iGEM 2024 — Team subcat-hs

{% 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 %}
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{% 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 %}
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Overview

Parkinson's disease (PD) is one of the common diseases in neurodegenerative diseases, so the early diagnosis of PD can effectively prevent the development process [1]. The pathophysiology of PD manifest as the loss of neurons in locus coeruleus and substantia nigra [2]. Studies have shown that PD pathogenesis is related to the accumulation of α-synuclein (α-syn) in substantia nigra, and the expression level of α-syn in cerebrospinal fluid can be used as an important specific biomarker for early diagnosis of PD [3-4]. Besides α-syn, Aβ42 show highly relevant in PD, because some PD patients exhibit amyloid plaques similar to those found in Alzheimer's diseases [5]. NF-L is a neuronal structural protein, which represents a key biomarker of nerve damage when its expression level elevating [5]. NF-L provides a quantitative indicator to track neurodegeneration [5]. Therefore, our project intends to obtain PD-specific markers NF-L, Aβ42, and α-syn by genetic engineering methods, and then verifies the accuracy of recombination proteins by ELISA assay. Finally, we expect to prepare for the acquisition of specific antibodies and the construction of early PD diagnosis kits.

Part 1 pET28a-NF-L (BBa\_K5190003)

**Design:**

The pET28a(+) plasmid was provided by the strain library of our unit, and the target gene NF-L was synthesized by a biotechnology company. The NF-L fragment was homologously recombined with the linearized plasmid to construct pET28a-NF-L.

![](https://static.igem.wiki/teams/5533/eng/1.png)

Fig 1. The plasmid map of pET28a-NF-L

**Build:**

We constructed pET28a-NF-L by homologous recombination. The pET28a vector backbone was obtained by enzyme digestion, with a length of 5329 bp. Figure 2B shows a band consistent with the target size. The NF-L gene was amplified by PCR, with a length of 1059 bp. Figure 2A shows a band consistent with the target size, indicating that both the target gene and the vector were successfully amplified. After agarose gel electrophoresis and gel recovery, the recombinant plasmid was obtained by homologous recombination.

![](https://static.igem.wiki/teams/5533/eng/2.png)

Fig. 2 Agarose gel electrophoresis of target genes and linearized pET-28a(+) vector

The recombinant plasmid was introduced into E. coli BL21, and different numbers of colonies were selected for colony PCR verification. Figure 3B shows that the length is consistent with the target fragment, Figure 3A is the clone on the plate, and the results in Figure 4 show that the NF-L gene has been successfully connected to the pET28a vector without obvious mutations, confirming that the recombinant plasmid pET28a-NF-L was successfully constructed.

![](https://static.igem.wiki/teams/5533/eng/3.png)

Fig. 3 Verification of the transformation results of pET-28a(+)-NF-L/Aβ-42/α-syn positive colony. A: The single colony on the plate; B: The amplification results of colony PCR.

![](https://static.igem.wiki/teams/5533/eng/4.png)

Fig. 4 Sanger sequencing results of pET-28a(+)-NF-L/Aβ-42/α-syn positive colony.

Part 2 pET28a-Aβ42 (BBa\_K5190005)

**Design:**

The pET28a(+) plasmid was provided by the strain library of our unit, and the target gene Aβ42 was synthesized by a biotechnology company. The Aβ42 fragment was homologously recombined with the linearized plasmid to construct pET28a-Aβ42.

![](https://static.igem.wiki/teams/5533/eng/5.png)

Fig 5. The plasmid map of pET28a-Aβ42

We constructed pET28a-Aβ42 by homologous recombination. The pET28a vector backbone was obtained by enzyme digestion, with a length of 5329 bp. Figure 2B shows a band consistent with the target size. The NF-L gene was amplified by PCR, with a length of 789 bp. Figure 2A shows a band consistent with the target size, indicating that both the target gene and the vector were successfully amplified. The recombinant plasmid was obtained by homologous recombination after agarose gel electrophoresis and gel recovery.

The recombinant plasmid was introduced into E. coli BL21, and different numbers of colonies were selected for colony PCR verification. Figure 3B shows that the length is consistent with the target fragment, Figure 3A is the clone on the plate, and the results in Figure 4 show that the Aβ42 gene has been successfully connected to the pET28a vector without obvious mutations, confirming that the recombinant plasmid pET28a- Aβ42 was successfully constructed.

Part 3 pET28a-α-syn (BBa\_K5190007)

**Design:**

The pET28a(+) plasmid was provided by the strain library of our unit, and the target gene α-syn was synthesized by a biotechnology company. The α-syn fragment was homologously recombined with the linearized plasmid to construct pET28a-α-syn.

![](https://static.igem.wiki/teams/5533/eng/6.png)

Fig 6. The plasmid map of pET28a-α-syn

**Build:**

We constructed pET28a-α-syn by homologous recombination. The pET28a vector backbone was obtained by enzyme digestion, with a length of 5329 bp. Figure 2B shows a band consistent with the target size. The NF-L gene was amplified by PCR, with a length of 522 bp. Figure 2A shows a band consistent with the target size, indicating that both the target gene and the vector were successfully amplified. The recombinant plasmid was obtained by homologous recombination after agarose gel electrophoresis and gel recovery.

The recombinant plasmid was introduced into E. coli BL21, and different numbers of colonies were selected for colony PCR verification. Figure 3B shows that the length is consistent with the target fragment, Figure 3A is the clone on the plate, and the results in Figure 4 show that the α-syn gene has been successfully connected to the pET28a vector without obvious mutations, confirming that the recombinant plasmid pET28a- α-syn was successfully constructed.

Part 4 pET28a-NF-L-mCherry (BBa\_K5190004)

**Design:**

The pET28a(+) plasmid and mCherry gene were provided by the strain library of our unit, and the target gene NF-L was synthesized by a biotechnology company. The mCherry fragment was homologously recombined with the NF-L fragment and the linearized plasmid to construct pET28a-NF-L-mCherry.

![](https://static.igem.wiki/teams/5533/eng/7.png)

Fig 7. The plasmid map of pET28a-NF-L-mChaerry

**Build:**

We constructed pET28a-NF-L-mCherry by homologous recombination. The pET28a vector backbone was obtained by enzyme digestion, with a length of 5329 bp, and Figure 2B shows a band consistent with the target size. The NF-L gene was amplified by PCR, with a length of 1059 bp, and Figure 8A shows a band consistent with the target size. The mCherry gene was amplified by PCR, with a length of 711 bp, and Figure 8B shows a band consistent with the target size, indicating that both the target gene and the vector were successfully amplified. After agarose gel electrophoresis and gel recovery, the recombinant plasmid was obtained by homologous recombination.

