iGEM 2024 — Team subcat-shanghai

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/5189/desc-banner.png)

Notebook

Date: 8.3

Date: 8.4

Date: 8.5

Date: 8.6

Date: 8.7

Date: 8.8

Date: 8.9

Date: 8.10

Date: 8.11

Date: 8.12

Date: 8.13-8.23

Date: 8.3

**Participants: Yi Zhang, Siyuan Zhou, Zhichen Jin, Bohan Zhu, Liyiyang Tao, Wenbo Xu, Xinyi Yan, Yuxin Wu, Leyao Li, XueSong Lu, Yuncong Dong, YuFei Li**

Procedures

* Rinsing Equipment
  inse all equipment with double-distilled water.
* LB broth Preparation
  Dissolve 25 g of LB powder in 1 liter of water, adjust pH to 7.0, and autoclave at 121°C for 15 minutes.
* Solid Medium Preparation
  Add 15 g of agar to the LB broth, pour into petri dishes, and allow to solidify.
* Sealing
  Seal conical flasks with parafilm and rubber bands.
* Sterilization
  Autoclave sealed flasks at 121°C for 20 minutes.

Date: 8.4

**Participants: Zhichen Jin, Wenbo Xu, Yixin Yan, Shuhan Hu, Yi Zhang, Yuxin Wu**

Procedures

* ET Bacterial Culture
  Inoculate *E. coli*  into shaking culture tubes containing LB liquid medium with ampicillin resistance under a clean bench. Incubate overnight in a 37°C shaking incubator. The following day, the LB broth should appear turbid, indicating successful amplification of E. coli.
* Gel Recovery
* Plasmid Extraction
* Configure PCR System
* Configuration of Agarose Gel
  Weigh 0.5 g of agarose powder and add it to a 50 mL conical flask containing 50 mL of TAE buffer.
* Run the Gel
  Turn on the electrophoresis tank, set it to 180 V, and run for 30 minutes. Then, turn off the electrophoresis tank.

  ![](https://static.igem.wiki/teams/5189/notebook/1.jpg)
* Gel Imaging
  Transfer the gel to the gel imager for UV irradiation and observe the band sizes. The results are as follows:

  ![](https://static.igem.wiki/teams/5189/notebook/2.jpg)
* Cutting Gel
  Excise the agarose containing the target gene under external light.

Date: 8.5

**Participants: Yi Zhang, Siyuan Zhou, Zhichen Jin, Bohan Zhu, Liyiyang Tao, Wenbo Xu, Xinyi Yan, Yuxin Wu, Leyao Li**

Procedures

* Measuring pRSF Sample Using Nanodrop

  ![](https://static.igem.wiki/teams/5189/notebook/3.jpg)

  Fig.1 At a wavelength of 260 nm, the absorbance of pRSF is 0.349; at 280 nm, it is 0.197; and at 230 nm, it is 0.322. The concentration of DNA in pRSF is 17.45 ng/µL.
* Cutting the pRSF Plasmid
* Digestion of the *metF*  Gene and *ftfL*  Gene
* Gel Electrophoresis
* Analysis of Gel Electrophoresis

  ![](https://static.igem.wiki/teams/5189/notebook/4.jpg)

  In addition to conventional annotation, the brightness of the electrophoretic image should be increased without any retouching.
  Fig 2. The first result is the marker; the second is pRSF, and the third and fourth results are for the *metF*  gene.
* Preparation for Recycling of Agarose
  Ensure that the washing buffer contains 45 mL of absolute ethanol.
* Recycling of Agarose
* Combining pRSF and *metF*  gene, pET and *ftfL*  Gene

Date: 8.6

**Participants: Yi Zhang, Siyuan Zhou, Zhichen Jin, Bohan Zhu, Liyiyang Tao, Wenbo Xu, Xinyi Yan, Yuxin Wu, Leyao Li**

Procedures

* Recombinant Plasmid Transformation in the *E. coli*  DH5α
  Put the solution on a petri dish with a solid LB broth with kanamycin
  Put in a 37°C incubator

Date: 8.7

**Participants: Yi Zhang, Siyuan Zhou, Zhichen Jin, Bohan Zhu, Liyiyang Tao, Wenbo Xu, Xinyi Yan, Yuxin Wu, Leyao Li, XueSong Lu, Yuncong Dong, YuFei Li**

Procedures

* Preparation of Mixture for 5 Single Colonies
  Add 90 μL of 2× Mix Star, 72 μL of ddH₂O, and 6 μL each of Met-F and Met-R to a small tube. Gently rotate the tube to mix. For the second mixture, combine 90 μL of 2× Mix Star, 72 μL of ddH₂O, and 6 μL each of FtfL-F and FtfL-R in a separate small tube. Gently rotate the tube to mix.
* Extraction of Single Colonies
  Transfer the colonies to the prepared mixtures.
* Polymerase Chain Reaction (PCR)
  Prepare the gel for electrophoresis.
* Gel Electrophoresis
  Perform gel electrophoresis to verify whether the target fragments have been successfully transferred into E. coli.

Date: 8.8

**Participants: Yi Zhang, Siyuan Zhou, Zhichen Jin, Bohan Zhu, Liyiyang Tao, Wenbo Xu, Xinyi Yan, Yuxin Wu, Leyao Li, XueSong Lu, Yuncong Dong, YuFei Li**

Procedures

* Plasmid Extraction and Enzyme Digestion Verification
  Combine 5 µL of pRSF-metF-1, pRSF-metF-2, and pRSF-metF-3 with 3 µL of 10× green buffer, 1 µL of BamHI, 1 µL of HindIII, and 20 µL of ddH₂O in separate PCR tubes (30 µL per tube) using pipettes.
* Selection of the Correct Plasmid and Target Gene folA for Double Enzyme Digestion
  Ligate folA to pRSF-metF and culture overnight at 16°C. Similarly, ligate mtdA-fchA to pET-ftfL and culture overnight at 16°C.
* Enzymatic Digestion and Gel Recovery

Date: 8.9

**Participants: Yi Zhang, Siyuan Zhou, Zhichen Jin, Bohan Zhu, Liyiyang Tao, Wenbo Xu, Xinyi Yan, Yuxin Wu, Leyao Li, XueSong Lu, Yuncong Dong, YuFei Li**

Procedures

* Transformation in *E. coli* BL21 (DE3)
* Preparation of the LB broth

Date: 8.10

**Participants: Yi Zhang, Siyuan Zhou, Zhichen Jin, Bohan Zhu, Liyiyang Tao, Wenbo Xu, Xinyi Yan, Yuxin Wu, Leyao Li, XueSong Lu, Yuncong Dong, YuFei Li**

Procedures

* Preparation of Mixtures for Single Colonies
  Add 90 μL of 2x Mix Star, 72 μL of ddH₂O, and 6 μL each of folA-F and folA-R to a small tube. Gently rotate the tube to mix. Prepare another mixture by adding 90 μL of 2x Mix Star, 72 μL of ddH₂O, and 6 μL each of mtdA-fchA-F and mtdA-fchA-R to a separate small tube.
* Extraction and Transfer of Single Colonies
  Extract single colonies and transfer them to the prepared mixtures.
* PCR
  Perform PCR as needed.
* Preparation of Gel for Electrophoresis
  Prepare the gel for electrophoresis.
* Gel Electrophoresis
  Conduct gel electrophoresis to verify the presence of target fragments transferred into E. coli.
* ET Bacterial Culture
  Inoculate *E. coli*  into shaking culture tubes containing LB liquid medium with ampicillin resistance under a clean bench. Incubate overnight in a 37°C shaking incubator. The following day, the LB broth should be turbid, indicating successful amplification of E. coli.

Date: 8.11

**Participants: Yi Zhang, Siyuan Zhou, Zhichen Jin, Bohan Zhu, Liyiyang Tao, Wenbo Xu, Xinyi Yan, Yuxin Wu, Leyao Li, XueSong Lu, Yuncong Dong, YuFei Li**

Procedures

* Preparation of SDS-PAGE
* One-step Growth Curve Measurement
  Sampling and measuring the bacterial culture's OD600 every two hours for 12 consecutive hours.
* A 250 mL culture of LB broth, supplemented with the appropriate antibiotics, was inoculated with the bacterial culture and incubated on a shaker at 37°C until the OD600 reached a value between 0.5 and 0.6. Subsequently, IPTG was added to the medium, and the culture was further incubated overnight on a shaker at 18°C.

Date: 8.12

**Participants: Yi Zhang, Siyuan Zhou, Zhichen Jin, Bohan Zhu, Liyiyang Tao, Wenbo Xu, Xinyi Yan, Yuxin Wu, Leyao Li, XueSong Lu, Yuncong Dong, YuFei Li**

Procedures

* Bacterial cultures were harvested by centrifugation.
* Ultrasonic cell disruption was performed for protein extraction.
* The protein samples were loaded onto an SDS-PAGE gel for separation, followed by staining with *Coomassie* Brilliant Blue and subsequent destaining.

