iGEM 2025 — Team subcat-shanghai

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Main Timeline:

The timeline is in monthly increments, with each month normalized to 30 days. Each grid cell corresponds to a
three-day interval. The shaded areas within the grid represent time periods designated as workdays.

April：

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Literature Reading |  |  |  |  |  |  |  |  |  |  |
| Theoretical knowledge learning |  |  |  |  |  |  |  |  |  |  |
| Project brain storm |  |  |  |  |  |  |  |  |  |  |

May：

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Literature Reading |  |  |  |  |  |  |  |  |  |  |
| Project introduction |  |  |  |  |  |  |  |  |  |  |
| Theoretical knowledge training |  |  |  |  |  |  |  |  |  |  |

June：

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Theoretical knowledge training |  |  |  |  |  |  |  |  |  |  |
| Project brain storm |  |  |  |  |  |  |  |  |  |  |
| Experimental design |  |  |  |  |  |  |  |  |  |  |
| Laboratory safety training |  |  |  |  |  |  |  |  |  |  |
| Implement experimental plan |  |  |  |  |  |  |  |  |  |  |

July:

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Implement experimental plan |  |  |  |  |  |  |  |  |  |  |
| Project problems discussion |  |  |  |  |  |  |  |  |  |  |
| Record experiment report |  |  |  |  |  |  |  |  |  |  |

August:

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Implement experimental plan |  |  |  |  |  |  |  |  |  |  |
| Project problems discussion |  |  |  |  |  |  |  |  |  |  |
| Analysis of experimental results |  |  |  |  |  |  |  |  |  |  |

September:

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Project problems discussion |  |  |  |  |  |  |  |  |  |  |
| Wiki writing |  |  |  |  |  |  |  |  |  |  |
| Education |  |  |  |  |  |  |  |  |  |  |
| Preparation of defense materials |  |  |  |  |  |  |  |  |  |  |

October:

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Preparation of defense materials |  |  |  |  |  |  |  |  |  |  |
| Education |  |  |  |  |  |  |  |  |  |  |
| Project review |  |  |  |  |  |  |  |  |  |  |

Record of Main Experimental Content

July-August:

1. Inoculate glycerol stock (Escherichia coli K12 containing the target gene fragment and host strain containing
the target vector)

2. Plasmid extraction

3. PCR amplification of the target fragment and vector

4. Visualization of the target fragment and vector by agarose gel electrophoresis

5. Gel extraction, separation, and purification of the fragment and vector

6. Homologous recombination to obtain the recombinant vector

7. Transform competent cells

8. Plate plating

9. Verify single clones by colony PCR

10. Inoculate seed stock from a single clone

11. Expand seed stock

12. Plasmid extraction

13. Send plasmid for sequencing

14. Replace the TyrA gene from different sources in plasmid 2 by PCR and homologous recombination

15. First-round fermentation test to select the optimal TyrA gene

16. Replace the RBS by loop PCR

17. Second-round fermentation test to select the optimal RBS sequence

18. Transform plasmid 1 and optimized plasmid 2 into BL21 competent cells

19. Plate plating

20. Verify single clones by colony PCR

21. Inoculate seed culture with a single clone

22. Expand seed culture

23. Induce protein expression with IPTG

24. Sampling during gradient time

25. SDS-PAGE

26. Inoculate LBG medium for large-scale expression

27. Protein expression

28. Purification by nickel column

29. SDS-PAGE

30. Inoculate LBG medium

31. Set inducer concentration and sampling time

32. Detect L-DOPA production using ELISA kit

'
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{
"title": 'Overview',
"content": '

Parkinson\'s disease (PD) is the second most common neurodegenerative disease after Alzheimer\'s disease[1]. It is most common
in people aged 65 and older and is closely associated with aging[2]. With the accelerating aging of the global population,
the number of PD patients is increasing dramatically, resulting in a significant economic and medical burden. The primary
pathological features of PD are abnormal aggregation of α-synuclein (α-syn) and loss of dopaminergic neurons in the
substantia nigra of the midbrain[3]. It is characterized by motor and non-motor symptoms, which severely impact patients\'
quality of life. Studies have found that most PD patients experience gastrointestinal symptoms, such as nausea,
constipation, and delayed gastric emptying[4]. Approximately 80% of PD patients experience constipation, and constipation
symptoms typically develop 10 years or more earlier than motor symptoms[5-6]. In recent years, increasing research has
demonstrated a close association between the gut microbiome and PD. Therefore, clarifying the mechanisms of action of the
gut microbiome in PD may provide new insights into PD intervention and treatment. The research and application of levodopa
also holds significant significance in the field of neuroscience. Combining beneficial probiotic strains with levodopa could
provide insights into the development of new treatments and medications[7-8].

',
"children": []
},
{
"title": 'Part 1 pCDFDuet1-TktA-AroF-AroE (BBa\_259TIJNK)',
"content": '',
"children": [
{
"title": 'Design:',
"content": '

The pCDFDuet1 plasmid was provided by the strain library of our unit, and the target genes TktA、AroF and AroE were all
fragments amplified from the genome using primers using Escherichia coli K12 as templates. The three target genes were
constructed together with the vector using the homologous recombination method.

![](https://static.igem.wiki/teams/5740/engineering/1.webp)

Figure 1. The plasmid map of pCDFDuet1-TktA-AroF-AroE

',
},
{
"title": 'Build:',
"content": '

We used homologous recombination to construct the pCDFDuet-tktA-aroF-aroE plasmid. First, using PCR technology, we designed
corresponding primers and amplified the target fragments of tktA, aroF, and aroE using Escherichia coli K12 as a template.
We also amplified the pCDFDuet plasmid backbone using a laboratory-preserved plasmid. As we can see from Figure 2A, the
agarose gel electrophoresis results after amplification showed that the amplified lengths of the backbone and target
fragments were all in line with the expected sizes. The pCDFDuet plasmid backbone is 3781 bp in length, tktA is 1995 bp in
length, aroF is 1071 bp in length, and aroE is 819 bp in length.

After obtaining the empty vector and fragments of corresponding lengths, we recovered the agarose gel to obtain the purified
vector and fragments, and obtained the concentration of these gene fragments through the nanodrop instrument. After
obtaining the corresponding concentration, we used homologous recombination to allow the vector and fragments to be
recombined together. Then we transformed the recombinant product into the E. coli cloning strain TOP10. As shown in Figure
2B, after the transformation and overnight growth, we saw that there were single colonies with successful transformation.
However, whether the transformation was successful or not, we need to perform corresponding verification.

First, by colony PCR, we randomly selected 4 independent single colonies of the same size on the plate and used colony PCR
primers for identification. The length was designed to be 2102bp. In PCR After amplification and identification by agarose
gel electrophoresis, we found that the size of the amplified fragments met our expectations. Therefore, these single clones
were considered to have successfully transformed with the recombinant plasmid.

Finally, we need to expand and culture the corresponding correct single clones, extract the plasmids, and send them to a
biological company for sequencing. The purpose of this step is to ensure the position and order of our vector and connector,
and whether there are any base deletions or mutations. Figure 2D shows that our construction results are correct. At this
point, our first recombinant plasmid was successfully constructed.

![](https://static.igem.wiki/teams/5740/engineering/2.webp)

Figure 2. A. Electrophoresis results of plasmid backbone and target fragment, B. Recombinant plasmid transformed into
cloning strain TOP10, C. Electrophoresis results after single clone colony PCR, D. Plasmid sequencing results

',
},
]
},
{
"title": 'Part 2 pETDuet1-TyrA(K12)-HpaB-HpaC (BBa\_25TX8LQL)',
"content": '',
"children": [
{
"title": 'Design:',
"content": '

The pETDuet1 plasmid was provided by the strain library of our unit, and the target genes TyrA(K12), HpaB and HpaC were all
fragments amplified from the genome using primers using Escherichia coli K12 as templates. The three target genes were
constructed together with the vector using the homologous recombination method.

![](https://static.igem.wiki/teams/5740/engineering/3.webp)

Figure 3. The plasmid map of pETDuet1-TyrA(K12)-HpaB-HpaC

',
},
{
"title": 'Build:',
"content": '

We used E. coli K12 as a template to amplify other target fragments. First, we designed corresponding primers and then used
cultured E. coli K12 as a template to amplify the target genes TyrA, hpaB, and hpaC. The plasmid backbone was also obtained
by inverse PCR. After the amplification reaction, the amplified products were identified by agarose gel electrophoresis. The
results showed that the plasmid backbone and the three target genes were successfully amplified. The backbone length was
5420 bp, the length of TyrA was 1122 bp, the length of hpaB was 1563 bp, and the length of hpaC was 513 bp, as can be seen
in Figure 4.

![](https://static.igem.wiki/teams/5740/engineering/4.webp)

Figure 4. Electrophoresis results of pETDuet and TyrA(K12), hpaB and hpaC

After obtaining the gene fragment of the correct size, we need to recover the purified vector and fragment by gel recovery.
Then, we use homologous recombination to construct the complete recombinant plasmid. Next, we need to transform it into a
cloning strain and, after overnight growth, select the grown single clones for colony PCR identification. As shown in Figure
5, we selected six single clones and amplified them using the designed primers. The results showed that the amplified length
was 1500bp, which is consistent with the expected length, indicating that the transformation process was successful.