![](https://static.igem.wiki/teams/5533/eng/8.png)

Fig. 8 Agarose gel electrophoresis of target genes and mcherry fragment

The recombinant plasmid was transferred into Escherichia coli BL21, and different numbers of colonies were selected for colony PCR verification. Figure 9B shows that the length is consistent with the target fragment, Figure 9A is the clone on the plate, and the results in Figure 10 show that the NF-L and mCherry gene has been successfully connected to the pET28a vector without obvious mutations, confirming that the recombinant plasmid pET28a-NF-L- mCherry was successfully constructed.

![](https://static.igem.wiki/teams/5533/eng/9.png)

Fig. 9 Verification of the transformation results of pET-28a(+)-NF-L-mcherry/Aβ-42-mcherry /α-syn-mcherry positive colony. A: The single colony on the plate; B: The amplification results of colony PCR.

![](https://static.igem.wiki/teams/5533/eng/10.png)

Fig. 10 Sanger sequencing results of pET-28a(+)-NF-L-mcherry/Aβ-42-mcherry /α-syn-mcherry positive colony.

Part 5 pET28a-Aβ42-mCherry (BBa\_K5190006)

**Design:**

The pET28a(+) plasmid and mCherry gene were provided by the strain library of our unit, and the target gene Aβ42 was synthesized by a biotechnology company. The mCherry fragment was homologously recombined with the Aβ42 fragment and the linearized plasmid to construct pET28a- Aβ42-mCherry.

![](https://static.igem.wiki/teams/5533/eng/11.png)

Fig 11. The plasmid map of pET28a- Aβ42-mCherry

We constructed pET28a- Aβ42-mCherry by homologous recombination. The pET28a vector backbone was obtained by enzyme digestion, with a length of 5329 bp, and Figure 2B shows a band consistent with the target size. The Aβ42 gene was amplified by PCR, with a length of 789 bp, and Figure 8A shows a band consistent with the target size. The mCherry gene was amplified by PCR, with a length of 711 bp, and Figure 8B shows a band consistent with the target size, indicating that both the target gene and the vector were successfully amplified. After agarose gel electrophoresis and gel recovery, the recombinant plasmid was obtained by homologous recombination.

The recombinant plasmid was transferred into Escherichia coli BL21, and different numbers of colonies were selected for colony PCR verification. Figure 9B shows that the length is consistent with the target fragment, Figure 9A is the clone on the plate, and the results in Figure 10 show that the Aβ42 and mCherry gene has been successfully connected to the pET28a vector without obvious mutations, confirming that the recombinant plasmid pET28a- Aβ42- mCherry was successfully constructed.

Part 6 pET28a-α-syn-mCherry (BBa\_K5190008)

**Design:**

The pET28a(+) plasmid and mCherry gene were provided by the strain library of our unit, and the target gene α-syn was synthesized by a biotechnology company. The mCherry fragment was homologously recombined with the α-syn fragment and the linearized plasmid to construct pET28a- α-syn -mCherry.

![](https://static.igem.wiki/teams/5533/eng/12.png)

Fig 12. The plasmid map of pET28a- α-syn -mCherry

We constructed pET28a- α-syn -mCherry by homologous recombination. The pET28a vector backbone was obtained by enzyme digestion, with a length of 5329 bp, and Figure 2B shows a band consistent with the target size. The α-syn gene was amplified by PCR, with a length of 522 bp, and Figure 8A shows a band consistent with the target size. The mCherry gene was amplified by PCR, with a length of 711 bp, and Figure 8B shows a band consistent with the target size, indicating that both the target gene and the vector were successfully amplified. After agarose gel electrophoresis and gel recovery, the recombinant plasmid was obtained by homologous recombination.

The recombinant plasmid was transferred into Escherichia coli BL21, and different numbers of colonies were selected for colony PCR verification. Figure 9B shows that the length is consistent with the target fragment, Figure 9A is the clone on the plate, and the results in Figure 10 show that the α-syn and mCherry gene has been successfully connected to the pET28a vector without obvious mutations, confirming that the recombinant plasmid pET28a- α-syn -mCherry was successfully constructed.

**Test:**

1. Protein expression

Based on the lac operator system in pET-28a(+) vector backbone, we used IPTG for protein prokaryotic expression. First, we used a final concentration of 1 mM/L IPTG for usual protein expression. Then, we set two temperature and different expression time for determining the optimal induction condition. The expected molecular weights of 6xHis-NF-L, 6xHis-NF-L-mcherry, 6xHis-Aβ-42, 6xHis-Aβ-42-mcherry, 6xHis-α-syn, and 6xHis-α-syn-mcherry are 44.8 kDa, 71.5 kDa, 34.0 kDa, 60.7 kDa, 23.3 kDa, and 50.0 kDa, respectively. As showed in Fig. 7, we can clearly observe protein bands of target genes. Interestingly, we found that the molecular weight of 6xHis-Aβ-42 and 6xHis-Aβ-42-mcherry are slightly larger than as predicted, suggesting that these two proteins might have some post-translational modifications. For 6xHis-NF-L and 6xHis-NF-L-mcherry, there was no significant difference in the bands between 25°C and 37°C, and the protein expression level was highest after 6 hours of induction. For 6xHis-Aβ-42 and 6xHis-Aβ-42-mcherry, there was no significant difference in the bands between 25°C and 37°C, and the protein expression level showed similar between after 3 hours and 6 hours induction, indicating that the protein synthesis rate had reached a plateau. Interestingly, 6xHis-α-syn showed different protein synthesis trends under 25°C and 37°C. Under 25°C, the protein expression level of 6xHis-α-syn was highest after 1 hours of induction, but as the induction time increased, the protein was further degraded. For 37°C, the protein expression level of 6xHis-α-syn was highest after 6 hours of induction. For 6xHis-α-syn-mcherry, the protein synthesis rate under 37°C is faster than that under 25°C, and the protein expression level was highest after 6 hours of induction.

![](https://static.igem.wiki/teams/5533/eng/13-abcd.png)

![](https://static.igem.wiki/teams/5533/eng/13-ef.png)

Fig. 13 SDS-PAGE analysis for small amount of target proteins expression.