Date: 8.13-8.23

**Participants: Yi Zhang, Siyuan Zhou, Zhichen Jin, Bohan Zhu, Liyiyang Tao, Wenbo Xu, Xinyi Yan, Yuxin Wu, Leyao Li, XueSong Lu, Yuncong Dong, YuFei Li**

Procedures

* Compiling and analyzing experimental data into a coherent format, followed by the composition of a comprehensive wiki entry detailing the findings and methodology

{% 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. This achievement should be distinct from your Contribution for Bronze.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/5189/desc-banner.png)

Engineering Success

Overview

Part 1: pRSFDuet-metF-folA

Part 2. *pETduet-ftfL-mtdA-fchA*

Functional Test

HPLC assay for L-5-MTHF yield

References

Overview

Our project focuses on enhancing the production of L-5-methyltetrahydrofolate (*L-5-MTHF*) by engineering E. coli strains through targeted modifications. We constructed two key plasmids, *pRSF-metF-folA* and *pET-ftfL-mtdA-fchA*, each designed to express the enzymes crucial for synthesizing *L-5-MTHF*. The conversion process begins with folic acid, which is reduced to THF by DHFR encoded by the folA gene. THF is then converted to N¹⁰-formyl-THF by FTFL, followed by a series of enzymatic transformations involving Formyl-THF Cyclohydrolase (FCHA, *fchA*) and Methylene-THF Dehydrogenase (MTDA, *mtdA*). The final step consists of the reduction of N⁵, N¹⁰-methylene-THF to the bioactive *L-5-MTHF* by Methylenetetrahydrofolate Reductase (MTHFR, metF). By constructing plasmids that drive the expression of these key enzymes, we aim to improve the efficiency of this conversion process, thereby boosting *L-5-MTHF* production in E. coli.

![](https://static.igem.wiki/teams/5189/engineer/1.jpg)

Figure 1. The engineering design schematic diagram

Part 1: pRSFDuet-metF-folA

Design 1:

The pRSFDuet-metF-folA plasmid was constructed by selecting and amplifying the metF and folA genes to optimize the *L-5-MTHF* production pathway. The pRSFduet-1 vector was chosen due to its capability to accommodate multiple gene insertions and its strong, regulated expression under the T7 promoter. The metF gene (894 bp) and folA gene (480 bp) were amplified via PCR and inserted into the pRSFDuet-1 vector.

![](https://static.igem.wiki/teams/5189/engineer/2.png)

Figure 2. The plasmid map of pRSFDuet-metF-folA.

Build 2:

The metF and folA genes were successfully amplified using PCR, yielding bands of 894 bp and 480 bp, respectively. The metF gene was inserted into the pRSFDuet-1 vector by digestion with BamHI and HindIII, while the folA gene was inserted into the vector by digestion with NdeI and XhoI. The recombinant plasmid was then transformed into E. coli DH5α. Validation was performed using colony PCR and enzyme digestion, and the results confirmed successful ligation, as evident from the expected band sizes in gel electrophoresis.

![](https://static.igem.wiki/teams/5189/engineer/3.png)

Figure 3. The gel electrophoresis validation of metF (Left) and folA (Right) nucleic acids.

Upon verifying the successful amplification of the targeted plasmid, they were then transformed into E.coli DH5α. We selected colonies and sequenced them.

![](https://static.igem.wiki/teams/5189/engineer/4.png)

Figure 4. Transformation plate of pRSFDuet-metF-folA (A); Enzyme digestion verification for DH5α: pRSF-metF (B), *pRSF-metF-folA* (C); Sequencing results of pRSFDuet-metF-folA (D).

Test 1:

The pRSFDuet-metF-folA plasmid was transformed into E. coli BL21 (DE3) to evaluate the co-expression of the metF and folA genes. Protein expression was induced using IPTG and analyzed via SDS-PAGE and Western Blot techniques. The SDS-PAGE results displayed distinct bands corresponding to the metF and folA proteins, particularly under induction at 37 ℃. Western Blot analysis confirmed the successful expression of all proteins, demonstrating effective co-expression.

![](https://static.igem.wiki/teams/5189/engineer/5.png)

Figure 5. Expression of metF and folA Proteins in BL21 (DE3) Analyzed by SDS-PAGE (left) and Western Blot (right)

Learn 1:

The enzyme digestion verification confirmed that the *pRSF-metF-folA* plasmid was successfully constructed, as evidenced by the expected target bands in the gel electrophoresis results. The successful amplification and ligation of the metF and folA genes demonstrate the effectiveness of the selected restriction sites and the compatibility of the pRSFDuet-1 vector for multi-gene insertion. Moving forward, the focus will be on optimizing the expression conditions for metF and folA in E. coli BL21 (DE3), with particular attention to factors such as induction temperature, IPTG concentration, and post-transformation growth conditions. Additionally, exploring different promoter strengths and ribosome binding site sequences could further enhance protein expression levels, ensuring a more efficient *L-5-MTHF* production pathway.

Part 2. *pETduet-ftfL-mtdA-fchA*

Design 2:

The *pETduet-ftfL-mtdA-fchA* plasmid was constructed with the objective of enhancing the *L-5-MTHF* synthesis pathway by co-expressing the *ftfL*, *mtdA*, and *fchA* genes. The pETduet-1 vector was selected for its dual-expression system, utilizing the T7 promoter to drive synchronized expression of these critical enzymes. The *ftfL* gene (1685 bp) was inserted first, followed by the *mtdA-fchA* fragment (1488 bp), ensuring that all genes were under the control of the T7 promoter for optimal co-expression in E. coli BL21 (DE3).

![](https://static.igem.wiki/teams/5189/engineer/6.png)

Figure 6. The plasmid map of *pETduet-ftfL-mtdA-fchA*.

Build 2:

The *ftfL* gene (1685 bp) and the *mtdA-fchA* fragment (1488 bp) were successfully amplified using PCR. The *ftfL* gene was inserted into the pETduet-1 vector by digestion with BamHI and HindIII, while the *mtdA-fchA* fragment was inserted by digestion with NdeI and KpnI. The resulting plasmid was transformed into E. coli DH5α. Validation was performed using colony PCR and enzyme digestion, and the results confirmed successful ligation, as indicated by the expected band sizes in gel electrophoresis.

![](https://static.igem.wiki/teams/5189/engineer/7.png)

Figure 7. The gel electrophoresis validation of *ftfL* (Left), *mtdA*, and *fchA* (Right) nucleic acids.

Upon verifying the successful amplification of the targeted plasmid, they were then transformed into E.coli DH5α. We selected colonies and sequenced them.

![](https://static.igem.wiki/teams/5189/engineer/8.png)

Figure 8. Transformation plate of pETDue-ftfL-mtdA-fchA (A);Enzyme digestion verification for DH5α: pETDue-ftfL F (B), pETDue-ftfL-mtdA-fchA (C);Sequencing results of pETDue-ftfL-mtdA-fchA (D).

Test 2:

The *pETduet-ftfL-mtdA-fchA* plasmid was transformed into E. coli BL21 (DE3) to evaluate the co-expression of the *ftfL*, *mtdA*, and *fchA* genes. Protein expression was induced using IPTG and analyzed via SDS-PAGE and Western Blot techniques. The SDS-PAGE results displayed distinct bands corresponding to the *ftfL*, MtdA, and *fchA* proteins, particularly under induction at 37 ℃. Western Blot analysis confirmed the successful expression of all three proteins, demonstrating effective co-expression.

![](https://static.igem.wiki/teams/5189/engineer/9.png)

Figure 9. Expression of metF, folA, *mtdA*, *fchA*, and *ftfL* Proteins in BL21 (DE3) Analyzed by SDS-PAGE (left) and Western Blot (right)

Functional Test

One-Step Growth Curve Analysis

A one-step growth curve was generated to compare the growth rates of the different strains. The control strain, BL21, initially entered a rapid growth phase, transitioning into the stationary phase after approximately 10 hours. In contrast, the strains *pRSF-metF-folA*, *pET-ftfL-mtdA-fchA*, and Strain A (which contains both the pRSF and pET plasmids) exhibited slower initial growth rates compared to the control strain. Notably, Strain A showed continued growth even at the 12-hour mark, indicating a prolonged exponential growth phase. This suggests that Strain A may have a higher potential for sustained growth, likely due to the combined effects of both plasmids, which enhance *L-5-MTHF* production and lead to a more robust metabolic capacity under the given conditions.

![](https://static.igem.wiki/teams/5189/engineer/10.png)

Figure 10: One-Step Growth Curve for BL21, *pRSF-metF-folA*, *pET-ftfL-mtdA-fchA*, and Strain A.

The successful co-expression of *ftfL*, *mtdA*, and *fchA* genes confirmed the effectiveness of the plasmid design and construction process. However, the data also suggested that the expression levels of the three proteins were not perfectly balanced, indicating room for optimization. The expression was notably more efficient at 37 ℃, which suggests that this temperature is more suitable for the co-expression of these genes.

HPLC assay for L-5-MTHF yield

To further determine the actual yield of L-5-MTHF, we utilised HPLC. Firstly, we inoculated the constructed host bacteria into 100 mL of LB medium at 1% inoculum, supplemented the medium with folic acid (0.13 g/L) and sodium formate (0.013 g/L), and incubated the medium at 37℃ and 220 rpm until the OD600 value was 0.8, then added IPTG at a final concentration of 0.8 mM, and the temperature was lowered to 18℃, and the rotational speed was lowered to 110 rpm. Induction culture was carried out for 12-48h.

