tube, and add 3 zirconia steel beads.
2．Freeze quickly in liquid nitrogen.
3．Grind with the grinder at 120 Hz for 60 seconds by oscillation.
4．Add 500 μL of CTAB extraction solution.
5．Invert to mix, place in a 65°C oven for 30 minutes, and mix the solution at intervals.
6．Add 450 μL of chloroform, mix thoroughly, and centrifuge at 12,700 rpm for 15 minutes.
7．Pipette 350 μL of supernatant into a new EP tube, add 650 μL of anhydrous ethanol, invert to mix, and place at -80°C for more than 1 hour.
8．Centrifuge at 12,700 rpm at 4°C for 15 minutes, then discard the supernatant.
9．Add anhydrous ethanol, mix, centrifuge for 1 minute, and pour off the supernatant; centrifuge briefly, then aspirate the residual liquid with a pipette tip.
10．Let the white precipitate dry naturally, then add 40 μL of ddH₂O to dissolve it. The DNA extraction is completed.

# Reagents and Instruments Required for Gene PCR Amplification

## Reagents

2×Phanta™ Master Mix, ddH₂O, Template DNA, Primers

## Instrument

PCR Thermocycler

## PCR Reaction System

| Component | Volume |
| --- | --- |
| 2×Phanta™ Master Mix | 25 μL |
| Template DNA | 2 μL |
| Primer 1 (10 μM) | 2 μL |
| Primer 2 (10 μM) | 2 μL |
| ddH₂O (to make up volume) | Up to 50 μL |

## PCR Amplification Conditions

| Step | Temperature | Time | Cycles |
| --- | --- | --- | --- |
| Pre-denaturation | 95°C | 3 min | 1 |
| Denaturation | 95°C | 15 sec | 34 |
| Annealing | 60°C | 15 sec | 34 |
| Extension | 72°C | 60 sec/kb | 34 |
| Final Extension | 72°C | 5 min | 1 |

16. Electrophoresis detection

*![](https://static.igem.wiki/teams/5818/wiki-image/down.webp)*

# Reagents and Instruments Required

## Reagents

5×TAE (Tris-Acetate-EDTA Buffer), Agarose, 5×Loading Buffer, Golden View (nucleic acid stain), DNA Marker

## Instruments

Electrophoresis Power Supply, Developer (Gel Imaging System), Microwave Oven, Electronic Balance, Pipette, Gel Mold, Comb (10 μL volume)

# Experimental Steps

1．Weigh 1 g of agarose with an electronic balance, add 100 mL of 5×TAE solution, and heat in a microwave oven until the agarose is fully dissolved.
2．When the solution cools to approximately 60°C, add 3.4 μL of Golden View and shake well.
3．Pour the solution into a gel mold, insert a 10 μL-volume comb, and cool at room temperature for 20 minutes (to solidify the gel).
4．Take the agarose gel out of the mold, place it in an electrophoresis tank, and submerge it in electrophoresis buffer.
5．Use a pipette to aspirate 10 μL of PCR amplification product (with loading buffer added in advance) respectively, and add them to the sample wells in order.
6．Use a pipette to aspirate 3 μL of DNA Marker and add it to a sample well.
7．Set the voltage to 180 V, and perform constant-voltage electrophoresis for 15 minutes.
8．After electrophoresis, place the agarose gel into the developer to observe whether the size of the target band is correct, so as to determine the positive status of the transformed seedlings.

17. Folate extraction and detection

*![](https://static.igem.wiki/teams/5818/wiki-image/down.webp)*

# Reagents and Instruments Required

## Reagents

Disodium hydrogen phosphate (Na₂HPO₄), Sodium dihydrogen phosphate (NaH₂PO₄), Ascorbic acid, β-Mercaptoethanol, α-Amylase, Protease, Rat serum, Pure water

## Instruments

50mL small glass bottles, Graduated cylinders, Beakers, Weighing paper, Weighing spoons, Precision balance, 2mL sterile centrifuge tubes, 3KD MWCO ultrafiltration tubes, Pipettes (with appropriate volume ranges), Sterile pipette tips, Small coffee bean grinder, Water bath/metal bath, Shaker, Refrigerated centrifuge

# Experimental Steps

## Step 1: Prepare Phosphate Buffer in Advance

Prepare a neutral buffer containing disodium hydrogen phosphate, sodium dihydrogen phosphate, ascorbic acid, and β-mercaptoethanol (100mL system):
1．Weigh 0.317g NaH₂PO₄ and 0.271g Na₂HPO₄, add approximately 80mL pure water, and adjust the pH to 7.
2．Add 0.5g ascorbic acid and 0.2mL β-mercaptoethanol, adjust the pH to 7 again, and bring the total volume to 100mL.

## Step 2: Extract Folate Components

Preliminarily extract folate-containing mixtures from soybeans:
1．On the day of the experiment, grind soybeans into powder using a small coffee bean grinder (freshly ground).
2．Add 50mg (0.05g) soybean powder and 1mL phosphate buffer to each 2mL centrifuge tube, shake well, and immediately transfer to a 100°C boiling water bath/metal bath for 10 minutes (during heating, take out and invert to shake evenly every 5 minutes to prevent clumping).
3．After boiling, open the tube cap to release gas, then close the cap and place the tube on ice for 10 minutes.