![](https://static.igem.wiki/teams/5740/engineering/5.webp)

Figure 5. Results of monoclonal plates and colony PCR after transformation

Finally, we inoculated the successful transformants into seed solution, cultured them, extracted the plasmids, and sent them
to a biotechnology company for sequencing. The results are shown in Figure 6. The sequencing results show that the
constructed recombinant plasmids have no base mutations or deletions.

![](https://static.igem.wiki/teams/5740/engineering/6.webp)

Figure 6. Plasmid sequencing results

',
},
]
},
{
"title": 'Part 3 pETDuet1-TyrA(SO)-HpaB-HpaC (BBa\_256I72YK)',
"content": '',
"children": [
{
"title": 'Design:',
"content": '

The pETDuet1 plasmid was provided by the strain library of our unit, and the target genes TyrA(SO) is synthesized by a
biological company, HpaB and HpaC were all fragments amplified from the genome using primers using Escherichia coli K12 as
templates. The three target genes were constructed together with the vector using the homologous recombination method.

![](https://static.igem.wiki/teams/5740/engineering/7.webp)

Figure 7. The plasmid map of pETDuet1-TyrA(SO)-HpaB-HpaC

',
},
{
"title": 'Build:',
"content": '

we constructed the pETDuet-TyrA(SO)-hpaB-hpaC plasmid. Compared with pETDuet-TyrA(K12)-hpaB-hpaC, the difference between the
two plasmids is the source of TyrA. Therefore, we used homologous recombination to replace TyrA based on
pETDuet-TyrA(K12)-hpaB-hpaC. We designed two pairs of primers, one to amplify the TyrA gene from the new source, and the
other pair of primers to amplify the rest of the pETDuet-TyrA(K12)-hpaB-hpaC except TyrA(K12). After amplification, agarose
gel electrophoresis was performed for identification, as shown in Figure 8A. As shown in Figure 8B , the plasmid backbone
and TyrA(SO) amplification results after electrophoresis were consistent with the expected lengths: the plasmid backbone was
7535 bp long, and TyrA(SO) was 1152 bp long. The two fragments were then recovered and purified from the gel. After
homologous recombination, the recombinant products were transformed into E. coli TOP10. As shown in Figure 8B , single
clones emerged after overnight growth. To verify the correctness of the transformed single clones, we performed colony PCR
identification. Twelve single clones were selected for identification. In Figure 8C , the colony PCR result showed a size of
2100 bp, demonstrating that the transformed single clones all contained the correct plasmid. The correct single clones were
then inoculated into seed solution, and the plasmids were extracted and sent to a biotechnology company for sequencing.
Sequencing results confirmed that the constructed plasmid was successful.

![](https://static.igem.wiki/teams/5740/engineering/8.webp)

Figure 8 A. Electrophoresis results of plasmid backbone and target gene fragments, B. Plate of recombinant plasmid
transformed into E. coli, C. Electrophoresis results of monoclonal colony after PCR, D. Sequencing results of recombinant
plasmid

',
},
]
},
{
"title": 'Part 4 pETDuet1-TyrA(HI)-HpaB-HpaC (BBa\_25L16LN5)',
"content": '',
"children": [
{
"title": 'Design:',
"content": '

The pETDuet1 plasmid was provided by the strain library of our unit, and the target genes TyrA(HI) is synthesized by a
biological company, HpaB and HpaC were all fragments amplified from the genome using primers using Escherichia coli K12 as
templates. The three target genes were constructed together with the vector using the homologous recombination method.

![](https://static.igem.wiki/teams/5740/engineering/9.webp)

Figure 9. The plasmid map of pETDuet1-TyrA(HI)-HpaB-HpaC

',
},
{
"title": 'Build:',
"content": '

When constructing pETDuet-TyrA(HI)-hpaB-hpaC, we used a similar approach to pETDuet-TyrA(SO)-hpaB-hpaC. We first designed
primers to amplify the TyrA(HI) target gene, and another pair of primers to amplify the backbone. After amplification, we
verified the success of the amplification by agarose gel electrophoresis. As shown in Figure 10A, the amplified vector and
target gene lengths were consistent with the expected values, with the backbone being 7535 bp and the target gene being 1146
bp. Following amplification, the two were ligated by homologous recombination and transformed into the corresponding E. coli
cloning strain. After overnight growth, as shown in Figure 10B, 12 single colonies were selected for colony PCR analysis to
confirm the success of the transformants.

![](https://static.igem.wiki/teams/5740/engineering/10.webp)

Figure 10 A. Plasmid backbone and target gene electrophoresis results. B.plate image of the transformed single colonies.

After selecting the corresponding single clone for colony PCR, we performed electrophoresis analysis on the amplified
product. Figure 11A shows that the colony PCR result met the expected length of 2000 bp. Furthermore, we selected a single
clone that successfully obtained colony PCR and inoculated it into seed solution. The plasmid was then extracted and sent to
a biotechnology company for sequencing. The results are shown in Figure 11B. The sequencing results indicate that the
recombinant plasmid construction was successful, with no base mutations or deletions.

![](https://static.igem.wiki/teams/5740/engineering/11.webp)

Figure 11 A. Plasmid backbone and target gene electrophoresis results. B.plate image of the transformed single colonies.

',
},
]
},
{
"title": 'Test1:',
"content": '',
"children": [
{
"title": '1. Target protein expression test',
"content": '

After constructing Plasmid 1 (pCDFDuet1-TktA-AroF-AroE) and the three TyrA plasmids 2, we need to transform two plasmids
into a new E. coli expression strain. Because the production of L-DOPA requires the simultaneous function of all six genes,
we first transform two plasmids into E. coli BL21 (DE3), as shown in Figure 12. After overnight growth, we see that the
double-transformed host bacteria have corresponding monoclonal colonies growing. We will then perform colony PCR to verify
whether the double plasmid transformation is successful.

![](https://static.igem.wiki/teams/5740/engineering/12.webp)

Figure 12. The corresponding single colony plate image after plasmid 1 and plasmid 2 were transformed into BL21 (DE3)
together

After the double plasmid transformation is completed, we need to perform colony PCR identification. Since there are two
plasmids, two pairs of primers need to be designed to identify each plasmid separately. Therefore, colony PCR is performed
on each single clone of strain1-3. The corresponding lengths are: plasmid 1 of strain1 is 1000bp, plasmid 2 is 800bp,
plasmid 1 of strain2 is 800bp, plasmid 2 is 1000bp, and plasmid 1 of strain3 is 1000bp, plasmid 2 is 1000bp. After the
amplification is completed, we perform agarose gel electrophoresis. The results are shown in Figure 13. The lengths of all
colony PCRs are in line with the expected size. Therefore, the double plasmid transformation is considered successful.

![](https://static.igem.wiki/teams/5740/engineering/13.webp)

Figure 13. Electrophoresis of colony PCR results of three strains after double plasmid transformation

Before conducting the first round of fermentation tests, we needed to perform protein expression tests on the two plasmids
to initially verify the normal expression of our six genes in E. coli. Therefore, we transformed BL21 (DE3) host cells with
plasmid 1 (pCDFDuet-tktA-aroF-aroE) and plasmid 2 (pETDuet-TyrA(K12)-hpaB-hpaC) for protein expression. This round of
protein expression testing employed rapid induction, using a temperature of 37°C, a rotational speed of 220 rpm, an inducer
concentration of 1 mM IPTG, and a duration of 6 hours.

After induction, we collected the protein and divided it into the supernatant and precipitate of crude protein for SDS-PAGE
identification to observe whether the expression of the target gene was successful. As shown in Figure 14, wells 1-4 are the
supernatant of plasmid 1, 5 is the precipitate of plasmid 1, wells 6-8 are the supernatant of plasmid 2, and 9-10 are the
precipitate of plasmid 2. Plasmid 2 contains inclusion bodies, and the proteins corresponding to the target genes are all
successfully expressed. Although the target protein corresponding to plasmid 2 has inclusion bodies during the collection
process, this may be related to the operational steps in the protein collection process. In addition, since we are
strengthening the L-dopa biosynthesis pathway in the cell, we confirm that the expression has achieved our goal.

![](https://static.igem.wiki/teams/5740/engineering/14.webp)

Figure 14. Target protein expression test using plasmids 1 (pCDFDuet-tktA-aroF-aroE) and 2 (pETDuet-TyrA(K12)-hpaB-hpaC).
Wells 1-4 contain the supernatant from plasmid 1, and well 5 contains the precipitate from plasmid 1. Wells 6-8 contain the
supernatant from plasmid 2, and wells 9-10 contain the precipitate from plasmid 2.

',
},
{
"title": '2. Detection of L-dopa production using an ELISA test kit',
"content": '

After confirming that our target proteins are expressed normally, we need to perform fermentation tests on the previously
constructed strains carrying TyrA from three sources in order to obtain the optimal TyrA. The fermentation method is
consistent with the protein expression conditions, and the fermentation time is 48 hours. Three replicates are set for each
strain fermentation, as shown in Figure 15A. After the fermentation is completed, we use the L-DOPA Elisa detection kit for
measurement. The sample preparation, reaction process, and reaction termination are shown in Figures 15BCD. Finally, we use
a microplate reader to measure the absorbance value at 450nm Figures 15E.