Subsequently, the samples were processed, 50 mL of bacterial solution was taken, centrifuged at 4°C, 6000 rpm for 10 min, the supernatant was discarded, and resuspended to the same volume in an anaerobic chamber with Tris-HCl (pH 7.2) containing 0.1% ascorbic acid and 0.1% mercaptoethanol, sealed in an anaerobic vial, and the cells were broken by boiling for 10 min in a 100°C water bath and immediately ice-bathed. 15, 000 rpm at 4°C, 15 min after centrifugation, and the supernatant was collected, fresh mouse serum (100 μl/mL)37 was added for 3 h, and the reaction was terminated by boiling for 5 min. 000 rpm, 4°C centrifugation for 15 min and collect the supernatant, add fresh mouse serum (100 μl/mL)37 reaction for 3 h, boil for 5 min to terminate the reaction, centrifugation to take the supernatant, filtered with 0.22 μm cellulose acetate filter membrane, and loaded into the liquid-phase vial. The product measured experimentally was L-5-MTHF, and its HPLC conditions were fluorescence detector Ex=290 nm, Em=356 nm.

As can be seen from Table 1, the actual amount of L-5-MTHF produced by strain A after co-transformation was higher compared to the control BL21 strain, which indicates that the addition of folic acid ultimately produces biologically active L-5-MTHF by the enzyme expressed by strain A.

Table 1 L-5-MTHF concentration of BL21 and Strains A

| Time | L-5-MTHF concentration of BL21 (16℃) | L-5-MTHF concentration of BL21 (37℃) | L-5-MTHF concentration of Strains A (16℃) | L-5-MTHF concentration of Strains A (37℃) |
| --- | --- | --- | --- | --- |
| 0h | 0.308 | 0.268 | 0.451 | 0.4 |
|  | 0.3 | 0.27 | 0.449 | 0.398 |
| 2h | 0.322 | 0.222 | 0.605 | 0.488 |
|  | 0.31 | 0.220 | 0.61 | 0.501 |
| 4h | 0.315 | 0.250 | 0.697 | 0.550 |
|  | 0.31 | 0.249 | 0.7 | 0.56 |
| 10h | 0.328 | 0.234 | 0.719 | 0.608 |
|  | 0.32 | 0.233 | 0.72 | 0.609 |
| 22h | 0.375 | 0.276 | 0.836 | 0.714 |
|  | 0.36 | 0.267 | 0.838 | 0.72 |
| 32h | 0.304 | 0.228 | 0.946 | 0.821 |
|  | 0.3 | 0.2 | 0.95 | 0.811 |
| 48h | 0.331 | 0.256 | 1.498 | 0.926 |
|  | 0.3 | 0.3 | 1.52 | 0.937 |

In addition, as can be seen from Fig. 11, the data measured for the control strain BL21 did not fluctuate significantly with the prolongation of the incubation time; whereas for the co-transformed strain A, the final production of active L-5-MTHF increased gradually and reached the maximum value at 48 h. Due to the oxidation of samples and depletion of samples during the experimental sampling process, the actual production of active L-5-MTHF should be slightly higher than the measured value. It is worth affirming that we successfully increased the yield of L-5-MTHF by genetically engineering the metabolic pathway of E. coli BL21.

![](https://static.igem.wiki/teams/5189/engineer/11.jpeg)

Figure 11. Variation of L-5-MTHF production over time.

**Summary**

In conclusion, we successfully increased the production of L-5-MTHF by genetically engineering the metabolic pathway of E. coli BL21. Notably, the product of methionine synthase MTRR (encoded by the metH gene) in the metabolic pathway inhibits the activity of MTHFR in the L-5-MTHF synthesis pathway, which further affects the yield of L-5-MTHF. Therefore, in the future, we will continue to study the metH gene in the metabolic pathway and explore the effect of its knockdown on the whole metabolic pathway and the strain itself.

References

[1]Ismail S, Eljazzar S, Ganji V. 2023. Intended and Unintended Benefits of Folic Acid Fortification—A Narrative Review. Foods 12:1612.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/5189/desc-banner.png)

Experiments

1.1.Disposition of LB Culture Base

2.The construction of plasmid

3.Protein expression

4.Functional Test

1.Disposition of LB Culture Base

**Goal:** To create a functional and nutritional liquid environment for growing bacteria. Liquid media serve various purposes, including the propagation of microorganisms and other experimental tests.

Materials:

1) Sterilized Duran bottle and cap
2) Autoclave
3) Tryptone
4) Yeast extract
5) Sodium chloride
6) Double-distilled H₂O

Procedure:

1) Create LB Medium (used for maintaining and cultivating recombinant strains of E.coli):
2) Prepare 14g of premix LB broth powder (Tryptone: Yeast extract: Sodium chloride = 2:1:1).
3) Add into a sterilised 1L Duran bottle.
4) Add double distilled H2O to a total volume of 700mL.
5) Close the cap and autoclave it for at least 20 minutes.
6) Wait until it cools.
7) Store at 4°C.

2.The construction of plasmid

2.1Construction of pRSFDuet-metF, pETDuet-ftfL

A.Obtaining target gene fragments by PCR

**Goal:** To amplify pTracK99 and RIP fragments for subsequent recombination.

Materials:

1) 2 × PrimeMixStar
2) metF and ftfL gene templates
3) double-distilled water (ddH₂O)
4) Primer-R
5) Primer-F
6) Centrifuge tube
7) PCR thermal cycler
\* PrimeMixStar includes buffer and dNTPs for PCR. The extension rate for PrimeMixStar (2 × ) is 2000 bp/min; the time varies with different templates.

Procedure:

1) Add 25 μL of 2 × PrimeMixStar, 1 μL of gene template, 1 μL of Primer-R, and 1 μL of Primer-F to a centrifuge tube. Add ddH₂O to the tube until the total volume reaches 50 μL. If bubbles are present in the mixture, centrifuge briefly to remove them.
2) Insert the mixture into a PCR thermal cycler. Denature the fragments at 95 ℃ for 3 minutes.
3) Perform 30 cycles consisting of 30 seconds of denaturation at 95 ℃, 30 seconds of annealing at 55 ℃, and 2 minutes of extension at 72 ℃.
4) Extend the fragments for 5 minutes at 72 ℃.
5) If not used immediately, store the fragments at 4 ℃.
6) Repeat steps 1-5 for RIP fragments, using the appropriate template and primers for RIP.

B.Agarose gel construction, deployment and recycle

**Goal:** To produce a functional and well-formed gel for electrophoresis.

Materials:

1) Casting tray
2) Well comb
3) Microwave
4) 1× TAE
5) Agarose
6) 10, 000× nucleic acid gel stain
7) Erlenmeyer flask

Procedure:

1) Dissolve 0.3g Agarose in a 30mL Erlenmeyer flask.
2) Add TAE (Running buffer).
3) Heat the material with a microwave.
4) Melt the mixture at 30-second intervals.
5) See whether bubbles are forming if successfully mixed.
6) Add 3μL nucleic acid gel stain (10000x).
7) Let it cool for 5-10 minutes.
8) Pour the agarose mixture into the casting tray.
9) Place the appropriate well comb to create the wells.
10) Wait to solidify for 7-10 minutes.
11) Remove the comb and place the gel in the gel box.

C.Agarose gel electrophoresis

**Goal:** To verify the lengths of DNA bands and prepare for gel extraction by isolating bands of the correct lengt h, thereby removing genetic fragments that did not successfully undergo PCR.

Materials:

1) Sample

| Sample | Amount |
| --- | --- |
| PCR result | 100+ |

2) Marker (DNA ladder)
3) Prepared gel (from the previous step)
4) 10 × Loading buffer (dye)
5) Horizontal gel electrophoresis system

Procedure:

1) Extract 50µL of every sample; add 5µL of loading buffer (10x) to each.
2) Place the gel in the horizontal electrophoresis system.
3) Extract 50µL of the mixed solution containing the sample and mix until the colours are almost uniform.
4) Add the marker to the first well and load 50µL of each sample into the wells.
5) The procedure runs at 160V for 30 minutes.
6) Afterwards, take out the gel from the device and proceed to the following steps.

D.DNA Gel Extraction

**Goal:** To recover DNA fragments from agarose gel.

Materials:

• Agarose gel with PCR result
• Clean scalpel
• 1.5 mL Eppendorf (EP) tube
• Pipettes and sterilised pipette heads
• Hot water bath
• Buffer B2
• Purification column and collection tube
• Centrifuge
• Wash solution containing pure ethanol
• Elution buffer

Procedure:

1) Cut out the portion of the gel containing the PCR result, remove as much excess gel as possible, and place it in a 1.5 mL Eppendorf (EP) tube.
2) Add 500 μL of buffer B2 to the tube and incubate in a 50 ℃ hot water bath until the gel has completely melted.
3) Transfer the solution containing the melted gel to a purification column placed in a collection tube, and centrifuge at 8, 000 × g for 30 seconds.
4) Discard the flow-through from the collection tube, place the purification column back in the tube, and add 500 μL of wash solution containing pure ethanol. Centrifuge at 9, 000 × g for 30 seconds, then discard the flow-through again.
5) Repeat the washing step.
6) Perform a final centrifugation at 9, 000 × g for 1 minute. Open the tube cap and allow it to air dry for 1 minute to ensure complete evaporation of ethanol.
7) Transfer the purification column to a fresh 1.5 mL EP tube. Add 20 μL of elution buffer to the center of the purification column.
8) Close the lid and let the tube sit for 1 minute. Centrifuge at 9, 000 × g for 1 minute.
9) Discard the purification column. Store the eluted DNA in the EP tube at 4 ℃.