We further used anti-His antibody for Western Blotting experiments and then quantified the induction amount of target proteins according to the relative gray value of the band. As showed in Fig. 14A, we found that the His antibody specificity is not particularly high, suggesting that we might need to optimize the relative concentrations of the primary and secondary antibodies, but we can still clearly see the bands of target proteins. Then, we used ImageJ software to calculate the relative gray value intensity of target proteins. The results were consistent with those of Coomassie Brilliant Blue staining in Fig. 14. Different temperatures have an effect on the correct folding and biological activity of proteins. Finally, except for 6xHis-α-syn, we chose 25°C and 37°C after 6 hours of induction for large-scale purification. For 6xHis-α-syn, we chose 25°C after 1 hours of induction and 37°C after 6 hours of induction for large-scale purification, respectively.

![](https://static.igem.wiki/teams/5533/eng/14-a.png)

![](https://static.igem.wiki/teams/5533/eng/14-b.png)

Fig. 14 Western Blotting analysis and the relative gray value intensity for small amount of target proteins expression.

2. Protein His-tag purification

As showed in Fig. 15, we could clearly observe that the combination proteins with mcherry fluorescent tag have visible red precipitate. We further used His-tag purification kit to purify target proteins and then stained protein bands by Coomassie Brilliant Blue staining. As showed in Fig. 16 and Fig. 17, target proteins were enriched in elution components. Compared with proteins induced under 25°C, proteins induced under 37°C had better purification efficiency and less miscellaneous bands. In addition, we also found that the efficiency of protein binding with Ni-NTA beads is not particularly high, so we might need to optimize the conditions to obtain higher recovery yield. Then, we collected elution components for functional test.

![](https://static.igem.wiki/teams/5533/eng/15.png)

Fig. 15 Representative images of large-scale protein expression.

![](https://static.igem.wiki/teams/5533/eng/16.png)

Fig. 16 SDS-PAGE analysis for large-scale His-tag protein purification of target proteins expressed under 25°C. T: Total protein; FT: Flow Through.

![](https://static.igem.wiki/teams/5533/eng/17.png)

Fig. 17 SDS-PAGE analysis for large-scale His-tag protein purification of target proteins expressed under 37°C. Total protein; FT: Flow Through.

2. Functional test

**2.1 Determination of protein fused with mcherry tag by fluorescence microscope**

First, we determined the effectivity of target protein fused with mcherry tag by fluorescence microscope. After fixing sample and adding anti-fading agent, we detected mcherry signal using an excited light wavelength of 587nm and an emitted light wavelength of 610nm. As showed in Fig. 18, E.coli BL21 expressing pET-28a(+)-NF-L-mcherry/Aβ-42-mcherry/α-syn-mcherry could be clearly observed with red fluorescence in cord-like structure.

![](https://static.igem.wiki/teams/5533/eng/18.png)

Fig. 18 Observation of E.coli BL21 expressing pET-28a(+)-NF-L-mcherry/Aβ-42-mcherry/α-syn-mcherry by fluorescence microscopy

**2.2 Verification of target recombination protein by ELISA assay**

We used ELISA assay to verify the accuracy of NF-L protein. As showed in Fig. 19A, the correlation between NF-L protein and absorbance 450nm is 0.9972, indicating that ELISA assay can accurately and effectively detect the content of NF-L in elution components. In elution components of protein expression under 25°C, the content of NF-L protein in E6 was the highest, reaching 25.31 ng/mL (Fig. 19B). Interestingly, in elution components of protein expression under 37°C, the content of NF-L protein in E2 was the highest, reaching 34.25 ng/mL (Fig. 19C).

![](https://static.igem.wiki/teams/5533/eng/19.png)

Fig. 19 Verification of NF-L by ELISA assay

Then, we used ELISA assay to verify the accuracy of Aβ-42 protein. As showed in Fig. 20A, the correlation between Aβ-42 protein and absorbance 450nm is 0.9983, indicating that ELISA assay can accurately and effectively detect the content of Aβ-42 in elution components. In elution components of protein expression under 25°C, the content of Aβ-42 protein in E6 was the highest, reaching 21.35 pg/mL (Fig. 20B). In elution components of protein expression under 37°C, the content of Aβ-42 protein in E6 was the highest, reaching 26.92 pg/mL (Fig. 20C).

![](https://static.igem.wiki/teams/5533/eng/20.png)

Fig. 20 Verification of Aβ-42 by ELISA assay

Finally, we used ELISA assay to verify the accuracy of α-syn protein. As showed in Fig. 21A, the correlation between α-syn protein and absorbance 450nm is 0.994, indicating that ELISA assay can accurately and effectively detect the content of α-syn in elution components. In elution components of protein expression under 25°C, the content of α-syn protein in E6 was the highest, reaching 97.54 ng/mL (Fig. 21B). In elution components of protein expression under 37°C, the content of α-syn protein in E6 was the highest, reaching 98.32 ng/mL (Fig. 21C).

![](https://static.igem.wiki/teams/5533/eng/21.png)

Fig. 21 Verification of α-syn by ELISA assay

Learn

- Our experiment did not optimize the protein expression conditions, and fermentation regulation plays a crucial role in yield. We can continue to optimize the expression conditions of the strains, such as oxygen levels, inoculum size, and pH, to improve production and to achieve the amount of protein required for antigen preparation.
- In our expression process, we added inducers IPTG. We can explore more environmentally friendly production methods by modifying promoter of vector backbone or replacing the inducers with external regulatory conditions, such as temperature and light exposure, to induce expression through these greener and harmless approaches.

Reference

- Goedert M. Alpha-synuclein and neurodegenerative diseases. Nat Rev Neurosci. (2001) 2:492–501.
- Braak H, Ghebremedhin E, Rüb U, Bratzke H, Del Tredici K. Stages in the development of Parkinson's disease-related pathology. Cell Tissue Res. (2004) 318(1):121-34.
- Giasson BI, Murray IV, Trojanowski JQ, Lee VM, A. hydrophobic stretch of 12 amino acid residues in the middle of alpha-synuclein is essential for filament assembly. J Biol Chem. (2001) 276:2380–6.
- Hoyer W, Cherny D, Subramaniam V, Jovin TM. Impact of the acidic C-terminal region comprising amino acids 109-140 on alpha-synuclein aggregation in vitro. Biochemistry. (2004) 43:16233–42.
- Xiang C, Cong S, Tan X, Ma S, Liu Y, Wang H, Cong S. A meta-analysis of the diagnostic utility of biomarkers in cerebrospinal fluid in Parkinson's disease. NPJ Parkinsons Dis. (2022) 29;8(1):165.