![](https://static.igem.wiki/teams/5740/engineering/15.webp)

Figure 15. A. Fermentation test of strain 123 (three replicates), B. Sample preparation, C. Reaction, D. End of reaction, E.
Absorbance test

We first developed a standard curve, as shown in Figure 16A. We then calculated the L-DOPA content in the sample based on
the standard curve and absorbance values. It can be seen that strain 1 had the highest L-DOPA production, as shown in Figure
16B. That is, TyrA derived from K12 showed better enzyme activity in the modified strain. For this modified strain, genes
derived from similar species have better adaptability, so this gene was selected for subsequent RBS screening.

![](https://static.igem.wiki/teams/5740/engineering/16.webp)

Figure 16. A. Standard curve of the ELISA test kit; B. L-DOPA production by three strains, where Strain 1 contains TyrA from
K12, Strain 2 contains TyrA from SO, and Strain 3 contains TyrA from HI.

',
},
]
},
{
"title": 'Learn1:',
"content": '

Our overall technical approach involves first constructing a complete L-DOPA production route. The construction of the
L-DOPA production pathway in E. coli is divided into two modules. Module 1, Plasmid 1, serves as an enhanced precursor
supply, strengthening the rate-limiting enzymes in the PPP and glycolysis pathways and enhancing the metabolic flux of PEP
and E4P to the shikimate pathway. Module 2, Plasmid 2, enhances the key precursors in the shikimate pathway that lead to
L-DOPA production and the key enzymes for L-DOPA synthesis. Product production is achieved by enhancing pathway carbon flux
and product synthesis, respectively.After finalizing the route, the second step involves comparing the effects of TyrA genes
from three different sources. This step aims to identify the optimal TyrA gene, as it determines the critical rate-limiting
step in L-DOPA synthesis. Fermentation testing will identify the optimal TyrA gene source and use this plasmid for
subsequent transformations.

The final results showed that TyrA from the same species exhibited better enzymatic activity in the modified strain. For
this modified strain, genes from similar species have better adaptability, but we still hope to improve this rate-limiting
step, so this gene was selected for subsequent RBS screening.

',
"children": []
},
{
"title": 'Part 5 pETDuet1-TyrA(K12)-RBS2-HpaB-HpaC (BBa\_256PUDZB)',
"content": '',
"children": [
{
"title": 'Design:',
"content": '

The construction of pETDuet1-TyrA(K12)-RBS2-HpaB-HpaC is based on pETDuet1-TyrA(K12)-HpaB-HpaC. It only needs to design the
new RBS sequence on the primer and amplify it with the original plasmid as the template. The completed plasmid is
pETDuet1-TyrA(K12)-RBS2-HpaB-HpaC with the RBS sequence replaced. We then sequenced the plasmid to ensure that there was no
deletion or mutation at the replaced position.

![](https://static.igem.wiki/teams/5740/engineering/17.webp)

Figure 17. The plasmid map of pETDuet1-TyrA(K12)-RBS2-HpaB-HpaC

',
},
{
"title": 'Build:',
"content": '

To replace the RBS at the start of the TyrA gene in the pETDuet-TyrA(K12)-hpaB-hpaC plasmid, we used inverse PCR. We
designed a pair of primers targeting the desired RBS sequence and amplified the plasmid using the original
pETDuet-TyrA(K12)-hpaB-hpaC plasmid as a template. Compared to the original plasmid, only the RBS sequence was altered in
the amplified plasmid. Because the newly amplified plasmid was essentially the same length as the original, we avoided
agarose gel electrophoresis and instead performed transformation after amplification and extracted the plasmid for
sequencing.The sequencing results in Figure 19 demonstrate the successful replacement of the new RBS sequence. The sequence
on the left represents the replacement of RBS2.

',
},
]
},
{
"title": 'Part 6 pETDuet1-TyrA(K12)-RBS3-HpaB-HpaC (BBa\_25ZKRZOR)',
"content": '',
"children": [
{
"title": 'Design:',
"content": '

The construction of pETDuet1-TyrA(K12)-RBS3-HpaB-HpaC is based on pETDuet1-TyrA(K12)-HpaB-HpaC. It only needs to design the
new RBS sequence on the primer and amplify it with the original plasmid as the template. The completed plasmid is
pETDuet1-TyrA(K12)-RBS2-HpaB-HpaC with the RBS sequence replaced. We then sequenced the plasmid to ensure that there was no
deletion or mutation at the replaced position.

![](https://static.igem.wiki/teams/5740/engineering/18.webp)

Figure 18. The plasmid map of pETDuet1-TyrA(K12)-RBS3-HpaB-HpaC

',
},
{
"title": 'Build:',
"content": '

The construction of this step is consistent with the method of replacing RBS2，The sequencing results in Figure 19
demonstrate the successful replacement of the new RBS sequence. The sequence on the right represents the replacement of
RBS3.

![](https://static.igem.wiki/teams/5740/engineering/19.webp)

Figure 19. Sequencing results after replacing the RBS sequence

',
},
]
},
{
"title": 'Test2:',
"content": '',
"children": [
{
"title": '1. Detection of L-dopa production using an ELISA test kit',
"content": '

After the RBS sequence replacement was completed, fermentation testing was repeated to determine which RBS sequence
performed best. Three strains were inoculated into LBG (20 g/L glucose) medium at an initial OD of 0.05. After fermentation,
product testing was performed to identify the optimal RBS sequence for the TyrA gene and improve expression of the key
enzyme.

The fermentation testing steps were similar to those described above. Three replicates of different RBS strains were tested,
as shown in Figure 20A. The assays were performed using a kit, with the reaction progressing and reaction completion times
(Figure 20BC). Finally, the fermentation yields were calculated using a standard curve.

![](https://static.igem.wiki/teams/5740/engineering/20.webp)

Figure 20. A Fermentation test of different RBS strains (three replicates), BC is divided into reaction progress and
reaction completion

By substituting the absorbance value into the standard curve for calculation, we can obtain the amount of L-dopa produced by
the strain under different RBS sequences. According to the bar graph in Figure 21, the three different RBS sequences do have
a certain effect on gene expression. Among the RBS sequences we screened, the optimal expression level increased the
strain\'s yield by 16.4%, reaching 500.3 mg/L.

![](https://static.igem.wiki/teams/5740/engineering/21.webp)

Figure 21. The yield of levodopa corresponding to different RBS sequences

',
},
{
"title": '2. Functional test',
"content": '

At this point, we have confirmed the optimal TyrA source (K12) and RBS sequence TCACACAGG. We then initially tested the
effect of different glucose concentrations on L-DOPA production by fermentation in the BL21 (DE3) host strain. We set
initial glucose concentrations of 10 g/L, 20 g/L, 30 g/L, 40 g/L, and 50 g/L, and the fermentation conditions were the same
as before, After this, we will add some new parameters to explore the factors affecting levodopa production, such as inducer
concentration and time.

Figure 22 shows that the yield of the strain increased with increasing glucose concentration, reaching a peak of 689.3 mg/L.
However, as the glucose concentration continued to increase to 50 g/L, the yield began to decline. We speculate that the
high glucose concentration affected the strain\'s metabolism, resulting in a decrease in yield.

![](https://static.igem.wiki/teams/5740/engineering/22.webp)

Figure 22. Analysis of results at different initial sugar concentrations (optimal RBS sequence strain)

To further explore the factors affecting levodopa, we simultaneously investigated the inducer concentration, glucose
concentration, and fermentation time. The glucose concentration was still set at 10 g/L to 50 g/L, with 5 gradients, and the
inducer concentration was 0, 0.2, 0.4, 0.6, 0.8, 1.0 mM, with 6 gradients, and the fermentation time was 36 h and 48 h. The
strain was inoculated into LBG medium with an initial OD of 0.05. After the fermentation was completed, the product was
tested.

![63080ce321ff2d29e92777e1eaebd1a4](https://static.igem.wiki/teams/5740/engineering/23.webp)

Figure 23. Effects of inducer concentration and glucose concentration on levodopa production at different times

The results showed significant differences in L-DOPA yields across different conditions, with overall yield levels
increasing over time. Results at 36 hours (Figure 23A) show that L-DOPA yields were concentrated in the 50–100 pg range
across most conditions. Yields generally increased with increasing IPTG concentrations, but there was significant
variability among glucose levels. At 0 mM IPTG, yields were generally low across all glucose conditions, ranging from 20–40
pg. At 0.2–0.4 mM IPTG, yields reached the highest levels at 20 g/L and 30 g/L glucose, reaching 90–100 pg, while yields at
0 g/L and 10 g/L were lower. At 0.8–1.0 mM IPTG, 40 g/L glucose performed exceptionally well, exceeding 100 pg, while other
conditions generally fell below this level. In summary, at 36 hours, at moderate IPTG levels (0.4–0.6 mM), 20–30 g/L glucose
was more favorable, while at high IPTG levels (0.8–1.0 mM), 40 g/L glucose performed best.