E.Digestion reaction of PCR products and Plasmids and ligand reaction

**Goal:** Creating the vector pRSFDuet-metF and pETDuet-ftfL.

Materials & Apparatus:

• Green digestion buffer (10x)
• Double distilled water (ddH2O)
• PCR products & plasmid
• Nanodrop
• Water bath

Procedure:

1 Test the concentration of PCR products and plasmids using the Nanodrop.
2 Prepare the digestion reaction system (30 μL).

| Component | Volume |
| --- | --- |
| Green digestion buffer (10 ×) | 3 μL |
| Vector (pRSFduet, pETduet) | 5 μL |
| BamHⅠ | 1 μL |
| HindⅢ | 1 μL |
| ddH2O | Fill to 30 μL |

| Component | Volume |
| --- | --- |
| Green digestion buffer (10 ×) | 3 μL |
| Target genes (metF, ftfL) | 25 μL |
| BamHⅠ | 1 μL |
| HindⅢ | 1 μL |
| ddH2O | Fill to 30 μL |

3 Place the system in a 37 ℃ water bath for 2 h
4 Agarose gel electrophoresis and DNA Gel Extraction
5 Prepare the ligand reaction system (10 μL)

| Component | Volume |
| --- | --- |
| Ligand reaction buffer (10 ×) | 1 µL |
| Vector (pRSFduet) | 3 µL |
| Ligase | 1 µL |
| Target genes (metF) | 5 µL |
| ddH2O | Fill to 10 µL |

| Component | Volume |
| --- | --- |
| Ligand reaction buffer (10 ×) | 1 µL |
| Vector (pETduet) | 3 µL |
| Ligase | 1 µL |
| Target genes (ftfL) | 5 µL |
| ddH2O | Fill to 10 µL |

6 Place the system in a 16 ℃ water bath overnight

F.Transformation

Materials:

Ligated samples, LB Agar plates and liquid solution

Procedure:

a) Ligated samples are added to E. coli samples.
b) Place on ice for 20 minutes.
c) Heat at 42 degrees Celsius for 45 seconds, then immediately put on ice for 2-3 minutes.
d) Add 900μL LB Agar solution and incubate for 30 minutes.
e) Centrifuge at 5000xg for 3 minutes, extract 900μL of supernatant, then discard.
f) Apply end product to agar plates, incubate for 12-16 hours at 37 degrees Celsius.
\* Possible mistake: It’s required to spread bacteria gently since harsh physical shock will damage their structure.

G.Monoclonal Antibody Validation

**Goal:** DH5α bacteria containing constructed plasmids have multiplied. Monoclonal Antibody Validation ensures that the plasmids have replicated during this process.

Procedure:

| PCR Program Step | Temperature (°C) | Period |
| --- | --- | --- |
| Pre-denaturation | 95 | 3min. |
| Denaturation | 95 | 30sec. |
| Annealing | 55 | 30sec. |
| Elongation | 72 | 2min. |
| Repetition-Denaturation, Annealing, Elongation | / | 30times |

Procedure:

A) Obtain a conical flask. Use a measuring cylinder to measure 100 mL of (1x) TAE solution and transfer it to the conical flask.
B) Place the agarose sample on weighing paper. Use a digital balance and spatula to measure 1 g of the agarose sample and add it to the flask.
C) Place the flask in the microwave oven and heat at high temperature for 2 to 3 minutes.
D) Use a pipette to measure 5μL of nucleic acid dye (Gel Red) and add it to the flask.
E) Add the sample in the flask to the electrophoresis instrument mode.
F) Wait for approximately 20 minutes until the gel coagulates.

2.2 Construction of pRSFuet-metF-folA, pETDuet-ftfL-mtdA-fchA

A.PCR of target gene fragments

**Goal:** To amplify fragments for later combination

Materials:

1) 2 × PrimeMixStar
2) folA and mtdA-fchA gene template
3) double distilled water (ddH2O)
4) Primer-R
5) Primer-F
6) centrifuge tube
7) PCR thermal cycler
PrimeMixStar includes buffer and dNTPs for PCR. The extension rate for PrimestarMix (2 × ) is 2000bp/min; the time varies with different templates

Procedure:

1) Add 2 × PrimerMixStar 25 μL, template 1 μL, primer-R 1 μL, and primer-F 1 μL to centrifuge tube. Add ddH2O to the centrifuge tube until it reaches 50 μL. If bubbles are observed in the mixture, centrifuge it for a few seconds to remove them.
2) Insert the mixture into a PCR thermal cycler. Denature the fragments at 95 ℃ for 3 minutes
3) Perform 30 cycles of 30 seconds of 95 ℃ denaturing, 30 seconds of 55 ℃ annealing, and 2 minutes of 72 ℃ extension.
4) Extend the fragments for 5 minutes at 72 ℃
5) If not used immediately, preserve the fragments at 4 ℃
6) Repeat steps 1-5 to amplify folA and mtdA-fchA. Replace the template and corresponding primers with those of folA and mtdA-fchA.

B. Digestion reaction of PCR products and Plasmids and ligand reaction

**Goal:** creating the vector pRSFDuet-metF-folA, pETDuet-ftfL-mtdA-fchA

Materials & Apparatus:

• Green digestion buffer (10x)
• Double distilled water (ddH2O)
• PCR products & plasmid
• Nanodrop
• Water bath

Procedure:

1. test the concentration on PCR products and Plasmids using Nanodrop
2. prepare the digestion reaction system (30 μL)

| Digestion of Vector (pRSFDuet-metF) | |
| --- | --- |
| Reagent | Volume |
| Green digestion buffer (10 µL) | 3 µL |
| Vector (pRSFDuet-metF) | 5 µL |
| NdeI | 1 µL |
| NotI | 1 µL |
| ddH2O | fill to 30 µL |

| Digestion of Target Gene (folA) | |
| --- | --- |
| Reagent | Volume |
| Green digestion buffer (10 µL) | 3 µL |
| Target gene (folA) | 25 µL |
| NdeI | 1 µL |
| NotI | 1 µL |
| ddH2O | fill to 30 µL |

| Digestion of Vector (pETDuet-ftfL) | |
| --- | --- |
| Reagent | Volume |
| Green digestion buffer (10 µL) | 3 µL |
| Vector (pETDuet-ftfL) | 5 µL |
| NdeI | 1 µL |
| KpnI | 1 µL |
| ddH2O | fill to 30 µL |

| Digestion of Target Gene (mtdA-fchA) | |
| --- | --- |
| Reagent | Volume |
| Green digestion buffer (10 µL) | 3 µL |
| Target gene (mtdA-fchA) | 25 µL |
| NdeI | 1 µL |
| KpnI | 1 µL |
| ddH2O | fill to 30 µL |

3. place the system in a 37 ℃ water bath for 2h
4. agarose gel electrophoresis and DNA Gel Extraction
5. prepare the ligand reaction system (10 μL)

| Reaction Mix for Target Gene folA | |
| --- | --- |
| Component | Volume |
| Ligand Reaction Buffer (10 µL) | 1 µL |
| Vector (pRSFDuet-metF) | 3 µL |
| Ligase | 1 µL |
| Target Genes (folA) | 5 µL |
| ddH2O | Fill to 10 µL |

| Reaction Mix for Target Genes mtdA-fchA | |
| --- | --- |
| Component | Volume |
| Ligand Reaction Buffer (10 µL) | 1 µL |
| Vector (pETDuet-ftfL) | 3 µL |
| Ligase | 1 µL |
| Target Genes (mtdA-fchA) | 5 µL |
| ddH2O | Fill to 10 µL |

6. place the system in a 16 ℃ water bath overnight

C.Transformation of pRSFDut-metF-folA, pETDuet-ftfL-mtdA-fchA into DH5α via heatshock

**Goal:**

By transforming the plasmids into competent cells, quick replication of the plasmid copies is enabled, which provides the amount of plasmid we need for future experiments. By resistance selection, we can utilise the resistance gene initially designed on the plasmid to filter out the competent cells that don't have the needed transformed plasmids.

Transformation is conducted via the heat shock method.

Materials:

The materials used are similar to the ones in the former transformation process
• LB liquid culture medium (K+, A+) (1 mL)
• LB solid culture medium with kanamycin (K+) and ampicillin (A+)
• A box of ice
• DH5α (50 μL)
• pRSFDut-metF-folA, pETDuet-ftfL-mtdA-fchA (10 μL)

Equipment

• Water bath machine
• EP tubes
• Shaking incubator
• Mini-centrifuge
• Clean bench

Procedure:

1 Take out E. coli DH5α competent cells from -80 ℃ refrigerator;
2 Add 10 μL of pRSFDut-metF-folA and 50 μL of DH5α cells into an EP tube, and 10 μL of , pETDuet-ftfL-mtdA-fchA and 50 μL of DB3.1 cells into another DH5α tube. The process is repeated two times in total, so 2 samples of each are acquired.
3 Mix the EP tubes with the mini-centrifuge for 5 seconds
4 Put the EP tubes on ice for 30 minutes
5 Heat shock the samples at 42'C for 60 seconds in the water
6 Put the EP tubes in ice for 2 minutes
7 Transfer the bacteria samples from the EP tubes into K- a K-liquid LB medium. This step is performed on a clean bench.
8 Shake the cultures for 1 hour at 37 ℃, at 220rpm
9 Spread coat 50 μL on the petri dishes with K+, A+ LB solid culture medium, respectively. This step is also performed on the clean bench.