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{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
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![](https://static.igem.wiki/teams/5533/desc-banner.png)

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1. Preparation of LB (Luria-Bertani) culture medium

Goal: To create a functional and nutritional liquid or solid medium for bacteria growing. Activation, cultivation, and fermentation for microorganisms.

Materials:
1) Conical flask
2) Autoclave
3) Tryptone
4) Yeast Extract
5) Sodium Chloride
6) Double distilled H2O
7) Agar
Procedures:
1) Add 10 g tryptone, 5 g yeast extract, and 10 g of sodium chloride per liter of the culture medium. (Note: 1.5% agar powder was added to the solid culture medium);
2) Add into 1L conical flask;
3) Maintain at 121 ℃ for 20 minutes in an autoclave for sterilization.

2. Construction of recombination plasmids

**pET-28a(+)-NF-L/Aβ-42/α-syn and pET-28a(+)-NF-L/Aβ-42/α-syn-mcherry**

**Ⅰ. Obtain target gene fragments by PCR**

Goal: To amplify target genes fragments for Infusion recombination.
Materials:
1) 2x PrimeStarMix
2) Gene template
3) Double distilled water (ddH2O)
4) Primer-F
5) Primer-R
6) PCR tube
7) PCR thermal cycler
\* PrimeStarMix includes buffer and dNTPs for PCR. The extention rate for PrimeStarMix (2x) is 1000bp/10 s.
Procedures:
1) Add 2x PrimerStarMix 25µl, gene template 1µl, primer-F 1µl, and primer-R 1µl to centrifuge tube. Add ddH2O to the centrifuge tube up to 50µl. If bubbles are observed in the mixture, centrifuge the mixture for a few seconds to remove the bubbles;
2) Insert PCR tubes into PCR thermal cycler. Pre-denature the fragments at 95˚C for 3 minutes;
3) Perform 35 cycles of 30 seconds 95˚C denaturing, 30 seconds 55˚C annealing, and 2 minutes extension 72˚C extension;
4) Extend the fragments for 1 minutes at 72˚C;
5) If not use immediately, preserve the fragments at 4˚C;
6) Recover PCR fragments by agarose gel electrophoresis.

**Ⅱ. Linearize pET-28a(+) backbone by restriction endonucleases**
Goal: To linearize pET-28a(+) backbone for Infusion recombination.
Materials:
1) pET-28a(+) backbone plasmid
2) 10x Cutting buffer
3) EcoRI endonuclease
4) XhoI endonuclease
5) Double distilled water (ddH2O)
Procedures:
1) Add 5µg backbone plasmid, 10x Cutting buffer 5µl, EcoRI endonuclease 5µl, and XhoI endonuclease 5µl in PCR tube. Add ddH2O to PCR tube up to 50µl;
2) Mix by pipette and remove the bubbles by low-speed centrifuge;
3) Insert PCR tubes into PCR thermal cycler. Digest the plasmid at 37˚C for 30 minutes;
4) Recover linearized pET-28a(+) backbone fragments by agarose gel electrophoresis.

**Ⅲ. Agarose gel construction, deployment and recycle**
Goal: To produce a functional and well-shaped gel for electrophoresis.
Materials:
1) Casting Tray
2) Well comb
3) Microwave
4) 1x TAE buffer
5) Agarose M
6) 10000x Nucleic acid gel stain
7) Conical flask
Procedures:
1) Dissolve 1.5g Agarose in 200mL Conical flask;
2) Add 100mL TAE buffer;
3) Heat the material with microwave;
4) Melt the mixture at 30 sec interval;
5) See whether bubbles are forming, if did succesfully 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 ~30 minutes;
11) Remove the comb and place the gel in the gel box.

**Ⅳ. Agarose gel electrophoresis**
Goal: To verify the DNA bands' lengths and thus preparing for the next step of gel extraction to recover the bands that are of the correct length.
Procedures:
1) Extract 50µl of every sample; add 5µl of loading buffer (10x) to each sample;
2) Place the gel in the horizontal electrophoresis system;
3) Extract 50µl of the mixed solution containing the sample, mix them till their colors 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 120V for 20min;
6) Take out the gel from the device and proceed to following steps.

**Ⅴ. DNA gel extraction**

Goal: To recover DNA fragments in agarose gel.
Materials:
1) Agarose gel with target genes fragments and linearized pET-28a(+) backbone;
2) 1.5 mL Eppendorf (EP) tube
3) Agarose Gel Electrophoresis Recovery Kit
Procedures:
1) Cut the slice of gel containing target genes fragments and linearized pET-28a(+) backbone, cutting off as much unneeded gel as possible, and place it in an 1.5 mL EP tube;
2) Add 500 microliters of buffer B2 and put the tube into a 50°C hot water bath until gel has completely melted;
3) Transfer the solution containing melted gel into a purification column that's in a collection tube and perform centrifugation at 12000 rpm for 30 seconds;
4) Empty the collection tube, put the purification column back in, and add 500 microliters of wash solution containing pure ethanol. Perform centrifugation at 12000 rpm for 30 seconds, and empty collection tube again;
5) Repeat steps 4;
6) Perform centrifugation one more time at 12000 rpm for 2 minutes, then open the cap of the tube and let it sit for one minute to allow the ethanol to evaporate;
7) Transfer purification column to a new 1.5 mL EP tube. Add 50 microliters of double distilled water (ddH2O) at the center of the purification column;
8) Cap the lid and let the tube sit for 1 minute. Perform centrifugation at 12000 rpm for 1 minute;
9) Discard the purification column. Store DNA fragments in the EP tube at 4°C.