The 48-hour results (Figure 23B) showed that overall yield levels at 48 hours increased further compared to 36 hours,
exceeding 120 pg in some conditions. Under 0 mM IPTG, the 40 g/L glucose group achieved the highest yield, approximately
75–80 pg, while the other groups ranged from 45–55 pg. Extreme differences were observed at 0.2 mM IPTG: Yields at 20–40 g/L
glucose levels were high, reaching 80–100 pg, but at 10 g/L, yields were nearly zero, the lowest of all conditions. At
0.4–0.6 mM IPTG, the yields at 30 g/L and 40 g/L glucose levels remained high (approximately 90–110 pg), significantly
higher than those at 0–10 g/L. At 0.8–1.0 mM IPTG, the 40 g/L glucose group reached its highest peak, reaching 120–130 pg,
while the 0 g/L and 10 g/L conditions were significantly lower, only around 50–70 pg.

Results Summary

1. A significant time effect: The overall yield at 48 hours was higher than at 36 hours, with the maximum increase being
even more significant.

2. The optimal condition was prominent: The yield reached its highest level (approximately 120–130 pg) at high IPTG
concentrations (0.8–1.0 mM) combined with high glucose concentrations (40 g/L), reaching the peak among all conditions.

3. Large variability at low IPTG concentrations: At 0.2 mM IPTG, glucose concentration significantly affected the results,
particularly at 10 g/L, where yields were extremely low, in stark contrast to the other groups.

4. Significant intra-group variability: At the same IPTG concentration, different glucose levels often exhibited significant
differences, particularly at 0.4 mM and 0.8 mM IPTG.

![](https://static.igem.wiki/teams/5740/engineering/24.webp)

Figure 24. Optimal inducer concentration and glucose concentration at different time points

Figure 24 shows the color distribution after 36 hours of fermentation. A broad warm (orange-red) region forms from the
center-left to the center-upper portion of the image (approximately IPTG 0.2–0.6 mM, glucose 20–35 g/L), indicating
medium-to-high yields within this band. A localized dark red region appears in the upper right corner (high IPTG ≈0.75–1.0
mM, high glucose ≈35–40 g/L), clearly demarcated from the surrounding area by a series of contour lines, indicating a local
peak or maximum. The vertical band at the left edge (IPTG near 0) is generally cooler, indicating generally low yields under
no or very low induction conditions. After 48 hours of fermentation, the high-yield zone significantly expanded and shifted
upward: a larger, darker red area with higher color intensity formed in the upper right quadrant (IPTG ≈0.6–1.0 mM, glucose
≈30–40 g/L). Compared to 36 hours, this region exhibited increased red intensity and area, indicating the overall highest
yield in both figures. The dark blue lowest depression, located at low to medium IPTG and low to medium glucose levels
(approximately IPTG ≈0.15–0.30 mM, glucose ≈5–12 g/L), formed a distinctly closed, cool-colored cavity. The color was darker
and more concentrated than the low-value portion at 36 hours.

In contrast, the highest color intensity at 48 hours was significantly higher than at 36 hours (the upper limit of the color
scale expanded to approximately 125 pg), and the high-yield zone at 48 hours was concentrated in the higher glucose and
slightly higher IPTG regions. At 36 hours, a broad warm plateau appeared to the left of the center, with a localized peak in
the upper right corner. Both figures show abrupt gradient changes, but the gradient is steeper (with denser contours) in the
high-yield region at 48 h, indicating that yield at this time point is more sensitive to changes in IPTG/glucose
concentrations at certain boundaries.

Overall, the highest yields in both figures occur in the high glucose (≈30–40 g/L) and medium-high to high IPTG (≈0.6–1.0
mM) regions. The peak intensity and area at 48 h are both greater than those at 36 h. From 36 to 48 h, the high-yield region
converges toward higher glucose and IPTG concentrations, with a significant increase in peak yields. Low-yield extremes are
more concentrated and have steeper boundaries at 48 h. The peak L-DOPA yield at 48 h is significantly higher than at 36 h,
and the high-yield region is concentrated to the upper right in the parameter space of IPTG and glucose.

Finally, we analyzed the inducer concentration, glucose concentration and time together. In the three-dimensional results of
Figure 25, we can see that 0.8-1.0mM, 40g/L, 36-48h, the fermentation results are better. Finally, we chose the conditions
of 0.8mM, 40g/L, 48h for the fermentation of E. coli ECN.

![af171a22dfe2162d17b224bb71f5419a](https://static.igem.wiki/teams/5740/engineering/25.webp)

Figure 25. Effects of different inducer concentrations, glucose concentrations, and time on levodopa

After we have explored the optimal inducer concentration, glucose concentration and time for L-dopa production in E. coli
BL21 (DE3), the next step is to transform plasmid 1 and the optimized plasmid 2 into E. coli ECN, because our ultimate goal
is to let probiotics produce L-dopa.

As shown in Figure 26, the transformation diagram on the left proves that plasmid 1 and plasmid 2 were successfully
transformed into ECN. In addition, in order to further verify that there is no problem with the transformed plasmid, we used
colony PCR for verification. The final amplified band length is 800bp, indicating that the transformation is successful. The
next step is to select the correct single clone, expand the culture, and then induce fermentation to produce levodopa.

![](https://static.igem.wiki/teams/5740/engineering/26.webp)

Figure 26. Plasmid 1 and plasmid 2 were transformed into E. coli ECN (left), and the electrophoresis results of colony PCR
identification were shown (right).

We then fermented the successfully transformed ECN using the optimal conditions obtained in the BL21(DE3) host strain. After
fermentation, we analyzed the fermentation samples using HPLC in triplicate. Figure 27 shows the peaks of the three
replicates, revealing the concentrations of 1.064 g/L, 1.065 g/L, and 1.069 g/L, respectively.

![](https://static.igem.wiki/teams/5740/engineering/27.webp)

Figure 27. Peak diagram of levodopa production after HPLC detection of ECN fermentation

',
},
]
},
{
"title": 'Learn2:',
"content": '

In this round of experiments, we first identified the optimal TyrA source and RBS sequence. Then, through fermentation
testing, we ultimately determined the optimal fermentation conditions in ECN: 40 g/L glucose, 0.8 mM IPTG, and a
fermentation time of 48 hours. The results revealed the following key insights:

First, the experimental data showed that increasing the fermentation time significantly increased the overall L-DOPA yield.
The yield under 48 hours was generally higher than that under 36 hours, confirming the importance of production time in the
system and suggesting that future optimization efforts will require attention to the kinetics of product
accumulation.Second, the coupling effect of parameters was significant. The optimal glucose concentration varied under
different IPTG levels. Under moderate IPTG conditions, moderate glucose concentrations yielded better results, while under
high IPTG conditions, high glucose concentrations were required to maintain high yields. This demonstrates that optimizing a
single variable is insufficient; there is an interaction between the inducer and the carbon source, and finding a balance is
more critical than simply increasing a single condition.Third, a distinct "yield valley" appeared in the experiment,
demonstrating the system\'s sensitivity to parameter changes. If conditions are slightly inappropriate, yield can drop
dramatically. This lesson teaches us that stability and reproducibility are crucial for subsequent process design, as
otherwise, fluctuations between batches are likely to occur.Fourth, by comparing the contour distributions at 36 and 48
hours, we also realized that plotting a response surface and identifying high-yield "islands" is more intuitive and
effective than simply comparing individual data points. This provides guidance for more intensive sampling within the local
optimal range and even for implementing response surface design experiments.

Finally, from a future perspective, the greatest value of our learning lies in: first, allowing us to further refine and
replicate the current optimal parameters, such as the 40 g/L and 0.8 mM combination; second, providing a foundation for
scale-up experiments and fed-batch strategies. After all, high yields under high sugar conditions are likely to be practical
in industrial settings.

',
"children": []
},
{
"title": 'References',
"content": '

1. Monzio Compagnoni G, Di Fonzo A, Corti S, et al. The role of mitochondria in neurodegenerative diseases: the lesson from
   Alzheimer’s disease and Parkinson’s disease[J]. Molecular neurobiology, 2020, 57(7): 2959-2980.
2. Xie A, Gao J, Xu L, et al. Shared mechanisms of neurodegeneration in Alzheimer’s disease and Parkinson’s disease[J].
   BioMed research international, 2014, 2014(1): 648740.
3. Chopade P, Chopade N, Zhao Z, et al. Alzheimer\'s and Parkinson\'s disease therapies in the clinic[J]. Bioengineering &
   Translational Medicine, 2023, 8(1): e10367.
4. Ramanan V K, Saykin A J. Pathways to neurodegeneration: mechanistic insights from GWAS in Alzheimer’s disease, Parkinson’s
   disease, and related disorders[J]. American journal of neurodegenerative disease, 2013, 2(3): 145.
5. Khan A U, Akram M, Daniyal M, et al. Awareness and current knowledge of Parkinson’s disease: a neurodegenerative
   disorder[J]. International Journal of Neuroscience, 2019, 129(1): 55-93.
6. Cui L, Hou N N, Wu H M, et al. Prevalence of Alzheimer\'s disease and Parkinson\'s disease in China: an updated systematical
   analysis[J]. Frontiers in aging neuroscience, 2020, 12: 603854.
7. Manoharan S, Guillemin G J, Abiramasundari R S, et al. The role of reactive oxygen species in the pathogenesis of
   Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease: a mini review[J]. Oxidative medicine and cellular
   longevity, 2016, 2016(1): 8590578.
8. Fernández-Ruiz J, Romero J, Ramos J A. Endocannabinoids and neurodegenerative disorders: Parkinson’s disease, Huntington’s
   chorea, Alzheimer’s disease, and others[J]. Handb Exp Pharmacol, 2015, 231: 233-259.