10 Inverted culture at 37 ℃ and 22 ℃ for 12-16 hours. This process will be explained in later sections

D.Plating and culturing of DH5α- pRSFDut-metF-folA and DH5α-pETDuet-ftfL-mtdA-fchA

**Goal:**Bacterial culture serves various roles in scientific research. In our project, our team performed sample plating to grow modified bacteria that will later be extracted to obtain the desired plasmid. In this case, we will culture DH5α- pRSFDut-metF-folA and DH5α-pETDuet-ftfL-mtdA-fchA.

Materials:

1) Disposable latex gloves
2) Pipette
3) Pipette tubes
4) 1L Duran bottle
5) Alcohol
6) LB medium
7) Black permanent marker
8) Sterile bacteria swabs
9) Sterile bacteria agar plates
10) Sterile collection tubes
11) Vertical clean flow bench
12) Bacteriological Incubator

Procedure:

1) Label all the materials
2) used correctly (empty plates, tubes)
3) Activate the UV light in the vertical clean flow bench to sterilise the environment inside all the materials, including the gloves used, go into a vertical clean flow bench sterilised through alcohol
4) Set up a bunsen burner--> creating a sterilised area created by the updraft of flame--> place to the right side of the bench--> every time using something, place the equipment near the fire
5) Use a pipette to pour 10 mL of LB medium onto plates and wait until it becomes solid
6) The 0.1 mL of bacteria sample (DH5α- pRSFDut-metF-folA and DH5α-pETDuet-ftfL-mtdA-fchA) pipetted onto the surface of the cooled agar plate.
7) Use sterilised bacteria swabs or spreader to spread the sample evenly over the surface of an agar plate
8) Use bacteriological incubation (37 degreelsis Celcius) to grow the desired modified bacteria

E.Bacterium picking ( monoclonal ) and shaking

**Goal:**To identify if the bacteria is correct and amplify the amount of the valid strain.

• Samples collected: RSF 1, 2, 3, 4 and ET 1, 2, 3, 4
• Performed under a vertical flow clean bench.

Protocol:

1. Add 5 mL of kanamycin-included LB medium into a 15 mL incubation tube.
2. Pick colonies using small pipette tips, and eject the tips into the incubation tubes containing the LB medium.
3. Incubate, shaking, at 37℃ for 12-16 hours.

F.Plasmid Extraction

**Goal:**Prepare the sample for electrophoresis to see whether it's correct.

Materials & Apparatus:

• Bacterial fluid of DH5α-pRSFDut-metF-folA and DH5α-pETDuet-ftfL-mtdA-fchA
• Centrifuge
• Various 1.5 mL EP tubes and absorption columns
• Sangon plasmid extraction kit
• Pipette & pipette tubes

Procedure:

1 Take all bacterial fluid into a centrifuge for 2 minutes at 8000×g and abandon all supernatant.
2 Add 250 μL Buffer P1 to the bacteria and let it resuspend.
3 Add 250 μL Buffer P2 and gently turn the centrifuge tube upside down 5-10 times to let it mix. Leave the centrifuge tube at room temperature for 2-4 minutes.
4 Add 350 μL Buffer P3, immediately and gently invert the centrifuge tube 5-10 times to mix thoroughly.
5 Centrifuge the tube at 12,000×g for 5-10 minutes. Carefully move all the supernatant into the adsorption column and centrifuge at 8000×g for 30 seconds. Drain the liquid in the collection tube.
6 Add 500 μL Wash Solution to the adsorption column, centrifuge at 9000×g for 30 seconds, and drain the liquid in the collection tube.
7 Repeat step 6 once.
8 Put the empty adsorption column and the collection tube together and centrifuge them at 9000×g for 1 minute.
9 Add 50-100 μL Elution Buffer to the center of the adsorption membrane. Leave the tube at room temperature for 1-2 minutes. Centrifuge it at 9000×g for 1 minute.

G.Verification of Extracted Plasmids

**Goal:**To produce a functional and well-shaped gel for electrophoresis

Materials:

1) Casting Tray
2) Well comb
3) Microwave
4) TAE
5) Agarose
6) Nucleic acid gel stain
7) Erlenmeyer flask

Procedure:

1 Dissolve 0.3g Agarose in 30 mL Erlenmeyer flask
2 Add TAE (Running buffer)
3 Heat the material with a microwave
4 Melt the mixture at 30-sec intervals
5 See whether bubbles are forming if successfully mixed
6 Add 3 μL nucleic acid gel stain (10000x)
7 Let it cool for 5-10 minutes
8 Pour the agarose mixture into the casting tray
9 Place the appropriate well comb to create the wells
10 Wait to solidify 7-10 minutes

H.Agarose gel electrophoresis

**Goal:**To verify the DNA bands' lengths of the plasmids extracted from the competent cells, thus preparing for the next step of gel extraction to extract the bands of the correct length.

Materials:

1) Sample

| Sample | Amount (µl) |
| --- | --- |
| pRSFDuet-metF-folA and pETDuet-ftfL-mtdA-fchA  (Plasmid extraction result) | 300+ |

2) Marker (DNA ladder)
3) Prepared gel (from the previous step)
4) 10 × Loading buffer (dye)
5) Horizontal gel electrophoresis system

Gel electrophoresis

1) Extract 50 μL of every sample; add 5 μL of loading buffer (10 ×) to each.
2) Place the gel in the horizontal electrophoresis system.
3) Extract 50 μL of the mixed solution containing the sample and mix them till their colours are almost uniform.
4) Add the marker to the first well and load 50 μL of each sample into the wells.
5) Run at 160V for 30 minutes.
6) Afterwards, take out the gel from the device and proceed to the following steps.

A.Transformation of pRSFDut-metF-folA and pETDuet-ftfL-mtdA-fchA into DB3.1 via heatshock

Goal

By transforming the plasmids into competent cells, quick replication of the plasmid copies is enabled, which provides the amount of plasmid we need for future experiments. By resistance selection, we can utilise the resistance gene initially designed on the plasmid to filter out the competent cells which don't have the needed transformed plasmids. Since DH5α cannot stand the toxicity the ccdB gene induces, DB3.1, an alternative competent cell that can withstand the toxicity, is used. Transformation is conducted via the heat shock method again.

Materials:

• LB liquid culture medium (K+, A+) (1ml)
• LB solid culture medium with kanamycin (K+)
• A box of ice
• pRSFDut-metF-folA and pETDuet-ftfL-mtdA-fchA (10μl)
• Equipment
• Water bath machine
• EP tubes
• Shaking incubator
• Mini-centrifuge
• Clean bench

Procedure:

1 Take out E. coli BL21 competent cells from -80°C refrigerator.
2 Add 10 μL of pRSFDut-metF-folA and pETDuet-ftfL-mtdA-fchA (K+, A+) and 50 μL of DB3.1 cells into an EP tube. Also, add 10 μL of pRSFDut-metF-folA (K+) and 50 μL of DB3.1 cells into another DB3.1 tube. Additionally, add pETDuet-ftfL-mtdA-fchA (A+) and 50 μL of DB3.1 cells into another DB3.1 tube. The process is repeated twice in total, so 2 samples of each are acquired.
3 Mix the EP tubes with the mini-centrifuge for 5 seconds.
4 Put the EP tubes on ice for 30 minutes.
5 Heat shock the samples at 42°C for 60 seconds in the water.
6 Put the EP tubes in ice for 2 minutes.
7 Transfer the bacterial samples from the EP tubes into a liquid LB medium containing K+, A+, and both, respectively. This step is performed on a clean bench.
8 Shake the cultures for 1 hour at 37°C, at 220 rpm.
9 Spread coat 50 μL on the Petri dishes with K+ LB solid culture medium, respectively. This step is also performed on the clean bench.
10 Inverted culture at 37 ℃ and 22 ℃ for 12-16 hours. This process will be explained in later sections

B.Plating and culturing of BL21-pRSFDuet-metF-folA, BL21-pETDuet-ftfL-mtdA-fchA and BL21-pRSFDuet-metF-folA-pETDuet-ftfL-mtdA-fchA

**Goal:**Bacterial culture serves various roles in scientific research. In our project, our team performed sample plating to grow modified bacteria that will later be extracted to obtain the desired plasmid. In this case, we will culture BL21-pRSFDuet-metF-folA, BL21-pETDuet-ftfL-mtdA-fchA and BL21-pRSFDuet-metF-folA-pETDuet-ftfL-mtdA-fchA.