## Step 3: First Enzyme Treatment

Add 20µL α-Amylase (0.1mg/mL) to the sample tube, and incubate in a 28°C shaker for 30 minutes.

## Step 4: Second Enzyme Treatment

1．Pre-cool the centrifuge and set the centrifugation temperature to 4°C.
2．Add 15µL protease to the sample tube, continue incubating in a 37°C shaker for 1 hour.
3．After incubation, boil the sample in a boiling water bath for 5 minutes, then transfer to the 4°C centrifuge and centrifuge at 13,000 rpm for 10 minutes.
4．Aspirate 400µL of the supernatant into a new 2mL sterile centrifuge tube.

## Step 5: Depolymerase Treatment

Add rat serum at a volume ratio of 2% to the supernatant, and incubate at 37°C for 4 hours to remove the polyglutamate tail.

## Step 6: Washing and Centrifugation

1．Boil the sample again for 10 minutes, then cool it on ice for 10 minutes.
2．Centrifuge at 13,000 rpm for 10 minutes at 4°C.

## Step 7: Sample Filtration and Preparation

1．Transfer 400µL of the supernatant to a 3KD MWCO ultrafiltration tube, centrifuge at 13,000 rpm for 20 minutes at 4°C, and aspirate 240µL of the supernatant into a new tube.
2．Finally, take 100µL of the filtrate for analysis, and store the remaining part at -80°C

18. Medium Preparation

*![](https://static.igem.wiki/teams/5818/wiki-image/down.webp)*

# Preparation of LB Antibiotic-Free Liquid Medium (1L)

## Components & Dosages

| Component | Dosage |
| --- | --- |
| TRYPTONE | 10 g |
| NaCl | 10 g |
| Yeast Extract | 5 g |
| ddH₂O | 1 L |

## Preparation Steps

1．Weigh 10 g TRYPTONE, 5 g Yeast Extract, and 10 g NaCl into a heat-resistant container, then add approximately 800 mL ddH₂O to dissolve them.
2．Bring the volume to 1 L, mix well, dispense into autoclave-resistant containers or bottles, and leave the lids loose.
3．Autoclave at 121°C for 15–20 minutes, then take out and cool to room temperature for later use.

# Preparation of LB Antibiotic-Free Solid Medium (1L)

## Components & Dosages

| Component | Dosage |
| --- | --- |
| TRYPTONE | 10 g |
| NaCl | 10 g |
| Yeast Extract | 5 g |
| Agar | 14 g |
| ddH₂O | 1 L |

## Preparation Steps

1．Weigh 10 g TRYPTONE, 5 g Yeast Extract, 10 g NaCl, and 14 g Agar into a container, add approximately 800 mL ddH₂O, heat and stir until fully dissolved (avoid local gelatinization).
2．Bring the volume to 1 L, dispense into containers, and autoclave at 121°C for 15–20 minutes.
3．Keep the sterilized medium warm for later use; cool it to approximately 55°C when antibiotics need to be added subsequently.

# Extraction of Kanamycin (50 mg/mL)

1．Take a 50 mL tube, add 1.5 g kanamycin sulfate powder (molecular weight: 582.58) to 30 mL sterile ddH₂O.
2．Transfer to a sterile operating bench, draw the liquid with a syringe, replace with a filter tip, and filter the liquid into 2 mL sterile centrifuge tubes or other containers.

# Preparation of LB Antibiotic-Containing Liquid Medium (1L)

Add 1 mL of kanamycin (Kana) working solution to the cooled LB antibiotic-free liquid medium.

# Preparation of LB Antibiotic-Containing Solid Medium (1L)

1．When the LB antibiotic-free solid medium cools to approximately 55°C, add 1 mL of kanamycin (Kana) working solution.
2．Mix well, immediately pour into petri dishes (about 10 mL per dish), and store in a sterile operating bench.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}Present the results of your project, along with a detailed analysis and discussion of their significance. Also outline future plans and reflections on the impact of your project.{% endblock %}
{% block extra\_css %}

{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/5818/wiki-image/logo1.webp)

# Results

# Results

* [Overview](#section1)
* [Part 1. Selection of Key components for High-folate Soybean Bio-system](#section2)
* [Part 2. Construction of Overexpression Vectors](#section3)
* [Part 3. Acquisition and Detection of Genetically Modified Soybeans](#section4)
* [Part 4. Extraction of Folate](#section5)
* [Part 5. Establishment of Detection Method for Active Components of Folate](#section6)
* [Part 6. Identification of Active Folate Components in Genetically Modified Soybeans](#section7)

# Overview

To develop soybeans with high folate content, a set of biosynthetic components was constructed and their feasibility was successfully confirmed. First, vector modules were built to overexpress key enzymes involved in folate biosynthesis specifically in soybean seeds. Second, these vector modules were introduced into soybeans using the Agrobacterium-mediated cotyledon node method, resulting in the generation of transgenic plants. Finally, it was verified that the introduced modules effectively led to a one-fold increase in the accumulation of active folate in soybean seeds.