',
"children": []
},
] %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Engineering{% endblock %}
{% block page\_content %}
{% endblock %}

{% extends "layout.html" %}
{% set info\_data = [
{
"title": '1. Preparation of LB (Luria-Bertani) culture medium',
"content": '

Goal: Liquid and solid culture media are formulated and optimized to support robust microbial growth and provide
essential nutrients. These media are designed to facilitate key processes in controlled bioprocessing
applications, including cell activation, proliferation, and fermentation.

Materials:

1. 1) Conical flask
2. 2) Autoclave
3. 3) Tryptone
4. 4) Yeast Extract
5. 5) Sodium Chloride
6. 6) Double distilled H2O
7. 7) Agar

Procedures:

1. 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. 2) Add into 1L conical flask;
3. 3) Maintain at 121 ℃ for 20 minutes in an autoclave for sterilization.

',
},
{
"title": '2. First round of recombinant plasmid construction',
"content": '

pCDFDuet-tktA-aroF-aroE，pETDuet-TyrA(K12/SO/HI)-hpaB-hpaC

**Obtain target genes and Vector fragments by PCR**

Goal: To amplify target genes and Vectorfragments for Infusion recombination.

Materials:

1. 1) 2x PrimeStar Mix
2. 2) Gene template
3. 3) Double distilled water (ddH2O)
4. 4) Primer-F
5. 5) Primer-R
6. 6) PCR tube
7. 7) PCR thermal cycler

PrimeStar Mix includes buffer and dNTPs for PCR. The extention rate for PrimeStar Mix (2x)
is 1000bp/10 s.

Procedures:

1. 1) Add 2x PrimerStar Mix 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. 2) Insert PCR tubes into PCR thermal cycler. Pre-denature the fragments at 95˚C for 3 minutes;
3. 3) Perform 40 cycles of 30 seconds 95˚C denaturing, 30 seconds 55˚C annealing, and 2 minutes extension 72˚C
   extension, For the two empty vectors pCDFDuet and pETDuet, the extension time was set at 6 minutes due to their
   longer lengths.
4. 4) Extend the fragments for 1 minutes at 72˚C;
5. 5) If not use immediately, preserve the fragments at 4˚C;
6. 6) Recover PCR fragments by agarose gel electrophoresis.

**Agarose gel construction, deployment and recycle**

Goal: To prepare an agarose gel with uniform consistency and optimal physical properties suitable for
high-resolution electrophoretic separation of nucleic acids or proteins.

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. 1) Dissolve 1.5g Agarose in 200mL Conical flask;
2. 2) Add 100mL TAE buffer;
3. 3) Heat the material with microwave;
4. 4) Melt the mixture at 30 sec interval;
5. 5) See whether bubbles are forming, if did succesfully mixed;
6. 6) Add 10 μl nucleic acid gel stain (10000x);
7. 7) Let it cool for 5~10 minutes;
8. 8) Pour the agarose mixture into the casting tray;
9. 9) Place the appropriate well comb to create the wells;
10. 10) Wait to solidify ~30 minutes;
11. 11) Remove the comb and place the gel in the gel box.

**Agarose gel electrophoresis**

Goal: To confirm the sizes of DNA fragments following electrophoresis, enabling precise excision and purification
of target bands for subsequent molecular procedures.

Procedures:

1. 1) Extract 50µl of every sample; add 5µl of loading buffer (10x) to each sample;
2. 2) Place the gel in the horizontal electrophoresis system;
3. 3) Extract 50µl of the mixed solution containing the sample, mix them till their colors are almost uniform;
4. 4) Add the marker to the first well and load 50µl of each sample into the wells;
5. 5) The procedure runs at 125V for 25min;
6. 6) Take out the gel from the device and proceed to following steps.

**DNA gel extraction**

Goal: The target DNA fragments are isolated and purified from the agarose gel matrix by electrophoretic
separation.

Materials:

1. 1) Agarose gel with target genes fragments and linearized pCDFDuet and pETDuet backbone;
2. 2) 1.5 mL Eppendorf (EP) tube
3. 3) Agarose Gel Electrophoresis Recovery Kit

Procedures:

1. 1) Cut the slice of gel containing target genes fragments and linearized pCDFDuet and pETDuet backbone, cutting off
   as much unneeded gel as possible, and place it in an 1.5 mL EP tube;
2. 2) Add 500 microliters of buffer B2 and put the tube into a 50°C hot water bath until gel has completely melted;
3. 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. 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. 5) Repeat steps 4;
6. 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. 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. 8) Cap the lid and let the tube sit for 1 minute. Perform centrifugation at 12000 rpm for 1 minute;
9. 9) Discard the purification column. Store DNA fragments in the EP tube at 4°C.

**Homologous recombination of** **linearized pCDFDuet and pETDuet backbone with target gene fragments**

Goal: Perform homologous recombination between the linearized pCDFDuet and pETDuetbackbone and
target gene fragments to generate the complete plasmid.

Materials:

1. 1) CloneExpress Mix (2x)
2. 2)PCR products of tktA, aroF, aroE, tyrA，hpaB，hpaC
3. 3)Linearized pCDFDuet and pETDuet backbone

Procedure:

1. 1) Measure the concentration of the gel-recovered fragments;
2. 2) Prepare the system for the construction of pCDFDuet-tktA-aroF-aroE in PCR tubes: 5μl CloneExpress Mix, 2.5μl
   Linearized pCDFDuet backbone, and 2.5μl tktA or aroF or aroE fragments;
3. 3) Prepare the system for the construction of pETDuet-TyrA-hpaB-hpaC in PCR tubes: 5μl CloneExpress Mix, 2.5μl
   Linearized pETDuet backbone, and 2.5μl TyrA or hpaB or hpaC fragments;
4. 4) Insert PCR tubes into PCR thermal cycler. Ligase the fragments at 50˚C for 30 minutes;

**Transformation**

Materials:

1. 1) Ligated recombination plasmid
2. 2) Liquid LB solution without antibiotics
3. 3) LB Agar plates with Ampicillin and spectinomycin antibiotics

Procedures:

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

**Colony PCR verification**

Goal: Following successful transformation and growth of the recombinant *E. coli* BL21 strain, colony PCR
was employed to screen for individual colonies containing the correctly constructed plasmid.

Materials:

1. 1) Taq Master Mix (2x)
2. 2) Double distilled water (ddH2O)
3. 3) Culture plates post-transformation

Procedures:

1. 1) Add 10μl Taq Master Mix, 1μl primer-F, 1μl primer-R, and 8μl ddH2O into PCR tubes;
2. 2) Pick a single colony with a 10μl sterile pipette tip and perform aspiration and dispensing in PCR tubes;
3. 3) Insert mixture into a PCR thermal cycler, design the PCR program according to the manufacturer\'s instructions.
4. 4) Perform agarose gel electrophoresis, and the presence of the correct bands indicates the successful construction
   of recombination plasmids.

',
},
{
"title": '3. Functional testing',
"content": '',
"children": [
{
"title": '3.1 Target protein expression test',
"content": '

**Ⅰ. Amplification culture for correct colony**

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

Materials:

1. 1) Liquid LB solution
2. 2) pCDFDuet-tktA-aroF-aroE，pETDuet-TyrA(K12/SO/HI)-hpaB-hpaC bacterial fluid
3. 3) Ampicillin and spectinomycin antibiotics
4. 4) Inducer: IPTG
5. 5) Iso-thermic shaker

Procedures:

1. 1) Add 1000μl of pCDFDuet-tktA-aroF-aroE，pETDuet-TyrA(K12/SO/HI)-hpaB-hpaC bacterial fluid and 100μl of Ampicillin
   and spectinomycin antibiotics into 100mL of liquid LB solutions, respectively;
2. 2) Culture bacterial liquid in iso-thermic shaker for 3 hours until absorbance OD600nm to 0.4-0.6;
3. 3) Add IPTG into bacterial liquid with 1mM final concentration;
4. 4) Sample for each bacterial liquid cultured at 37°C for 6h, respectively.

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

Goal: To verify the expression of desired proteins.

Materials for protein crude extraction:

1. 1) 500μl bacterial liquid for different induced conditions
2. 2) Centrifuge
3. 3) Double distilled water (ddH2O)
4. 4) 6x Protein loading buffer

Procedures for protein crude extraction:

1. 1) Centrifuge samples at 12000 rpm for 1 minute;
2. 2) Discard supernatant and resuspend precipitation with 50μl 1x Protein loading buffer diluted by ddH2O;
3. 3) Boiled samples at 95°C for 15 minutes in PCR thermal cycler.