Materials:

1) Disposable latex gloves
2) Pipette
3) Pipette tips
4) 1L Duran bottle
5) Alcohol
6) LB medium
7) Black permanent marker
8) Sterile bacteria swabs
9) Sterile bacteria agar plates
10) Sterile collection tubes
11) Bunsen burner
12) Vertical clean flow bench
13) Bacteriological Incubator

Procedures

1) Label all the materials used correctly (empty plates, tubes)
2) Activate the UV light in the vertical clean flow bench to sterilise the environment inside
3) All the materials, including the gloves used, go into a vertical clean flow bench sterilised through alcohol
4) Set up a bunsen burner → creating a sterilised area created by the updraft of flame → place to the right side of the bench → every time using something, place the equipment near the fire
5) Use a pipette to pour 10ml of LB medium onto plates and wait until it becomes solid
6) The 0.1 ml of bacteria sample (BL21-pRSFDuet-metF-folA, BL21-pETDuet-ftfL-mtdA-fchA, and BL21-pRSFDuet-metF-folA-pETDuet-ftfL-mtdA-fchA)
7) Pipetted onto the surface of the cooled agar plate
8) Use sterilised bacteria swabs or spreader to spread the sample evenly over the surface of an agar plate
9) Use bacteriological incubation (37°C) to grow the desired modified bacteria

C.Bacterium picking and shaking

**Goal:** To identify if the bacteria is correct and amplify the amount of the valid strain.

Samples collected: RSF 1, 2, 3, 4; ET 1, 2, 3, 4; Stain A 1, 2, 3, 4.
Performed under a Vertical flow clean bench.

Protocol:

1. Add 5 mL of kanamycin-included LB medium into a 15 mL incubation tube.
2. ick colonies using small pipette tips, and eject the tips into the incubation tubes containing the LB medium.
3. Incubate, shaking, at 37 ℃ for 12-16 hours.

D.Plasmid Extraction

**Goal:**preparing the sample so that it can go through electrophoresis to see whether it's correct.

Materials & Apparatus:

• Bacterial fluid of BL21-pRSFDuet-metF-folA, BL21-pETDuet-ftfL-mtdA-fchA, and BL21-pRSFDuet-metF-folA-pETDuet-ftfL-mtdA-fchA
• Centrifuge
• Various 1.5 mL EP tubes and absorption columns
• Sangon plasmid extraction kit
• Pipette & pipette tips

Procedures:

1 Take all bacterial fluid into a centrifuge for 2 minutes at 8000g and abandon all supernatant.
2 Add 250 μL Buffer P1 to the bacteria and let it resuspend.
3 Add 250 μL Buffer P2 and gently turn the centrifuge tube upside down 5-10 times to let it mix. Leave the centrifuge tube at room temperature for 2-4 minutes.
4 Add 350 μL Buffer P3 immediately and gently invert the centrifuge tube 5-10 times to mix thoroughly.
5 Centrifuge the tube at 12,000×g for 5-10 minutes. Carefully move all the supernatant into the adsorption column and centrifuge at 8000×g for 30 seconds. Drain the liquid in the collection tube.
6 Add 500 μL Wash Solution to the adsorption column, centrifuge at 9000×g for 30 seconds, and drain the liquid in the collection tube.
7 Repeat step 6 once.
8 Put the empty adsorption column and the collection tube together and centrifuge them at 9000×g for 1 minute.
9 Add 50-100 μL Elution Buffer to the center of the adsorption membrane. Leave the tube at room temperature for 1-2min. Centrifuge it at 9, 000× g for 1min.

E.Verfication of Extracted Plasmids

**Goal:**To produce a functional and well-shaped gel for electrophoresis

Materials:

1) Casting Tray
2) Well comb
3) Microwave
4) TAE
5) Agarose
6) Nucleic acid gel stain
7) Erlenmeyer flask

Procedure:

1) Dissolve 0.3 g Agarose in 30 mL Erlenmeyer flask.
2) Add TAE (Running buffer).
3) Heat the material with a microwave.
4) Melt the mixture at 30 sec intervals.
5) See whether bubbles are forming; if so, it has mixed successfully.
6) Add 3 μL nucleic acid gel stain (10000 ×).
7) Let it cool for 5-10 minutes.
8) Pour the agarose mixture into the casting tray.
9) Place the appropriate well comb to create the wells.
10) Wait to solidify for 7-10 minutes.
11) Remove the comb and place the gel in the gel box

G.Agarose gel electrophoresis

**Goal:**To verify the DNA bands' lengths of the plasmids extracted from the competent cells, thus preparing for the next step of gel extraction to extract the bands of the correct length.

Materials:

1) Sample

| Sample | Amount (µl) |
| --- | --- |
| DB3.1-pRSFDuet-metF-folA,  DB3.1-pETDuet-ftfL-mtdA-fchA,  and DB3.1-pRSFDuet-metF-folA-pETDuet-ftfL-mtdA-fchA  (Plasmid extraction result) | 400+ |

2) Marker (DNA ladder)
3) Prepared gel (from the previous step)
4) 10x Loading buffer (dye)
5) Horizontal gel electrophoresis system

Gel electrophoresis

1) Extract 50 µL of every sample; add 5 µL of loading buffer (10x) to each.
2) Place the gel in the horizontal electrophoresis system.
3) Extract 50 µL of the mixed solution containing the sample, and mix them till their colors are almost uniform.
4) Add the marker to the first well and load 50 µL of each sample into the wells.
5) The procedure runs at 160 V for 30 min.
6) Afterwards, take out the gel from the device and proceed to the following steps.

I. Snap the gel to identify the length of the PCR fragment

**Goal:**After running electrophoresis, we need to visualise the position of the DNA bands on the gel. We used a gel transilluminator to visualise the bands and collect the gel's image.

Materials:

• Agarose Gel containing DNA samples
• Gel transilluminator

Procedure:

1) Place the Gel inside the transilluminator
2) On a computer with software connected to the transilluminator, visualise the results
3) On the software, take the image of the gel under UV light and save the image
4) Take the gel out and dispose of it with regards to safety requirements.

J. DNA Gel Extraction

**Goal:**To recover DNA fragments in agarose gel.

Materials:

agarose gel with pRSFDuet-metF-folA, pETDuet-ftfL-mtdA-fchA and pRSFDuet-metF-folA-pETDuet-ftfL-mtdA-fchA
• clean scalpel
• 1.5 mL Eppendorf (EP) tube
• pipettes and sterilised pipette heads
• hot water bath
• buffer B2
• purification column and collection tube
• centrifuge
• wash solution containing pure ethanol
• elution buffer

Procedure:

1) Cut the slice of gel containing pRSFDuet-metF-folA, pETDuet-ftfL-mtdA-fchA, and pRSFDuet-metF-folA-pETDuet-ftfL-mtdA-fchA, cutting off as much unneeded gel as possible, and place it in a 1.5 mL Eppendorf (EP) tube.
2) Add 500 microliters of buffer B2 and put the tube into a 50°C hot water bath until the gel completely melts.
3) Transfer the solution containing melted gel into a purification column in a collection tube and perform centrifugation at 8000g for 30 seconds.
4) Empty the collection tube, put the purification column back in, and add 500 microliters of wash solution containing pure ethanol. Centrifuge at 9000g for 30 seconds, then empty the collection tube again.
5) Repeat the above step.
6) Perform centrifugation at 9000g for 1 minute, then open the tube cap and let it sit for one minute to allow the ethanol to evaporate.
7) Transfer the purification column to a 1.5 mL EP tube. Add 20 microliters of elution buffer at the center of the purification column.
8) Cap the lid and let the tube sit for 1 minute. Perform centrifugation at 9000g for 1 minute.
9) Discard the purification column. Store DNA in the EP tube at 4°C.
10) Induction of protein expression.

3.Induction of protein expression

A.pRSFDuet-metF-folA, pETDuet-ftfL-mtdA-fchA and pRSFDuet-metF-folA-pETDuet-ftfL-mtdA-fchA Amplification culture

**Goal:** to acquire large amounts of target proteins for SDS-PAGE

Materials & Apparatus:

• liquid LB
• pRSFDuet-metF-folA, pETDuet-ftfL-mtdA-fchA and pRSFDuet-metF-folA-pETDuet-ftfL-mtdA-fchA bacterial fluid
• kana amp
• inducer: Isopropyl-beta-D-thiogalactopyranoside (IPTG)
• isothermic shaker
• microplate reader

Procedure:

1) Add 100 μL of pRSFDuet-metF-folA bacterial fluid and 100 μL of kana into 100 mL of liquid LB,
Add 100 μL of pETDuet-ftfL-mtdA-fchA bacterial fluid and 100 μL of amp into 100 mL of liquid LB,
Add 100 μL of pRSFDuet-metF-folA and pETDuet-ftfL-mtdA-fchA bacterial fluid and 100 μL of amp, Kana into 100 mL of liquid LB,
2) place the sample in the isothermic shaker 0.5 h for culturing
3) Add inducers to the sample with concentrations of 0.2 mmol, 0.4 mmol, 0.6 mmol, 0.8 mmol, and 1 mmol respectively
4) Samples for each concentration are cultured at 22 ℃ and 37 ℃ for 2.5 h, 5 h, 10 h, 20 h, and 30h respectively
5) Test the concentration of all samples under the microplate reader

B. Protein Extraction and SDS-PAGE

**Goal:**to verify the expression of desired proteins Protein Extraction:

Materials:

• bacteria culture containing desired protein
• 50 mL centrifuge tubes
• beaker containing ice
• centrifuge
• sonicator

Procedure:

1) add samples into 50 mL centrifuge tubes, perform centrifugation at 4000 rpm for 20 minutes.
2) partially bury a centrifuge tube into a beaker filled with ice, then put the beaker and tube into the sonicator.
3) sonicate for 15 minutes.
4) repeat steps 2 and 3 for all samples.
5) perform centrifugation of samples at 4000 rpm for 15 minutes.
6) remove the cell fragments that have precipitated at the bottom of the tubes.
7) the remaining liquid will contain desired proteins.

