iGEM 2024 — Team squirrel-beijing-i

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# PDF for Lab Notebook protocols

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![](https://static.igem.wiki/teams/5081/wetlab/engineer/engineer0.jpg)

- [Design](#Design)

- [Build](#BUILD)

- [Test](#TEST)

- [Learn](#Learn)

- [Reference](#Reference)

## Design

Our research aims to investigate the binding interactions of vitamin B6 and B12 with the PLP-1 protein using molecular docking techniques (Agu et al., 2023). We divided the project into three modules: the protein structure modeling module, the small molecule preparation module, and the docking analysis module. We utilized AlphaFold3 to model the target protein PLP-1 and employed Maestro computational software to optimize the structures of the protein and vitamins B6 and B12. The predicted protein structure and small molecules were optimized to ensure stability and minimize energy, significantly increasing the likelihood and accuracy of successful docking. Subsequently, we employed a semi-flexible docking method to analyze the binding interactions. We expect to identify the optimal binding modes of vitamin B6 and B12 with PLP-1, contributing to our understanding of their anti-inflammatory effects.

![](https://static.igem.wiki/teams/5081/wetlab/engineer/engineer1.jpg)

## BUILD

1.Pheochromacytoma (PC12) cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 5% horse serum (HS), and 1% penicillin/streptomycin(Kiernan et al., 2016). The cells were maintained in a humidified incubator at 37℃ with 5% CO2. The growth medium was replaced daily, and the cells were passaged every two days to ensure optimal growth conditions. To establish a cell injury model, the normal culture medium was replaced with a sugar-free medium (2 mL per well in a 6-well plate) and placed in a sealed anaerobic glass container for 2 hours. This anaerobic treatment simulates conditions that induce cellular stress and damage. Following this incubation, the cells were switched back to the normal culture medium to assess recovery. Concurrently, vitamin B6, vitamin B12, and their combinations were added in varying ratios: 1:1 (group 1), 1:10 (group 2), 10:1 (group 3), and 20:1 (group 4). The vitamins were allowed to act for a duration of 24 hours.

![](https://static.igem.wiki/teams/5081/wetlab/engineer/engineer2.png)

2. For the overexpression of PLP1, a membrane protein, we can select either a mammalian or insect cell expression system, depending on the desired post-translational modifications and protein yield (Ikuo et al., 1998). The plasmid designed for this purpose incorporates essential elements for effective expression. The design includes a strong promoter to drive high levels of transcription, a suitable multiple cloning site for the insertion of the PLP1 gene, and a selection marker to facilitate the identification of successfully transformed cells. The PLP1 gene is linked to the expression vector, ensuring that the protein is produced in sufficient quantities for subsequent analysis. The canonical sequence of PLP1, identified as P60201-1, serves as a reference for the design, ensuring that all positional information corresponds to this isoform. This sequence is also provided in downloadable formats for further research. The successful construction of this plasmid will enable the study of PLP1's functional roles and its potential implications in cellular processes.

![](https://static.igem.wiki/teams/5081/wetlab/engineer/engineer3.png)

3.Introduce the overexpression vector into expression-deficient cells for the purpose of interaction validation or to apply stress treatment to the overexpressing cells.

## TEST

1. Through Western blot analysis, we successfully validated the interaction between vitamins B6 and B12 with the PLP-1 protein. The results indicated that the combinations of vitamins B6 and B12 at ratios of 10:1 and 20:1 exhibited the most potent anti-inflammatory effects, characterized by the up-regulation of PLP-1 and PGC-1α proteins, alongside the down-regulation of TNF-α protein.

![](https://static.igem.wiki/teams/5081/wetlab/engineer/engineer4.png)

2. Conduct morphological, protein, and molecular level assessments on cells successfully transfected with the plasmid to confirm the successful construction of the vector and the functionality of the protein pathway.

## Learn

mins B6 and B12 and the PLP-1 protein. This pathway exhibits a nourishing effect on neurons and vestibular functional areas (Baltrusch, 2021), manifested as the alleviation of neuronal edema symptoms, thereby mitigating damage to key functional regions, including vision, hearing, and taste.
2. We found that combinations of vitamins B6 and B12 at ratios of 10:1 and 20:1 significantly upregulated the expression of PLP-1 and PGC-1α proteins while downregulating TNF-α protein. Based on these characteristics, we formulated a soothing nasal suction complex containing PLP-1 protein and vitamins.

## Reference

1. Agu P C , Afiukwa C A , Orji O U ,et al.Molecular docking as a tool for the discovery of molecular targets of nutraceuticals in diseases management[J].Scientific Reports, 2023, 13(1).DOI:10.1038/s41598-023-40160-2.

2. Kiernan, E. A. , Smith, S. M. C. , Mitchell, G. S. , & Watters, J. J. . (2016). Mechanisms of microglial activation in models of inflammation and hypoxia: implications for chronic intermittent hypoxia. Journal of Physiology, 594(6), 1563-1577.

3. Ikuo, T. , Li-Qing, K. , Tolley, N. D. , Lindsay, W. J. , & Fujinami, R. S. . (1998). Enhancement of experimental allergic encephalomyelitis (eae) by dna immunization with myelin proteolipid protein (plp) plasmid dna. Journal of Neuropathology & Experimental Neurology(8), 758-67.

4. Baltrusch, S. . The role of neurotropic b vitamins in nerve regeneration. BioMed research international, 2021, 9968228.

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- [PCR](#Plan)

- [Western Blot (WB) Overview](#phase1)

## PCR

Purpose: to amplify the target gene using beta active.