Materials for SDS-PAGE:

1. 1) Sangon 12.5% SDS-PAGE Color Preparation kit
2. 2) Electrophoresis buffer (Tris-Glycine)
3. 3) Protein ladder
4. 4) 6x Protein loading buffer
5. 5) Vertical electrophoresis system
6. 6) Coomassie blue staining solution

Procedures for SDS-PAGE：

1. 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. 2) Slowly pipetting mixture into casting stand and frame to avoid bubbles;
3. 3) Add 1 mL ddH2O at the top of separating gel;
4. 4) Wait the separating gel to solidify for 8 minutes;
5. 5) Discard the water that was added previously;
6. 6) Add and mix 825μL 2x stacking gel solution, 825μL 2X stacking gel buffer, and 11μL catalyst in centrifugation
   tubes for stacking gel;
7. 7) Add stacking gel mixture until the cast is completely filled, then slowly insert comb without producing any air
   bubbles;
8. 8) Wait the stacking gel to solidify for 12 minutes, then carefully remove the comb, and wash the wells with
   electrophoresis buffer;
9. 9) Add 10μL protein ladder into the first well, then load samples in each successive well;
10. 10) Transfer gel into vertical electrophoresis system;
11. 11) Run at 120V for 90 minutes;
12. 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.

'
},
{
"title": '3.2 Detection of L-dopa production using an ELISA test kit',
"content": '

Goal: The kit uses a double-antibody one-step sandwich enzyme-linked immunosorbent assay (ELSA). To a coating
medium pre-coated with a levodopa antibody, the standard, then the HBP-labeled detection antibody are added
sequentially. After incubation and washing, the sample is assayed using the substrate TMB. TMB is converted to
blue under the catalysis of a peroxidase and to yellow under the action of acid. The intensity of the color is
positively correlated with the levodopa content of the sample. The absorbance (OD) is measured at 450 nm using a
microplate reader to calculate the levodopa concentration in the sample.

Materials：

1. 1) Microwell ELISA plate
2. 2) Standards
3. 3) Sample diluent
4. 4) Detection antibody-HRP
5. 5) 20x wash buffer
6. 6) Substrate A
7. 7) Substrate B
8. 8) Stop solution
9. 9) Seal film
10. 10) Instructions
11. 11) Ziplock bag

Procedures:

Note: The concentrations of the standards (S0-S5) are: 0, 10, 20, 40, 80, and 160 pg/mL. Reagent Preparation: 20x
Wash Buffer Dilution: Dilute distilled water 1:20, i.e., 1 part 20x Wash Buffer with 19 parts distilled water.

Plate Washing Method：

1. Manual Plate Washing: Shake off all liquid from the wells, fill each well with wash buffer, let stand for 1
minute, shake off all liquid from the wells, and tap onto absorbent paper. Wash the plate five times.

2. Automatic Plate Washer: Add 350μL of wash buffer to each well, soak for 1 minute, and wash the plate five
times.

Specific Steps：

1. Remove the desired plate strips from the aluminum foil bag after equilibration at room temperature for 20
   minutes. Seal the remaining strips in a ziplock bag and return them to 4°C.
2. Set up standard and sample wells. Add 50 μL of the standard of different concentrations to each standard well.
3. Add 10 μL of the sample to be tested to the sample well, followed by 40 μL of the sample diluent. Leave blank
   wells empty.

4. Add 100 μL of horseradish peroxidase (HRP)-labeled detection antibody to each well, including the standard and
sample wells, except for the blank well. Seal the wells with sealing film and incubate at 37°C in a waterbath or
incubator for 60 minutes.

5. Discard the liquid, pat dry on absorbent paper, fill each well with wash solution, let stand for 1 minute,
discard the wash solution, pat dry on absorbent paper, and repeat this process five times (a plate washer can also
be used).

6. Add 50μL each of substrates A and B to each well and incubate at 37°C in the dark for 15 minutes.

7. Add 50μL of stop solution to each well. Within 15 minutes, measure the OD value of each well at a wavelength of
450 nm.

8. Draw a standard curve: In Excel spreadsheet, plot the standard concentration on the horizontal axis and the
corresponding OD value on the vertical axis. Calculate the concentration of each sample using the curve equation.

'
}
]
},
{
"title": '4. Second round of recombinant plasmid construction',
"content": '

Based on the first round of plasmid construction, two new recombinant plasmids were constructed, namely
pETDuet-TyrA(K12)-RRS2-hpaB-hpaC and pETDuet-TyrA(K12)-RRS3-hpaB-hpaC，The only difference between these two
plasmids and the first round of construction is the sequence of RBS, So the materials and steps are the same as
step 2.

',
},
{
"title": '5. Second round of fermentation testing',
"children": [
{
"title": "5.1 Detection of L-dopa production using an ELISA test kit. (Temperature, time, inducer concentration, glucose
concentration）",
"content": '

This step is consistent with the content of Section 3.2

',
},
{
"title": '5.2 HPLC determination of levodopa content in ECN',
"content": '

Goal: Quantitatively detect levodopa (L-DOPA) in samples, generate a linear calibration curve, and determine the
concentration of L-DOPA in the sample. This method utilizes a reversed-phase C18 column using HPLC-UV (or optional
electrochemical detection (ECD)). The steps include mobile phase preparation, standard preparation, sample
pretreatment, sample loading and testing, data processing, and post-column maintenance.

Materials:

1. HPLC system (with autosampler, UV detector; optional: electrochemical detector (ECD))

2. Reversed-phase C18 column (4.6 × 150 mm, 5 µm)

3. Mobile phase A: 50 mM phosphate buffer (NaH2PO4/Na2HPO4), pH 2.5 (adjusted with phosphoric acid), with 0.1%
antioxidant (optional)

4. Mobile phase B: Acetonitrile (HPLC grade) or methanol (HPLC grade)

5. L-DOPA standard (accurately weighed)

6. Internal standard (optional, a compound with known concentration that does not overlap with the sample)

7. Antioxidant: Ascorbic acid (analytical grade) to prevent oxidation of catechols

8. Acidifier: 1.0 M HCl or 0.1 M HCl (for sample stabilization)

9. Organic solvent: Methanol (HPLC grade) for protein precipitation; acetonitrile for mobile phase and rinsing

10. 0.22 µm syringe filter (polytetrafluoroethylene/polyether or PTFE/nylon) and syringe

11. Autosampler vials (amber, light-proof) and silicone caps

12. Micropipette and pipette tips, sterile centrifuge tubes (1.5 mL/2 mL), centrifuge, vortex mixer, ultrasonic
cleaner (optional)

13. Spectrometer (for preparing standards), ice bath and cold chain equipment (for keeping samples cold)

14. Waste container and chemical waste labeling

Procedures:

1. Mobile Phase Preparation and Handling (Pre-processing)

a. Prepare 50 mM phosphate buffer: Prepare according to the recipe and dissolve in sufficient deionized water.
Adjust the pH to 2.5 (adjust slightly with phosphoric acid or dilute HCl).

b. Add 0.1% (w/v) ascorbic acid or 0.1 mM EDTA to inhibit L-DOPA oxidation (recommended for long-term sample
storage). c. Mix the buffer and acetonitrile in the desired ratio (recommended starting ratio: A:B = 95:5,
isocratic). If using a gradient, design a time-ratio schedule. d. Filter the mobile phase through a 0.22 µm filter
and degas by sonication or use an in-line degasser. e. Place the mobile phase in a labeled bottle and place it in
the HPLC feed. 2. HPLC Conditions (recommended starting conditions, which can be optimized based on column and
detector) a. Column: C18 4.6 × 150 mm, 5 µm; Column temperature: 30°C (can be adjusted to 25–35°C to stabilize
retention time). b. Flow rate: 1.0 mL/min (can be adjusted to 0.6–1.2 mL/min for smaller column diameters or
detector requirements). c. Injection volume: 10 µL (can be increased to 20 µL for low-concentration samples). d.
Detection: UV at 280 nm (L-DOPA absorbs at ~280 nm); if using an ECD, set the operating potential to an
appropriate level (typically +0.6–+0.8 V) and use conditions compatible with the deionized mobile phase.

e. Run time: 10–15 min (depending on the retention time setting, to ensure complete peak elution); 10 min is
generally sufficient under isocratic conditions; longer may be used if interferences occur.

3. Standard Preparation and Calibration Curve

a. Accurately weigh L-DOPA (e.g., 10.0 mg) and dissolve it in ice-cold 0.1 M HCl + 0.1% ascorbic acid solution to
10.00 mL to prepare a 1000 µg/mL (or 1 mg/mL) stock solution. Protect from light and work on ice.

b. Prepare a series of working standards (e.g., 0.5, 1, 2, 5, 10, 20, 50 µg/mL) from the stock solution using
mobile phase or 0.1 M HCl + ascorbic acid. Prepare QC samples (low, medium, and high concentrations, e.g., 1, 10,
and 40 µg/mL, respectively). If using an internal standard, add a constant amount of internal standard to all
standards.

c. Use the standard solution as soon as possible. For long-term storage, refrigerate and protect from light
(recommended for no more than 24–48 hours).