C.SDS-PAGE

Materials:

• Sangon 12.5% SDS-PAGE Color Preparation kit
• pipette
• distilled water
• electrophoresis buffer
• protein ladder
• vertical electrophoresis system
• Coomassie blue

Procedure:

1) using a pipette, add and mix 2.2 mL of 2 × separating gel solution, 2.2 mL of 2 × separating gel buffer, and 44 μL of the catalyst in a plastic cup.
2) slowly inject the mixture into the casting stand and frame to avoid bubbles.
3) add 1 mL water to flatten out the top.
4) wait 8 minutes for the gel to set.
5) discard the water that was added previously.
6) using a pipette, add and mix 825 μL of 2 × stacking gel solution, 825 μL of 2 × stacking gel buffer, and 11 μL of catalyst in a plastic cup.
7) Add the stacking gel mixture until the cast is completely filled, then slowly insert the comb without producing any air bubbles.
8) wait 12 minutes for the gel to set, carefully remove the comb, and wash the wells with electrophoresis buffer.
9) add the protein ladder into the first well, then load samples in each successive well.
10)transfer gel into a vertical electrophoresis system.
11) set the voltage to 120V and run it for 90 minutes.
12) stain the gel with Coomassie blue by submerging it for 30 minutes.

D.Western Blot

Materials:

• Sample: Recombinant protein extract containing His-tag
• Primary Antibody: Anti-His-tag antibody
• Secondary Antibody: HRP-conjugated secondary antibody (corresponding to the primary antibody)
• Gel: SDS-PAGE gel
• Transfer Material: PVDF membrane
• Blocking Solution: BSA (Bovine Serum Albumin) solution
• Washing Solution: TBST (TBS with 0.1% Tween-20)

Procedure:

1.Sample Preparation:
Cell Lysis: Extract His-tagged recombinant protein using an appropriate lysis buffer (e.g., RIPA buffer).
Centrifugation: Centrifuge the lysate to collect the supernatant and remove cell debris.

2.SDS-PAGE Electrophoresis
Gel Preparation: Prepare a 10% SDS-PAGE gel for protein separation.
Sample Loading: Mix the protein sample with SDS loading buffer (containing reducing agents such as BME/Beta-mercaptoethanol) and heat to denature proteins before loading onto the gel.
Electrophoresis: Perform electrophoresis at an appropriate voltage (e.g., 80-120V) until the dye front reaches the bottom of the gel.

3.Protein Transfer (Dry Transfer)
Protein Transfer: Transfer proteins from the gel to a PVDF membrane using electroblotting. Transfer time is typically 1 hour (adjust depending on current and membrane thickness).

4.Blocking
Block the Membrane: Incubate the PVDF membrane in BSA blocking solution at room temperature for 1 hour to reduce non-specific binding.

5.Primary Antibody Incubation
Dilute Primary Antibody: Dilute the anti-His-tag primary antibody according to the manufacturer's instructions (typically 1:1000 to 1:5000).
Incubate Membrane: Incubate the membrane with the diluted primary antibody at room temperature for approximately 1.5 hours.

6.Washing the Membrane
Wash the Membrane: Wash the membrane 3-5 times with TBST, each for 5-10 minutes, to remove unbound primary antibody.

7.Secondary Antibody Incubation
Dilute Secondary Antibody: Dilute the HRP-conjugated secondary antibody according to the manufacturer's instructions (usually 1:2000 to 1:10000).
Incubate Membrane: Incubate the membrane with the diluted secondary antibody at room temperature for 1 hour.

8.Washing the Membrane Again
Wash the Membrane: Wash the membrane 3-5 times with TBST to remove unbound secondary antibody.

9.Detection
Chemiluminescent Detection: Use an appropriate substrate (e.g., ECL) to develop the HRP enzyme signal.
Imaging: Use a gel imaging system to capture the signal.

10.Data Analysis
Analyze the Data: Use image analysis software (e.g., ImageJ) to quantify the intensity of the bands and compare the expression levels of the target protein across different samples.

Important Notes:

• Antibody Dilution: Always dilute antibodies according to the manufacturer’s recommendations to ensure optimal specificity and sensitivity.
• Transfer Conditions: The efficiency of protein transfer is critical for accurate results. Adjust voltage and transfer time based on your equipment and membrane thickness.
• Washing Steps: Thorough washing is essential to reduce background signals and improve the reliability of results.

4.Functional testing

4.1 The growth ability of pRSFDuet-metF-folA, pETDuet-ftfL-mtdA-fchA and pRSFDuet-metF-folA-pETDuet-ftfL-mtdA-fchA in E.coil BL21.

Materials:

• E.coil BL21 (1000 μL)

Procedure:

1) In 100 mL LB medium, add 100 μL bacteria liquid.
2) Add 100 μL kan.
3) Culture 0D equal to 2 hours.
4) Adding different concentrations of inducers (N-(3-oxohexanoyl)-L-homoserinelactone(AI)), the concentrations were 0.2 mmol, 0.4 mmol, 0.6 mmol, 0.8 mmol, 1 mmol ;
5) Cultured at 18 degrees for 2 h, 4 h, 6 h, 8 h, 10 h, 120h; the concentration of bacterial solution was measured by microplate reader at OD600.

4.2 HPLC assay for the yield of active folic acid

Cultivation Conditions: Inoculate 100 mL of LB medium with 1% of the prepared host strain. Supplement the medium with folic acid (0.13 g/L) and sodium formate (0.013 g/L). Cultivate at 37°C and 220 rpm until the OD600 reaches 0.8. Then, add IPTG to a final concentration of 0.8 mM, reduce the temperature to 18°C, decrease the speed to 110 rpm, and induce for 12 hours.

Sample Processing Method: Take 50 mL of the cell culture and centrifuge at 4°C and 6000 rpm for 10 minutes. Discard the supernatant and resuspend the pellet in an anaerobic chamber with Tris-HCl (pH 7.2) containing 0.1% ascorbic acid and 0.1% β-mercaptoethanol, to the original volume. Seal in anaerobic vials and boil in a water bath at 100°C for 10 minutes to lyse the cells, then immediately place on ice. Collect the supernatant after centrifugation at 15,000 rpm and 4°C for 15 minutes. Add fresh mouse serum (100 μL/mL) and incubate at 37°C for 3 hours. Boil for 5 minutes to terminate the reaction, centrifuge to obtain the supernatant, filter through a 0.22 μm acetate cellulose membrane, and transfer to a liquid chromatography vial.

HPLC Conditions: Use a fluorescence detector with Ex = 290 nm and Em = 356 nm.

Preparation of Methyl-THF Standard: Accurately weigh 20 mg of methyl-THF standard powder and dissolve it in the mobile phase. Adjust the pH to complete dissolution with 1 M NaOH solution. Make up to 100 mL to obtain a 0.2 mg/mL methyl-THF solution and perform gradient dilution of this solution.

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{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
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![](https://static.igem.wiki/teams/5189/desc-banner.png)

Results

1. The construction of plasmid

2. Protein expression

3. Functional Test

4. Summary

Overview

*L-5-methyltetrahydrofolate* (L-5-MTHF) is the only biologically active form of folate in the human body. The production of L-5-MTHF using microbes is gaining attention as a method for green synthesis. However, microbes naturally produce only a small amount of L-5-MTHF. In this study, *Escherichia coli* BL21(DE3) was engineered to enhance the production of L-5-MTHF by overexpressing the intrinsic genes for dihydrofolate reductase *(folA)* and methylenetetrahydrofolate reductase *(metF)*, which were cloned into the pRSFDuet-1 vector to create the *pRSFDuet-metF-folA* plasmid. Additionally, genes encoding formate-THF ligase *(ftfL)*, formyl-THF cyclohydrolase *(fchA)*, and methylene-THF dehydrogenase *(mtdA)* were cloned into the *pETDuet-1* vector to form the *pETDuet-ftfL-mtdA-fchA* plasmid. By constructing these plasmids to drive the expression of key enzymes, we aim to improve the efficiency of the conversion process and thereby enhance L-5-MTHF production in *E. coli*.

1. The construction of plasmid

1.1 *pRSFDuet-metF-folA*

The *pRSFDuet-metF-folA* plasmid was constructed by selecting and amplifying the *metF* and *folA* genes to optimize the L-5-MTHF production pathway. The pRSFDuet-1 vector was chosen due to its capability to accommodate multiple gene insertions and its robust and regulated expression under the T7 promoter. The *metF* gene (894 bp) and *folA* gene (480 bp) were amplified via PCR and inserted into the pRSFDuet-1 vector.

![](https://static.igem.wiki/teams/5189/result/1.png)

*Figure 1: The gel electrophoresis validation of *metF*(Left) and *folA*(Right) nucleic acids.*

The *metF* and *folA* genes were successfully amplified using PCR, yielding bands of 894 bp and 480 bp, respectively. The *metF* gene was inserted into the pRSFDuet-1 vector by digestion with BamHI and HindIII, while the *folA* gene was inserted into the vector by digestion with NdeI and XhoI. The recombinant plasmid was then transformed into *E. coli* DH5α. Validation was performed using colony PCR and enzyme digestion, and the results confirmed successful ligation, as evident from the expected band sizes in gel electrophoresis.