For gene expressive production

1. Determine two types of cDNA templates, each with two small tubes

2. Mark the centrifuge tube properly; One is wt and the other is active genes

3. Configure a 20 microliter system and calculate the corresponding reagent dosage

CDNA template 2ul

Primers are divided into f and r primers, each with 1 μ l added

DNTPs plus 2ul

TAQ enzyme added 0.5ul

PCR buffer 2ul

Add 11.5ul of ddH2O (double distilled water)

Add according to the volume from large to small, with small volume added to the pipe wall

（All things need to be prepared more in order to eliminate the error）

4. Place 4 PCR systems into a centrifuge for centrifugation (centralized machine requires center of gravity balance), then place them into a PCR instrument for annealing and elongation

94 degrees Celsius for 3 minutes, 55 degrees Celsius for 30 seconds, 72 degrees Celsius for 1 minute

Then repeat 30 times at 94 degrees Celsius for 30 seconds, 55 degrees Celsius for 30 seconds, and 72 degrees Celsius for 1 minute, followed by 10 minutes at 72 degrees Celsius

Last 4 degrees Celsius storage

## Western Blot (WB) Overview

Purpose: Identification and quantification of a specific protein in a mixture of proteins extracted from cells or tissues.

1. Sample Collection: In our experiment, we used a cell lysis buffer.

2. Preparation of WB Gel:

Separation Gel (10%):

· Buffer: 625 µL × 4

· DD Water: 820 µL × 5

· 30% Acrylamide: 825 µL × 4

· 10% Ammonium Persulfate: 100 µL

· TEMED: 10 µL

Concentration Gel (5%):

· Buffer: 625 µL × 2

· DD Water: 710 µL × 5

· 30% Acrylamide: 830 µL

· 10% Ammonium Persulfate: 100 µL

· TEMED: 10 µL

Gel Casting:

· Stack the concentration gel on top of the separation gel and insert the comb.

· Smooth the separation gel with a methanol solution.

· Place the small glass plate on top of the large glass plate, and insert it into the electrophoresis chamber, adding running buffer to the chamber.

Sample Loading: Load your samples into the wells.

Gel Running: Run the gel according to your protocol.

Transfer: Sandwich Structure:

· Sponge + Filter Paper + Gel + PVDF Membrane (note to distinguish front and back) + Filter Paper + Sponge

Blocking: Use skim milk as the blocking solution. Prepare a 5% skim milk solution in 1× TBST.

Blocking Procedure:

1. After adding the blocking solution, let it block for 1 hour.

Primary Antibody Incubation:

1. Add the primary antibody and incubate overnight at 4°C.

Secondary Antibody Incubation:

1. Recover the primary antibody, wash with TBST for 15 minutes (repeat three times).

2. Add the corresponding secondary antibody and incubate on a shaker at room temperature for 2 hours, then wash with TBST for 15 minutes (repeat three times).

Detection:

1. Mix developing solution A and B in a 1:1 ratio, then immerse the membrane in the developing solution for 5 seconds before exposing it on the exposure machine.

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- [Analyzing results](#Intro)

- [Conclusion](#InSilico)

## Analyzing results

In the first index, -β-actin is used as the internal reference protein to ensure that the loading amount of the sample is the same. The results show that the fringe brightness of β-actin is relatively consistent among all groups, indicating that the loading amount of each sample is basically the same, which ensures the reliability of the experimental results, and can exclude the result error caused by the different loading amount of samples.

The indicator PLP1 in band 2 is a protein associated with nerve function, and its expression is associated with mechanisms related to myelination, neurological health, or neuroinflammation. Previous studies have shown that changes in PLP1 expression may be related to the neuroprotective effect of vitamins or the improvement of vertigo symptoms. The expression level of PLP1 protein in the control group was similar to that in the M+3 and M+4 groups, which proved that the vitamin ratio used in these two groups was more conducive to the recovery of nerve function or the prevention of injury.

Besides, the changes of PGC-1a stripes reflect the effects of different treatments on cellular metabolic function or mitochondrial activity. The brighter stripes, the higher PGC-1α expression, which suggest the enhanced mitochondrial activity or metabolism. Its expression did not differ significantly in all groups, indicating that different vitamin ratios did not affect mitochondrial activity or cellular metabolic capacity.

The last but not least, fringe brightness of TNF-α reflects the effect of different treatments on the inflammatory response. The brighter the stripes, the higher the expression of TNF-α, indicating a stronger inflammatory response. The results showed that with the increase of B6/B12 ratio (M+3 and M+4 groups), the expression of TNF-α decreased. This means that the high proportion of B6 may play a role in reducing the inflammatory response and thus the symptoms of vertigo associated with inflammation. The reduction of inflammation may be a key mechanism that vitamin B6 plays in preventing vertigo.

## Conclusion

From the changes of stripes in the results, the combination of the ratio of vitamin B6 to B12 in the drug of 10:1 (M+3) and 20:1 (M+4) was optimal. The expression of PLP1 and PGC-1α was significantly increased in these combinations, while the expression of TNF-α was significantly decreased. This suggests that a higher proportion of B6 may not only promote energy metabolism in mitochondria, but also effectively inhibit the inflammatory response, thereby improving symptoms associated with vertigo. At the same time, the results show that vitamin B6 may play a role in preventing vertigo by enhancing nervous system function, promoting mitochondrial production and reducing inflammatory response. While B12 failed to show significant effects at low levels, high levels of B6 seem to play a key role in this model. Whether this combination has a similar effect in larger animal or clinical studies could be further explored in the future to verify its potential application in the prevention and treatment of vertigo.

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