4. Sample Pretreatment (for fermentation supernatant/culture medium)

a. Centrifugation: Take a sample (e.g., 1 mL) of fermentation/culture broth, cool it (4°C), and centrifuge it at
10,000 × g for 10 minutes. Collect the supernatant.

b. Acidification Stabilization: Add 1–10 µL of 1.0 M HCl (or an amount to bring the pH to ~2–3) to the
supernatant. Add ascorbic acid to a final concentration of 0.1% (w/v) to prevent L-DOPA oxidation. Mix gently.

c. Protein precipitation: Add 3 volumes of cold methanol equal to the sample volume (e.g., 300 µL of cold methanol
for 100 µL sample), vortex mix for 30 seconds, place on ice for 10 minutes, and centrifuge at 10,000 × g for 10
minutes.

d. Collect the supernatant. If the concentration is low, perform solid-phase extraction (SPE) or concentrate with
nitrogen purge before reconstitution with mobile phase. If using directly on the instrument, filter the
supernatant through a 0.22 µm syringe filter into an autosampler vial (amber vials are recommended to prevent
photodegradation). If using an internal standard, add a known amount of internal standard before protein
precipitation.

e. For tissue or cell samples: Homogenize with ice-cold 0.1 M HCl + 0.1% ascorbic acid, centrifuge to remove
cellular debris, and then perform protein precipitation and filtration as above. 5. System Suitability and Sample
Loading Order

a. Equilibrate the column in the target mobile phase for 20–30 minutes (or at least 10 column volumes) before
loading.

b. Recommended injection sequence: Blank → Solvent Blank → Lowest Standard → Stepping Standard → Intermediate QC →
Sample (Batch) → High QC → Duplicate Samples → Final Blank; insert a standard every 10 samples to monitor drift.

c. System Suitability Assessment (Example Criteria): 5 replicate injections of the standard with a relative
standard deviation (RSD) of < 2% (or < 5%) area, a retention time RSD of < 0.5%, a number of theoretical
plates > 2000, and a peak tailing factor < 2. If these values ​​are not met, adjust the mobile phase or
column temperature and re-equilibrate.

6. Data Processing and Quantitation Methods

a. Peak Integration: Manually check the automated integration to ensure a reasonable baseline and no false peaks
are misidentified; record peak heights/areas and retention times. b. Calibration curve construction: Plot peak
area (or peak area ratio: target/internal standard) against standard concentration. Use linear regression
(weighting 1/x or 1/x^2 is recommended to minimize low-concentration bias). Calculate the slope, intercept, and
R^2 (target R^2 ≥ 0.995).

c. Sample concentration calculation: Back-calculate the injected sample concentration from the regression equation
and multiply by the dilution factor and volume conversion to obtain the actual concentration in the original
sample (µg/mL or mg/L).

d. LOD/LOQ assessment (optional): Use the signal-to-noise ratio (S/N = 3 for LOD, S/N = 10 for LOQ) or estimate
the standard error of the calibration curve.

7. Quality Control and Reproducibility

a. Each batch of samples should include at least one set of low, mid, and high quality control (QCs). If a QC
exceeds the acceptable range (e.g., ±15%), the batch should be retested or reprocessed.

b. It is recommended that samples be run in duplicate and analyzed, with intra- and inter-batch variation
documented.

c. Be sure to record sample collection and pretreatment times and temperatures, as catechol compounds are
extremely sensitive to the environment (light, oxygen, and metals).

8. Post-column Care and Storage

a. After completing the day\'s testing, flush the column with a mobile phase containing 50% acetonitrile (or
methanol) for 10–15 minutes to remove adhering matter, then flush with the original mobile phase for 5–10 minutes
to restore the column to storage conditions.

b. If the column will not be used for an extended period, store it in a buffer containing 20% ​​organic phase as
recommended by the column manufacturer (avoid long-term storage in pure water) and store at 4°C in the dark.

c. Regularly check column pressure and background noise (based on frequency of use), record column mileage, and
replace as needed.

9. Precautions (Quick Tips)

a. L-DOPA is susceptible to oxidation and polymerization: Maintain the column in the dark, cool, and quickly
throughout the entire process, and add ascorbic acid for stabilization.

b. Prepare samples and standards using the same solvent system or acidification conditions to minimize matrix
effects. c. Filtration and centrifugation must be thorough; microparticles can clog the injection needle or cause
baseline noise.

d. If background interference is severe, consider switching to an electrochemical detector (ECD), adjusting the
mobile phase pH, or adding an organic phase to improve separation.

e. All solvents and reagents must be HPLC or analytical grade. Wastewater should be collected separately and
disposed of according to laboratory standards.

'
}
],
},
] %}
{% block title %}Experiments{% endblock %}
{% block lead %}Experiments{% endblock %}
{% block page\_content %}
{% endblock %}

{% extends "layout.html" %}
{% set info\_data = [
{
"title": 'Overview',
"content": '

Parkinson\'s disease is mainly caused by the gradual loss of dopamine neurons in the brain that control movement,
resulting in a decrease in dopamine levels, which affects movement coordination and control[1]. In addition to
motor symptoms, Parkinson\'s disease may also cause non-motor symptoms such as sleep disorders, depression, and
cognitive dysfunction[2]. The treatment of Parkinson\'s disease mainly adopts surgical treatment and drug
treatment. Among them, the use of compound levodopa is the most basic and effective drug[3]. However, after taking
levodopa, the most common adverse reactions are gastrointestinal symptoms such as nausea, vomiting, anorexia and
constipation.

As the research progresses, the use of prebiotics and probiotics in Parkinson\'s disease may provide new ideas for
changing various non-motor symptoms[4]. A randomized controlled trial by an Italian research team demonstrated
that consuming fermented milk containing probiotic strains and prebiotic fiber significantly increased the
frequency of complete bowel movements in Parkinson\'s disease patients. Iranian researchers divided 60 Parkinson\'s
disease patients into two groups, administering either probiotics (containing strains of Lactobacillus and
Bifidobacterium) or a placebo. They found that after 12 weeks, probiotic intervention improved Parkinson\'s disease
symptoms[5]. Although the specific mechanism of action remains unclear, researchers believe that probiotics may
benefit Parkinson\'s disease patients through multiple pathways, including strengthening intestinal epithelial
integrity, preventing barrier breakdown, stimulating a healthy mucosal immune system, and inhibiting the growth of
pathogenic bacteria[6]. This project, by overexpressing an optimized levodopa production pathway in probiotics and
combining their inherent probiotic properties, will contribute to the development of new therapies targeting
host-gut microbial drug metabolism and other novel health interventions.

',
},
{
"title": 'Results of our work',
"content": '

|  |
| --- |
| **Outline**  **1.First round of recombinant plasmid construction**  1.1 pCDFDuet-tktA-aroF-aroE  1.2 pETDuet-TyrA(K12)-hpaB-hpaC  1.3 pETDuet-TyrA(SO)-hpaB-hpaC  1.4 pETDuet-TyrA(HI)-hpaB-hpaC  **2.Functional testing**  2.1 Target protein expression test  2.2 Detection of L-dopa production using an ELISA test kit  **3. Second round of recombinant plasmid construction**  3.1 pETDuet-TyrA(K12)-RRS2-hpaB-hpaC  3.2 pETDuet-TyrA(K12)-RRS3-hpaB-hpaC  **4. Second round of fermentation testing**  4.1 Detection of L-dopa production using an ELISA test kit. (Temperature, time, inducer concentration, glucose concentration）  4.2 HPLC determination of levodopa content in ECN |

',
},
{
"title": '1. The construction of plasmid',
"content": '',
"children": [
{
"title": '1.1 Construction for pCDFDuet-tktA-aroF-aroE',
"content": '

We used homologous recombination to construct the pCDFDuet-tktA-aroF-aroE plasmid. First, using PCR technology, we
designed corresponding primers and amplified the target fragments of tktA, aroF, and aroE using Escherichia coli
K12 as a template. We also amplified the pCDFDuet plasmid backbone using a laboratory-preserved plasmid. As we can
see from Figure 1A, the agarose gel electrophoresis results after amplification showed that the amplified lengths
of the backbone and target fragments were all in line with the expected sizes. The pCDFDuet plasmid backbone is
3781 bp in length, tktA is 1995 bp in length, aroF is 1071 bp in length, and aroE is 819 bp in length.

After obtaining the empty vector and fragments of corresponding lengths, we recovered the agarose gel to obtain
the purified vector and fragments, and obtained the concentration of these gene fragments through the nanodrop
instrument. After obtaining the corresponding concentration, we used homologous recombination to allow the vector
and fragments to be recombined together. Then we transformed the recombinant product into the E. coli cloning
strain TOP10. As shown in Figure 1B, after the transformation and overnight growth, we saw that there were single
colonies with successful transformation. However, whether the transformation was successful or not, we need to
perform corresponding verification.

First, by colony PCR, we randomly selected 4 independent single colonies of the same size on the plate and used
colony PCR primers for identification. The length was designed to be 2102bp. In PCR After amplification and
identification by agarose gel electrophoresis, we found that the size of the amplified fragments met our
expectations. Therefore, these single clones were considered to have successfully transformed with the recombinant
plasmid.

Finally, we need to expand and culture the corresponding correct single clones, extract the plasmids, and send
them to a biological company for sequencing. The purpose of this step is to ensure the position and order of our
vector and connector, and whether there are any base deletions or mutations. Figure 1D shows that our construction
results are correct. At this point, our first recombinant plasmid was successfully constructed.