**Ⅵ. Homologous recombination of linearized pET-28a(+) backbone with target gene fragments**
Goal: Perform homologous recombination between the linearized backbone and target gene fragments to generate the complete plasmid.
Materials:
1) CloneExpress Mix (2x) br
2) PCR products of NF-L, Aβ-42, α-syn, and mcherry
3) Linearized pET-28a(+) backbone
Procedure:
1) Measure the concentration of the gel-recovered fragments;
2) Prepare the system for the construction of pET-28a(+)-NF-L/Aβ-42/α-syn in PCR tubes: 5μl CloneExpress Mix, 2.5μl Linearized pET-28a(+) backbone, and 2.5μl NF-L or Aβ-42 or α-syn fragments;
3) Prepare the system for the construction of pET-28a(+)-NF-L/Aβ-42/α-syn-mcherry in PCR tubes: 5μl CloneExpress Mix, 2μl Linearized pET-28a(+) backbone, 1.5μl NF-L or Aβ-42 or α-syn fragments, and 1.5μl mcherry fragment;
4) Insert PCR tubes into PCR thermal cycler. Ligase the fragments at 50˚C for 30 minutes;

**Ⅶ. Transformation**

Materials:
1) Ligated recombination plasmid
2) Liquid LB solution without antibiotics
3) LB Agar plates with Kanamycin antibiotics
Procedures:
1) Add recombination plasmid into E.Coli BL21 strain;
2) Placed on ice for 20 minutes;
3) Heat shock at 42˚C for 45 seconds, then immediately put on ice for 2-3 minutes;
4) Add 400μL liquid LB solution without antibiotics, incubate at 37 ˚C for 30 minutes;
5) Centrifuge at 5000 rpm for 3 minutes, extract 300μL of supernatant then discard;
6) Apply bacterial liquid to LB Agar plates with Kanamycin antibiotics, incubate for 12-16 hours at 37˚C.

**Ⅷ. Colony PCR verification**
Goal: E.Coli BL21 strain containing constructed plasmids has multiplied. We aimed to identify mono-colony E.coli BL21 containing correct recombination plasmids by colony PCR.
Materials:
1) Taq Master Mix (2x)
2) Double distilled water (ddH2O)
3) Culture plates post-transformation
Procedures:
1) Add 10μl Taq Master Mix, 1μl primer-F, 1μl primer-R, and 8μl ddH2O into PCR tubes;
2) Pick a single colony with a 10μl sterile pipette tip and perform aspiration and dispensing in PCR tubes;
3) Insert mixture into a PCR thermal cycler, design the PCR program according to the manufacturer's instructions.
4) Perform agarose gel electrophoresis, and the presence of the correct bands indicates the successful construction of recombination plasmids.

3.Protein expression and purification

**3.1 Induction of protein expression for 6xHis-NF-L, 6xHis-Aβ-42, 6xHis-α-syn, 6xHis-NF-L-mcherry, 6xHis-Aβ-42-mcherry, and 6xHis-α-syn-mcherry**

**Ⅰ. Amplification culture for correct colony**

Goal: To acquire large amounts of target proteins for SDS-PAGE
Materials:
1) Liquid LB solution
2) pET-28a(+)-NF-L/Aβ-42/α-syn and pET-28a(+)-NF-L/Aβ-42/α-syn-mcherry bacterial fluid
3) Kanamycin antibiotics
4) Inducer: IPTG
5) Iso-thermic shaker
Procedures:
1) Add 100μl of pET-28a(+)-NF-L/Aβ-42/α-syn and pET-28a(+)-NF-L/Aβ-42/α-syn-mcherry bacterial fluid and 100μl of Kanamycin antibiotics into 100mL of liquid LB solutions, respectively;
2) Culture bacterial liquid in iso-thermic shaker for 3 hours until absorbance OD600nm to 0.4-0.6;
3) Add IPTG into bacterial liquid with 1mM final concentration;
4) Sample for each bacterial liquid cultured at 25°C and 37°C for 0.5h, 1h, 3h, and 6h, respectively.

**Ⅱ: Protein crude extraction and SDS-PAGE**

Goal: To verify the expression of desired proteins.
Materials for protein crude extraction:
1) 500μl bacterial liquid for different induced conditions
2) Centrifuge
3) Double distilled water (ddH2O)
4) 6x Protein loading buffer
Procedures for protein crude extraction:
1) Centrifuge samples at 12000 rpm for 1 minute;
2) Discard supernatant and resuspend precipitation with 50μl 1x Protein loading buffer diluted by ddH2O;
3) Boiled samples at 95°C for 15 minutes in PCR thermal cycler.
Materials for SDS-PAGE:
1) Sangon 12.5% SDS-PAGE Color Preparation kit
2) Electrophoresis buffer (Tris-Glycine)
3) Protein ladder
4) 6x Protein loading buffer
5) Vertical electrophoresis system
6) Coomassie blue staining solution
Procedures for SDS-PAGE：
1) Add and mix 2.2mL 2x separating gel solution, 2.2mL 2X separating gel buffer, and 44μL catalyst in centrifugation tubes for separating gel;
2) Slowly pipetting mixture into casting stand and frame to avoid bubbles;
3) Add 1 mL ddH2O at the top of separating gel;
4) Wait the separating gel to solidify for 8 minutes;
5) Discard the water that was added previously;
6) Add and mix 825μL 2x stacking gel solution, 825μL 2X stacking gel buffer, and 11μL catalyst in centrifugation tubes for stacking gel;
7) Add stacking gel mixture until the cast is completely filled, then slowly insert comb without producing any air bubbles;
8) Wait the stacking gel to solidify for 12 minutes, then carefully remove the comb, and wash the wells with electrophoresis buffer;
9) Add 10μL protein ladder into the first well, then load samples in each successive well;
10) Transfer gel into vertical electrophoresis system;
11) Run at 120V for 90 minutes;
12) Stain the gel with Coomassie blue staining solution for 10 minutes, then destain the gel using destaining solution repeatedly until gel background become transparent.