# Part 1. Selection of Key components for High-folate Soybean Bio-system

Four enzymes, GCH1 (GTP cyclohydrolase-1), ADCS (Aminodeoxychorismate Synthase), HPPK (Hydroxyphenylpyruvate Dioxygenase Kinase), and DHFR (Dihydrofolate Reductase), were selected as candidate genes to design elements for increasing folate content in soybeans. These enzymes act on different nodes of the folate biosynthesis pathway, respectively (de La Garza et al., 2007; Liang et al., 2019; Zamberlan, 2024). All of these enzymes are expressed at certain levels during soybean seed development.

In addition, four genes with high expression in the middle and late stages of seed maturation were screened, and their promoters were used to drive the expression of the candidate folate synthesis enzymes. These four genes are Oleosin-1, Oleosin-2, LEA5, and LEA7.

![](https://static.igem.wiki/teams/5818/wiki-image/results1.webp)

![](https://static.igem.wiki/teams/5818/wiki-image/results2.webp)

![](https://static.igem.wiki/teams/5818/wiki-image/results3.webp)

# Part 2. Construction of Overexpression Vectors

We constructed the screened promoters and candidate genes into the pTF-Flag-35S expression vector, and obtained the following 4 vector constructions: GmOleosin-1p:GmGCH1, GmOleosin-2p:GmADCS, GmLEA5p:GmHPPK, GmLEA7:GmDHFR. The accuracy of the inserted sequences was verified by sequencing.

![](https://static.igem.wiki/teams/5818/wiki-image/results4.webp)

![](https://static.igem.wiki/teams/5818/wiki-image/engineering6-2.webp)

# Part 3. Acquisition and Detection of Genetically Modified Soybeans

Soybean genetic transformation was carried out using the constructed expression vector, with HC6 selected as the recipient variety. This variety not only has high genetic transformation efficiency but is also a widely cultivated spring-planted soybean in South China. The half-cotyledon node method was employed for the transformation process, which includes explant preparation, infection and co-cultivation, induction and screening of clustered buds, stem elongation culture, and rooting culture.

A total of 149 transgenic seedlings were obtained from 4 transformation events, and PCR verification confirmed 115 of these as positive. Among the individual transformation events, 37 positive seedlings were obtained from GmOleosin-1p:GmGCH1 transformation, 29 from GmOleosin-2p:GmADCS transformation, 21 from GmLEA5p:GmHPPK transformation, and 28 from GmLEA7:GmDHFR transformation.

![](https://static.igem.wiki/teams/5818/wiki-image/results6.webp)

![](https://static.igem.wiki/teams/5818/wiki-image/results7.webp)

# Part 4. Extraction of Folate

The process for extracting folate from soybean dry seeds was optimized based on the published extraction steps (Sun et al., 2022), and a folate extract with high purity was obtained.

![](https://static.igem.wiki/teams/5818/wiki-image/results8.webp)

# Part 5. Establishment of Detection Method for Active Components of Folate

Five forms of folate standards, including folic acid (FA), tetrahydrogen folic acid (THF), 5-methyl tetrahydrofolate (5M-THF), 5,10-methylene-tetrahydrofolic acid (5,10M-THF), and 10-formyl folic acid (10F-THF), were selected and detected by HPLC-MS using an Agilent 1290UPLC-6470A triple quadruple mass spectrometer. All these five folate components were found to be accurately identifiable.

![](https://static.igem.wiki/teams/5818/wiki-image/results9.webp)

# Part 6. Identification of Active Folate Components in Genetically Modified Soybeans

The five folate components mentioned above were detected in genetically modified positive seedlings, and it was found that different candidate genes exert varying effects on the final folate content. Overexpression of the DHFR enzyme doubled the 5M-THF content in soybean seeds, rising from 410 μg/100g seeds in the control group to 867 μg/100g seeds in the transgenic plants. This marked increase was confirmed across multiple transgenic plant lines. Other folate components showed slight increases, though these changes were not statistically significant.

![](https://static.igem.wiki/teams/5818/wiki-image/results10.webp)

References
*![](https://static.igem.wiki/teams/5818/wiki-image/down.webp)*

Garza R, Gregory J F, Hanson A D. Folate biofortification of tomato fruit[J]. Proceedings of the National Academy of Sciences of the United States of America, 2007,104(10):4218-4222.
Liang Q J, Wang K, Liu X N et al. Improved folate accumulation in genetically modified maize and wheat[J]. Journal of Experimental Botany, 2019,70(5):1539-1551.
Waller J C, Akhtar T A, Lara-Nunez A et al. Developmental and feedforward control of the expression of folate biosynthesis genes in tomato fruit[J]. Molecular Plant, 2010,3(1):66-77.
Zamberlan F. Metabolic engineering Forming folate-fortified rice[J]. Nature Chemical Biology, 2024,20(11):1388.
Agyenim-Boateng K G, Zhang S, Islam M S, et al. Profiling of naturally occurring folates in a diverse soybean germplasm by HPLC-MS/MS. Food Chem. 2022 Aug 1;384:132520

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