Upon verifying the successful amplification of the targeted plasmid, they were then transformed into E.coli DH5α. We selected colonies and sequenced them.

![](https://static.igem.wiki/teams/5189/result/2.png)

*Figure 2: Transformation plate of *pRSFDuet-metF-folA*(A);Enzyme digestion verification for DH5α: pRSF-metF (B),pRSF-metF-folA(C);Sequencing results of *pRSFDuet-metF-folA* (D).*

The connected vectors were verified by enzyme digestion, and the exact target bands were obtained respectively. The first two lanes show successful enzyme digestion verification for pRSF-metF-folA, Which indicates that the destination segment is successfully connected.

1.2 *pETDuet-ftfL-mtdA-fchA*

The *pETDuet-ftfL-mtdA-fchA* plasmid was constructed with the objective of enhancing the L-5-MTHF synthesis pathway by co-expressing *the ftfL*, *mtdA*, and *fchA* genes. The *pETDuet-1* vector was selected for its dual-expression system, utilizing the T7 promoter to drive synchronized expression of these critical enzymes. The *ftfL*gene (1685 bp) was inserted first, followed by the *mtdA-fchA*fragment (1488 bp), ensuring that all genes were under the control of the T7 promoter for optimal co-expression in E.coli BL21(DE3).

![](https://static.igem.wiki/teams/5189/result/3.png)

*Figure 3: The gel electrophoresis validation of ftfL(Left), *mtdA*, and *fchA*(Right) nucleic acids.*

The *ftfL*gene (1685 bp) and the *mtdA-fchA* fragment (1488 bp) were successfully amplified using PCR. The *ftfL*gene was inserted into the *pETDuet-1* vector by digestion with BamHI and HindIII, while the *mtdA-fchA* fragment was inserted by digestion with NdeI and KpnI. The resulting plasmid was transformed into *E. coli* DH5α. Validation was performed using colony PCR and enzyme digestion, and the results confirmed successful ligation, as indicated by the expected band sizes in gel electrophoresis.

![](https://static.igem.wiki/teams/5189/result/4.png)

*Figure 4: Transformation plate of pETDue-ftfL-mtdA-fchA (A);Enzyme digestion verification for DH5α: pETDue-ftfL F (B), pETDue-ftfL-mtdA-fchA (C);Sequencing results of pETDue-ftfL-mtdA-fchA (D).*

2. Protein Expression

To further investigate the production pathway of L-5-MTHF, we co-transformed the constructed pETduet-ftfL-fchA-mtdA recombinant plasmid, which was completed, with the pRSFduet-metF-folA recombinant plasmid into E. coli BL21 (DE3). It was verified by colony PCR to prove that strain A was constructed successfully.

![](https://static.igem.wiki/teams/5189/result/7.jpeg)

*Figure 5. Construction of co-transformed strains; A: Colony PCR validation pRSFduet-metF-folA; B: Colony PCR validation pETduet-ftfL-mtdA-fchA; C: Colony A plate diagram.*

The *pRSFDuet-metF-folA*, *pETDuet-ftfL-mtdA-fchA* plasmid was transformed into *E. coli* BL21(DE3) to evaluate the co-expression of the ftfL, *mtdA*, and fchA genes. Protein expression was induced using IPTG and analyzed via SDS-PAGE and Western Blot techniques. The SDS-PAGE results displayed distinct bands corresponding to the FtfL, *mtdA*, and *fchA*proteins, particularly under induction at 37°C. Western Blot analysis confirmed the successful expression of all three proteins, demonstrating effective co-expression.

![](https://static.igem.wiki/teams/5189/result/5.png)

*Figure 6: Expression of *metF*, *folA*, *mtdA*, *fchA*, and ftfL Proteins in BL21(DE3) Analyzed by SDS-PAGE (left) and Western Blot (right)*

3. Functional Test

3.1 One-Step Growth Curve Analysis

A one-step growth curve was generated to compare the growth rates of the different strains. The control strain, BL21, initially entered a rapid growth phase, transitioning into the stationary phase after approximately 10 hours. In contrast, the strains pRSF-metF-folA, pET-ftfL-mtdA-fchA, and Strain A (which contains both the pRSF and pET plasmids) exhibited slower initial growth rates compared to the control strain. Notably, Strain A showed continued growth even at the 12-hour mark, indicating a prolonged exponential growth phase. This suggests that Strain A may have a higher potential for sustained growth, likely due to the combined effects of both plasmids, which enhance L-5-MTHF production and lead to a more robust metabolic capacity under the given conditions.

![](https://static.igem.wiki/teams/5189/result/6.png)

*Figure 7: One-Step Growth Curve for BL21, pRSF-metF-folA, pET-ftfL-mtdA-fchA, and Strain A.*

3.2 HPLC assay for L-5-MTHF yield

To further determine the actual yield of L-5-MTHF, we utilised HPLC. Firstly, we inoculated the constructed host bacteria into 100 mL of LB medium at 1% inoculum, supplemented the medium with folic acid (0.13 g/L) and sodium formate (0.013 g/L), and incubated the medium at 37℃ and 220 rpm until the OD600 value was 0.8, then added IPTG at a final concentration of 0.8 mM, and the temperature was lowered to 18℃, and the rotational speed was lowered to 110 rpm. Induction culture was carried out for 12-48h.

Subsequently, the samples were processed, 50 mL of bacterial solution was taken, centrifuged at 4°C, 6000 rpm for 10 min, the supernatant was discarded, and resuspended to the same volume in an anaerobic chamber with Tris-HCl (pH 7.2) containing 0.1% ascorbic acid and 0.1% mercaptoethanol, sealed in an anaerobic vial, and the cells were broken by boiling for 10 min in a 100°C water bath and immediately ice-bathed. 15, 000 rpm at 4°C, 15 min after centrifugation, and the supernatant was collected, fresh mouse serum (100 μl/mL)37 was added for 3 h, and the reaction was terminated by boiling for 5 min. 000 rpm, 4°C centrifugation for 15 min and collect the supernatant, add fresh mouse serum (100 μl/mL)37 reaction for 3 h, boil for 5 min to terminate the reaction, centrifugation to take the supernatant, filtered with 0.22 μm cellulose acetate filter membrane, and loaded into the liquid-phase vial. The product measured experimentally was L-5-MTHF, and its HPLC conditions were fluorescence detector Ex=290 nm, Em=356 nm.

As can be seen from Table 1, the actual amount of L-5-MTHF produced by strain A after co-transformation was higher compared to the control BL21 strain, which indicates that the addition of folic acid ultimately produces biologically active L-5-MTHF by the enzyme expressed by strain A.

Table 1 L-5-MTHF concentration of BL21 and Strains A

| Time | L-5-MTHF concentration of BL21 (16℃) | L-5-MTHF concentration of BL21 (37℃) | L-5-MTHF concentration of Strains A (16℃) | L-5-MTHF concentration of Strains A (37℃) |
| --- | --- | --- | --- | --- |
| 0h | 0.308 | 0.268 | 0.451 | 0.4 |
|  | 0.3 | 0.27 | 0.449 | 0.398 |
| 2h | 0.322 | 0.222 | 0.605 | 0.488 |
|  | 0.31 | 0.220 | 0.61 | 0.501 |
| 4h | 0.315 | 0.250 | 0.697 | 0.550 |
|  | 0.31 | 0.249 | 0.7 | 0.56 |
| 10h | 0.328 | 0.234 | 0.719 | 0.608 |
|  | 0.32 | 0.233 | 0.72 | 0.609 |
| 22h | 0.375 | 0.276 | 0.836 | 0.714 |
|  | 0.36 | 0.267 | 0.838 | 0.72 |
| 32h | 0.304 | 0.228 | 0.946 | 0.821 |
|  | 0.3 | 0.2 | 0.95 | 0.811 |
| 48h | 0.331 | 0.256 | 1.498 | 0.926 |
|  | 0.3 | 0.3 | 1.52 | 0.937 |

In addition, as can be seen from Fig. 8, the data measured for the control strain BL21 did not fluctuate significantly with the prolongation of the incubation time; whereas for the co-transformed strain A, the final production of active L-5-MTHF increased gradually and reached the maximum value at 48 h. Due to the oxidation of samples and depletion of samples during the experimental sampling process, the actual production of active L-5-MTHF should be slightly higher than the measured value. It is worth affirming that we successfully increased the yield of L-5-MTHF by genetically engineering the metabolic pathway of E. coli BL21.

![](https://static.igem.wiki/teams/5189/result/8.jpeg)

*Figure 8. Variation of L-5-MTHF production over time.*

4. Summary

In conclusion, we successfully increased the production of L-5-MTHF by genetically engineering the metabolic pathway of E. coli BL21. Notably, the product of methionine synthase MTRR (encoded by the metH gene) in the metabolic pathway inhibits the activity of MTHFR in the L-5-MTHF synthesis pathway, which further affects the yield of L-5-MTHF. Therefore, in the future, we will continue to study the metH gene in the metabolic pathway and explore the effect of its knockdown on the whole metabolic pathway and the strain itself.

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