**Ⅲ: Western blotting for target proteins using anti-His tag antibody**

Goal: To verify the correctness of recombination proteins by Western blotting, to calculate the relative gray intensity value of protein bands and finally to confirm the best induced conditions for target proteins.
Materials:
1) SDS-PAGE gel
2) Transfer buffer (Tris-Glycine added methanol)
3) PVDF membrane
4) TBST buffer (Tris-buffered saline with Tween-20)
5) Blocking buffer (5% non-fat dry milk in TBST)
6) Anti-His tag primary antibody from mouse
7) Goat anti-mouse HRP-conjugated secondary antibody
8) Chemiluminescent substrate
9) Imaging system
Procedures:
1) Gel electrophoresis: same as SDS-PAGE described at step 3.1 II;
2) After electrophoresis, carefully remove the gel and equilibrate it in transfer buffer for 10-15 minutes;
3) Assemble the transfer sandwich: place the PVDF membrane between the gel and filter papers in the transfer apparatus;
4) Transfer proteins from the gel to the PVDF membrane at 100 V for 2 hours on ice;
5) After transfer, rinse the membrane briefly with TBST;
6) Block non-specific binding sites by incubating the membrane in blocking buffer (5% non-fat dry milk in TBST) at room temperature for 1 hour;
7) Dilute the Anti-His tag primary antibody in blocking buffer for 1:5000;
8) Incubate the membrane with the diluted primary antibody at room temperature for 2 hours;
9) Wash the membrane 3 times with TBST (5 minutes each wash) to remove unbound antibodies;
10) Dilute the Goat anti-mouse HRP-conjugated secondary antibody in blocking buffer for 1:10000;
11) Incubate the membrane with the diluted secondary antibody at room temperature for 1 hours;
12) Wash the membrane 3 times with TBST (5 minutes each wash) to remove unbound secondary antibodies;
13) Prepare the chemiluminescent substrate A and B solutions according to the manufacturer's instructions;
14) Incubate the membrane with substrate solutions for 5 minutes until the membrane is fully covered;
15) Capture the signal using CCD imaging system to visualize the bands;
16) Analyze and calculate the relative gray intensity value of protein bands by ImageJ software.

**3.2 Protein His-tag purification**

Goal: To recover target proteins using His-tag and Ni-NTA beads affinity purification kit for antigen preparation.
Materials:
1) Bacteria culture containing desired protein
2) 50 mL centrifuge tubes
3) Beaker containing ice
4) Centrifuge
5) Sonicator
6) Beyotime Ni-NTA agarose beads purification kit
Procedures:
1) Add samples into 50 mL centrifuge tubes and then perform centrifugation at 4000 rpm for 20 minutes;
2) Put centrifuge tube into a beaker filled with ice and then insert the sonicator probe into centrifuge tube;
3) Sonicate for 15 minutes;
4) Repeat steps 2 and 3 for all samples;
5) Centrifugate samples at 10000 rpm for 30 minutes;
6) Remove cell fragments that have precipitated at the bottom of centrifuge tubes;
7) Transfer supernatant containing desired proteins to a new tube for remaining steps;
8) Add 500μl Ni-NTA agarose beads to a 2.0mL centrifuge tube, then centrifuge at 500g for 5 minutes;
9) Discard the supernatant, add 1.5mL PBS buffer to wash Ni-NTA agarose beads, and then centrifuge at 500g for 5 minutes;
10) Repeat step 9 for 3 times;
11) Add pre-treated Ni-NTA agarose beads to protein supernatant samples and then incubate at 4℃ for 60 minutes;
12) Add incubated protein to absorption column, stand for 2 minutes, and then drain the supernatant liquid;
13) Add 5mL Wash buffer to wash agarose beads;
14) Repeat step 13 for 5 times;
15) Add 500μl Elution buffer containing 250mM imidazole to elute protein;
16) Repeat step 15 for 5 times;
17) From step 13 to 16, collect all the fractions draining from absorption column;
18) Transfer 50μl sample and then add 6x Protein loading buffer;
19) Run and detect the purified protein by SDS-PAGE and Coomassie blue staining assay.

4. Functional testing

**4.1 Determination of protein fused with mcherry tag by fluorescence microscope**

Goal: To test the effectiveness of NF-L/Aβ-42/α-syn antigens using corresponding sandwich ELISA kits.
Materials:
1) Purified NF-L/Aβ-42/α-syn proteins using His-tag purification kits
2) Sandwich ELISA kits for NF-L
3) Sandwich ELISA kits for Aβ-42
4) Sandwich ELISA kits for α-syn
5) Coating PBS buffer
6) Blocking buffer
7) Sample diluent
8) Standard sample
9) Substrate solution
10) Wash buffer
11) Detection antibody conjugated to HRP
12) Stop solution (sulfuric acid for HRP)
13) Microplate Reader
Procedures:
1) Dilute Purified NF-L/Aβ-42/α-syn proteins in sample diluent as necessary;
2) Dilute standard samples in sample diluent according to the instruction;
3) Add 100 µL of each diluted sample and standard samples to 96-well microplate;
4) Cover and incubate for 1-2 hours at room temperature;
5) Wash the wells 3-5 times with washing buffer;
6) Prepare a dilution of the detection antibody in sample diluent;
7) Add 100 µL of the diluted detection antibody to each well;
8) Incubate for 1-2 hours at room temperature;
9) Wash the wells 3-5 times with washing buffer;
10) Add 100 µL of the substrate solution to each well. This substrate solution will react with HRP enzyme linked to the detection antibody;
11) Allow the color to develop for 30 minutes at room temperature in the dark;
12) Add 50 µL of stop solution to each well to terminate the enzymatic reaction;
13) Measure the absorbance of each well at 450 nm wavelength using microplate reader;
14) Determine the concentration of the target antigen in samples based on their absorbance values using the standard curve.

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![](https://static.igem.wiki/teams/5533/desc-banner.png)

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Overview

Parkinson's disease (PD) is one of the common diseases in neurodegenerative diseases, so the early diagnosis of PD can effectively prevent the development process [1]. The pathophysiology of PD manifest as the loss of neurons in locus coeruleus and substantia nigra [2]. Studies have shown that PD pathogenesis is related to the accumulation of α-synuclein (α-syn) in substantia nigra, and the expression level of α-syn in cerebrospinal fluid can be used as an important specific biomarker for early diagnosis of PD [3-4]. Besides α-syn, Aβ42 show highly relevant in PD, because some PD patients exhibit amyloid plaques similar to those found in Alzheimer's diseases [5]. NF-L is a neuronal structural protein, which represents a key biomarker of nerve damage when its expression level elevating [5]. NF-L provides a quantitative indicator to track neurodegeneration [5]. Therefore, our project intends to obtain PD-specific markers NF-L, Aβ42, and α-syn by genetic engineering methods, and then verifies the accuracy of recombination proteins by ELISA assay. Finally, we expect to prepare for the acquisition of specific antibodies and the construction of early PD diagnosis kits.

Results of our work

1.The construction of plasmid

**1.1 Construction for pET-28a(+)-NF-L/Aβ-42/α-syn**

We constructed pET-28a(+)-NF-L/Aβ-42/α-syn using Infusion recombination. First, we amplified NF-L, Aβ-42, and α-syn fragments by high-fidelity PCR, with a length of 1059 bp, 789 bp, and 522 bp, respectively. Fig. 1A showed correct bands with target size of three genes. Then, we used restriction endonuclease EcoRI and XhoI to linearize the pET-28a(+) vector backbone. Fig. 1B showed the pET-28a(+) vector. After agarose gel and bands recovery, we next used Infusion reaction to obtain recombinant plasmids.

![](https://static.igem.wiki/teams/5533/result/1.png)

Fig. 1 Agarose gel electrophoresis of target genes and linearized pET-28a(+) vector

After Infusion reaction assay, we directly the recombination plasmids into expression strain E.coli BL21. Fig. 2A showed the single colony on the plate. We then selected 3, 12, and 8 colonies for pET-28a(+)-NF-L, pET-28a(+)-Aβ-42, and pET-28a(+)-α-syn, respectively. Next, we used vector universal primers T7-Forward and T7-Terminal to detect target genes insertion by colony PCR. As showed in Fig. 2B, NF-L, Aβ-42, and α-syn gene fragments has been successfully inserted into the pET-28a(+) backbone.

![](https://static.igem.wiki/teams/5533/result/2.png)

Fig. 2 Verification of the transformation results of pET-28a(+)-NF-L/Aβ-42/α-syn positive colony. A: The single colony on the plate; B: The amplification results of colony PCR.

Finally, we used universal primer T7-Forward to verify the positive colonies by Sanger sequencing. Fig.3 showed the correct sequences of recombination plasmids. We chose correct colony for further IPTG induction.

![](https://static.igem.wiki/teams/5533/result/3.png)

Fig. 3 Sanger sequencing results of pET-28a(+)-NF-L/Aβ-42/α-syn positive colony.

**1.2 Construction for pET-28a(+)-NF-L-mCherry/Aβ-42-mCherry/α-syn-mCherry**

In order to visualize the recombination proteins, we further constructed pET-28a(+)-NF-L-mCherry, pET-28a(+)-Aβ-42-mCherry, and pET-28a(+)-α-syn-mCherry plasmids by fusing mCherry fluorescent gene to the C-terminal of target genes. We constructed recombination plasmid by three fragments Infusion ligation, including target genes, mCherry gene, and linearized vector backbone. Thus, we designed PCR primer for amplification with adding appropriate overlap adaptor sequences. First, we amplified NF-L, Aβ-42, and α-syn fragments by high-fidelity PCR with specific adaptor primers, with a length of 1059 bp, 789 bp, and 522 bp, respectively. Fig. 4A showed correct bands with target size of three genes. Then, we amplified mCherry fluorescent gene by high-fidelity PCR with a length of 711 bp. Fig. 4B showed correct bands with target size of mCherry gene. The linearized pET-28a(+) vector backbone was the same as Fig. 1B.

![](https://static.igem.wiki/teams/5533/result/4.png)

Fig. 4 Agarose gel electrophoresis of target genes and mCherry fragment

After Infusion reaction assay, we directly the recombination plasmids into expression strain E.coli BL21. Fig. 5A showed the single colony on the plate. We then selected 3, 12, and 11 colonies for pET-28a(+)-NF-L-mCherry, pET-28a(+)-Aβ-42-mCherry, and pET-28a(+)-α-syn-mCherry, respectively. Next, we used vector universal primers T7-Forward and T7-Terminal to detect target genes insertion by colony PCR. As showed in Fig. 5B, NF-L, Aβ-42, α-syn , and mCherry gene fragments has been successfully inserted into the pET-28a(+) backbone.

![](https://static.igem.wiki/teams/5533/result/5.png)

Fig. 5 Verification of the transformation results of pET-28a(+)-NF-L-mCherry/Aβ-42-mCherry /α-syn-mCherry positive colony. A: The single colony on the plate; B: The amplification results of colony PCR.

Finally, we used universal primer T7-Forward to verify the positive colonies by Sanger sequencing. Fig.6 showed the correct sequences of the fusion junction between target genes and mCherry fluorescent gene. We chose correct colony for further IPTG induction.

![](https://static.igem.wiki/teams/5533/result/6.png)

Fig. 6 Sanger sequencing results of pET-28a(+)-NF-L-mCherry/Aβ-42-mCherry /α-syn-mCherry positive colony.

2. Protein expression and purification

**2.1 Protein expression**

Based on the lac operator system in pET-28a(+) vector backbone, we used IPTG for protein prokaryotic expression. First, we used a final concentration of 1 mM/L IPTG for usual protein expression. Then, we set two temperature and different expression time for determining the optimal induction condition. The expected molecular weights of 6xHis-NF-L, 6xHis-NF-L-mCherry, 6xHis-Aβ-42, 6xHis-Aβ-42-mCherry, 6xHis-α-syn, and 6xHis-α-syn-mCherry are 44.8 kDa, 71.5 kDa, 34.0 kDa, 60.7 kDa, 23.3 kDa, and 50.0 kDa, respectively. As showed in Fig. 7, we can clearly observe protein bands of target genes. Interestingly, we found that the molecular weight of 6xHis-Aβ-42 and 6xHis-Aβ-42-mCherry are slightly larger than as predicted, suggesting that these two proteins might have some post-translational modifications. For 6xHis-NF-L and 6xHis-NF-L-mCherry, there was no significant difference in the bands between 25°C and 37°C, and the protein expression level was highest after 6 hours of induction. For 6xHis-Aβ-42 and 6xHis-Aβ-42-mCherry, there was no significant difference in the bands between 25°C and 37°C, and the protein expression level showed similar between after 3 hours and 6 hours induction, indicating that the protein synthesis rate had reached a plateau. Interestingly, 6xHis-α-syn showed different protein synthesis trends under 25°C and 37°C. Under 25°C, the protein expression level of 6xHis-α-syn was highest after 1 hours of induction, but as the induction time increased, the protein was further degraded. For 37°C, the protein expression level of 6xHis-α-syn was highest after 6 hours of induction. For 6xHis-α-syn-mCherry, the protein synthesis rate under 37°C is faster than that under 25°C, and the protein expression level was highest after 6 hours of induction.

![](https://static.igem.wiki/teams/5533/result/7.png)

![](https://static.igem.wiki/teams/5533/result/7-1.png)
Fig. 7 SDS-PAGE analysis for small amount of target proteins expression.

We further used anti-His antibody for Western Blotting experiments and then quantified the induction amount of target proteins according to the relative gray value of the band. As showed in Fig. 8A, we found that the His antibody specificity is not particularly high, suggesting that we might need to optimize the relative concentrations of the primary and secondary antibodies, but we can still clearly see the bands of target proteins. Then, we used ImageJ software to calculate the relative gray value intensity of target proteins. The results were consistent with those of Coomassie Brilliant Blue staining in Fig. 7. Different temperatures have an effect on the correct folding and biological activity of proteins. Finally, except for 6xHis-α-syn, we chose 25°C and 37°C after 6 hours of induction for large-scale purification. For 6xHis-α-syn, we chose 25°C after 1 hours of induction and 37°C after 6 hours of induction for large-scale purification, respectively.

![](https://static.igem.wiki/teams/5533/result/8-a.png)

![](https://static.igem.wiki/teams/5533/result/8-b.png)
Fig. 8 Western Blotting analysis and the relative gray value intensity for small amount of target proteins expression.

**2.2 Protein His-tag purification**

As showed in Fig. 9, we could clearly observe that the combination proteins with mCherry fluorescent tag have visible red precipitate. We further used His-tag purification kit to purify target proteins and then stained protein bands by Coomassie Brilliant Blue staining. As showed in Fig. 10 and Fig. 11, target proteins were enriched in elution components. Compared with proteins induced under 25°C, proteins induced under 37°C had better purification efficiency and less miscellaneous bands. In addition, we also found that the efficiency of protein binding with Ni-NTA beads is not particularly high, so we might need to optimize the conditions to obtain higher recovery yield. Then, we collected elution components for functional test.

![](https://static.igem.wiki/teams/5533/result/9.png)

Fig. 9 Representative images of large-scale protein expression.

![](https://static.igem.wiki/teams/5533/result/10.png)

Fig. 10 SDS-PAGE analysis for large-scale His-tag protein purification of target proteins expressed under 25°C. T: Total protein; FT: Flow Through.

![](https://static.igem.wiki/teams/5533/result/11.png)

Fig. 11 SDS-PAGE analysis for large-scale His-tag protein purification of target proteins expressed under 37°C. Total protein; FT: Flow Through.

3. Functional test

**3.1 Determination of protein fused with mCherry tag by fluorescence microscope**

First, we determined the effectivity of target protein fused with mCherry tag by fluorescence microscope. After fixing sample and adding anti-fading agent, we detected mCherry signal using an excited light wavelength of 587nm and an emitted light wavelength of 610nm. As showed in Fig. 12, E.coli BL21 expressing pET-28a(+)-NF-L-mCherry/Aβ-42-mCherry/α-syn-mCherry could be clearly observed with red fluorescence in cord-like structure.

![](https://static.igem.wiki/teams/5533/result/12.png)

Fig. 12 Observation of E.coli BL21 expressing
 pET-28a(+)-NF-L-mCherry/Aβ-42-mCherry/α-syn-mCherry by fluorescence microscopy

**3.2 Verification of target recombination protein by ELISA assay**

We used ELISA assay to verify the accuracy of NF-L protein. As showed in Fig. 13A, the correlation between NF-L protein and absorbance 450nm is 0.9972, indicating that ELISA assay can accurately and effectively detect the content of NF-L in elution components. In elution components of protein expression under 25°C, the content of NF-L protein in E6 was the highest, reaching 25.31 ng/mL (Fig. 13B). Interestingly, in elution components of protein expression under 37°C, the content of NF-L protein in E2 was the highest, reaching 34.25 ng/mL (Fig. 13C).

![](https://static.igem.wiki/teams/5533/result/13.png)

Fig. 13 Verification of NF-L by ELISA assay

Then, we used ELISA assay to verify the accuracy of Aβ-42 protein. As showed in Fig. 14A, the correlation between Aβ-42 protein and absorbance 450nm is 0.9983, indicating that ELISA assay can accurately and effectively detect the content of Aβ-42 in elution components. In elution components of protein expression under 25°C, the content of Aβ-42 protein in E6 was the highest, reaching 21.35 pg/mL (Fig. 14B). In elution components of protein expression under 37°C, the content of Aβ-42 protein in E6 was the highest, reaching 26.92 pg/mL (Fig. 14C).

![](https://static.igem.wiki/teams/5533/result/14.png)

Fig. 14 Verification of Aβ-42 by ELISA assay

Finally, we used ELISA assay to verify the accuracy of α-syn protein. As showed in Fig. 15A, the correlation between α-syn protein and absorbance 450nm is 0.994, indicating that ELISA assay can accurately and effectively detect the content of α-syn in elution components. In elution components of protein expression under 25°C, the content of α-syn protein in E6 was the highest, reaching 97.54 ng/mL (Fig. 15B). In elution components of protein expression under 37°C, the content of α-syn protein in E6 was the highest, reaching 98.32 ng/mL (Fig. 15C).

![](https://static.igem.wiki/teams/5533/result/15.png)

Fig. 15 Verification of α-syn by ELISA assay

Future plans

- Our experiment did not optimize the protein expression conditions, and fermentation regulation plays a crucial role in yield. We can continue to optimize the expression conditions of the strains, such as oxygen levels, inoculum size, and pH, to improve production and to achieve the amount of protein required for antigen preparation.
- In our expression process, we added inducers IPTG. We can explore more environmentally friendly production methods by modifying promoter of vector backbone or replacing the inducers with external regulatory conditions, such as temperature and light exposure, to induce expression through these greener and harmless approaches.

Reference

- Goedert M. Alpha-synuclein and neurodegenerative diseases. Nat Rev Neurosci. (2001) 2:492–501.
- Braak H, Ghebremedhin E, Rüb U, Bratzke H, Del Tredici K. Stages in the development of Parkinson's disease-related pathology. Cell Tissue Res. (2004) 318(1):121-34.
- Giasson BI, Murray IV, Trojanowski JQ, Lee VM, A. hydrophobic stretch of 12 amino acid residues in the middle of alpha-synuclein is essential for filament assembly. J Biol Chem. (2001) 276:2380–6.
- Hoyer W, Cherny D, Subramaniam V, Jovin TM. Impact of the acidic C-terminal region comprising amino acids 109-140 on alpha-synuclein aggregation in vitro. Biochemistry. (2004) 43:16233–42.
- Xiang C, Cong S, Tan X, Ma S, Liu Y, Wang H, Cong S. A meta-analysis of the diagnostic utility of biomarkers in cerebrospinal fluid in Parkinson's disease. NPJ Parkinsons Dis. (2022) 29;8(1):165.

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