![](https://static.igem.wiki/teams/5740/result/1.webp)

Figure 1. A. Electrophoresis results of plasmid backbone and target fragment, B. Recombinant plasmid transformed
into cloning strain TOP10, C. Electrophoresis results after single clone colony PCR, D. Plasmid sequencing results

'
},
{
"title": '1.2 pETDuet-TyrA(K12)-hpaB-hpaC',
"content": '

We used E. coli K12 as a template to amplify other target fragments. First, we designed corresponding primers and
then used cultured E. coli K12 as a template to amplify the target genes TyrA, hpaB, and hpaC. The plasmid
backbone was also obtained by inverse PCR. After the amplification reaction, the amplified products were
identified by agarose gel electrophoresis. The results showed that the plasmid backbone and the three target genes
were successfully amplified. The backbone length was 5420 bp, the length of TyrA was 1122 bp, the length of hpaB
was 1563 bp, and the length of hpaC was 513 bp, as can be seen in Figure 2.

![](https://static.igem.wiki/teams/5740/result/2.webp)

Figure 2. Electrophoresis results of pETDuet and TyrA(K12), hpaB and hpaC

After obtaining the gene fragment of the correct size, we need to recover the purified vector and fragment by gel
recovery. Then, we use homologous recombination to construct the complete recombinant plasmid. Next, we need to
transform it into a cloning strain and, after overnight growth, select the grown single clones for colony PCR
identification. As shown in Figure 3, we selected six single clones and amplified them using the designed primers.
The results showed that the amplified length was 1500bp, which is consistent with the expected length, indicating
that the transformation process was successful.

![](https://static.igem.wiki/teams/5740/result/3.webp)

Figure 3. Results of monoclonal plates and colony PCR after transformation

Finally, we inoculated the successful transformants into seed solution, cultured them, extracted the plasmids, and
sent them to a biotechnology company for sequencing. The results are shown in Figure 4. The sequencing results
show that the constructed recombinant plasmids have no base mutations or deletions.

![](https://static.igem.wiki/teams/5740/result/4.webp)

Figure 4. Plasmid sequencing results

',
},
{
"title": '1.3 pETDuet-TyrA(SO)-hpaB-hpaC',
"content": '

Next, we constructed the pETDuet-TyrA(SO)-hpaB-hpaC plasmid. Compared with pETDuet-TyrA(K12)-hpaB-hpaC, the
difference between the two plasmids is the source of TyrA. Therefore, we used homologous recombination to replace
TyrA based on pETDuet-TyrA(K12)-hpaB-hpaC. We designed two pairs of primers, one to amplify the TyrA gene from the
new source, and the other pair of primers to amplify the rest of the pETDuet-TyrA(K12)-hpaB-hpaC except TyrA(K12).
After amplification, agarose gel electrophoresis was performed for identification, as shown in Figure 5A. As shown
in Figure 5B , the plasmid backbone and TyrA(SO) amplification results after electrophoresis were consistent with
the expected lengths: the plasmid backbone was 7535 bp long, and TyrA(SO) was 1152 bp long. The two fragments were
then recovered and purified from the gel. After homologous recombination, the recombinant products were
transformed into E. coli TOP10. As shown in Figure 5B , single clones emerged after overnight growth. To verify
the correctness of the transformed single clones, we performed colony PCR identification. Twelve single clones
were selected for identification. In Figure 5C , the colony PCR result showed a size of 2100 bp, demonstrating
that the transformed single clones all contained the correct plasmid. The correct single clones were then
inoculated into seed solution, and the plasmids were extracted and sent to a biotechnology company for sequencing.
Sequencing results confirmed that the constructed plasmid was successful.

![](https://static.igem.wiki/teams/5740/result/5.webp)

Figure 5 A. Electrophoresis results of plasmid backbone and target gene fragments, B. Plate of recombinant plasmid
transformed into E. coli, C. Electrophoresis results of monoclonal colony after PCR, D. Sequencing results of
recombinant plasmid

',
},
{
"title": '1.4 pETDuet-TyrA(HI)-hpaB-hpaC',
"content": '

When constructing pETDuet-TyrA(HI)-hpaB-hpaC, we used a similar approach to pETDuet-TyrA(SO)-hpaB-hpaC. We first
designed primers to amplify the TyrA(HI) target gene, and another pair of primers to amplify the backbone. After
amplification, we verified the success of the amplification by agarose gel electrophoresis. As shown in Figure 6A,
the amplified vector and target gene lengths were consistent with the expected values, with the backbone being
7535 bp and the target gene being 1146 bp. Following amplification, the two were ligated by homologous
recombination and transformed into the corresponding E. coli cloning strain. After overnight growth, as shown in
Figure 6B, 12 single colonies were selected for colony PCR analysis to confirm the success of the transformants.

![](https://static.igem.wiki/teams/5740/result/6.webp)

Figure 6 A. Plasmid backbone and target gene electrophoresis results. B.plate image of the transformed single
colonies.

After selecting the corresponding single clone for colony PCR, we performed electrophoresis analysis on the
amplified product. Figure 7A shows that the colony PCR result met the expected length of 2000 bp. Furthermore, we
selected a single clone that successfully obtained colony PCR and inoculated it into seed solution. The plasmid
was then extracted and sent to a biotechnology company for sequencing. The results are shown in Figure 7B. The
sequencing results indicate that the recombinant plasmid construction was successful, with no base mutations or
deletions.

![](https://static.igem.wiki/teams/5740/result/7.webp)

Figure 7 A. Plasmid backbone and target gene electrophoresis results. B.plate image of the transformed single
colonies.

',
},
]
},
{
"title": '2. Functional testing',
"content": '',
"children": [
{
"title": '2.1 Target protein expression test',
"content": '

After constructing Plasmid 1 and the three TyrA plasmids 2, we need to transform two plasmids into a new E. coli
expression strain. Because the production of L-DOPA requires the simultaneous function of all six genes, we first
transform two plasmids into E. coli BL21 (DE3), as shown in Figure 8. After overnight growth, we see that the
double-transformed host bacteria have corresponding monoclonal colonies growing. We will then perform colony PCR
to verify whether the double plasmid transformation is successful.

![](https://static.igem.wiki/teams/5740/result/8.webp)

Figure 8. The corresponding single colony plate image after plasmid 1 and plasmid 2 were transformed into BL21
(DE3) together

After the double plasmid transformation is completed, we need to perform colony PCR identification. Since there
are two plasmids, two pairs of primers need to be designed to identify each plasmid separately. Therefore, colony
PCR is performed on each single clone of strain1-3. The corresponding lengths are: plasmid 1 of strain1 is 1000bp,
plasmid 2 is 800bp, plasmid 1 of strain2 is 800bp, plasmid 2 is 1000bp, and plasmid 1 of strain3 is 1000bp,
plasmid 2 is 1000bp. After the amplification is completed, we perform agarose gel electrophoresis. The results are
shown in Figure 9. The lengths of all colony PCRs are in line with the expected size. Therefore, the double
plasmid transformation is considered successful.

![](https://static.igem.wiki/teams/5740/result/9.webp)

Figure 9. Electrophoresis of colony PCR results of three strains after double plasmid transformation

Before conducting the first round of fermentation tests, we needed to perform protein expression tests on the two
plasmids to initially verify the normal expression of our six genes in E. coli. Therefore, we transformed BL21
(DE3) host cells with plasmid 1 (pCDFDuet-tktA-aroF-aroE) and plasmid 2 (pETDuet-TyrA(K12)-hpaB-hpaC) for protein
expression. This round of protein expression testing employed rapid induction, using a temperature of 37°C, a
rotational speed of 220 rpm, an inducer concentration of 1 mM IPTG, and a duration of 6 hours.

After induction, we collected the protein and divided it into the supernatant and precipitate of crude protein for
SDS-PAGE identification to observe whether the expression of the target gene was successful. As shown in Figure
10, wells 1-4 are the supernatant of plasmid 1, 5 is the precipitate of plasmid 1, wells 6-8 are the supernatant
of plasmid 2, and 9-10 are the precipitate of plasmid 2. Plasmid 2 contains inclusion bodies, and the proteins
corresponding to the target genes are all successfully expressed. Although the target protein corresponding to
plasmid 2 has inclusion bodies during the collection process, this may be related to the operational steps in the
protein collection process. In addition, since we are strengthening the L-dopa biosynthesis pathway in the cell,
we confirm that the expression has achieved our goal.

![](https://static.igem.wiki/teams/5740/result/10.webp)

Figure 10. Target protein expression test using plasmids 1 (pCDFDuet-tktA-aroF-aroE) and 2
(pETDuet-TyrA(K12)-hpaB-hpaC). Wells 1-4 contain the supernatant from plasmid 1, and well 5 contains the
precipitate from plasmid 1. Wells 6-8 contain the supernatant from plasmid 2, and wells 9-10 contain the
precipitate from plasmid 2.

',
},
{
"title": '2.2 Detection of L-dopa production using an ELISA test kit',
"content": '

After confirming that our target proteins are expressed normally, we need to perform fermentation tests on the
previously constructed strains carrying TyrA from three sources in order to obtain the optimal TyrA. The
fermentation method is consistent with the protein expression conditions, and the fermentation time is 48 hours.
Three replicates are set for each strain fermentation, as shown in Figure 11A. After the fermentation is
completed, we use the L-DOPA Elisa detection kit for measurement. The sample preparation, reaction process, and
reaction termination are shown in Figures 11BCD. Finally, we use a microplate reader to measure the absorbance
value at 450nm Figures 11E.

![](https://static.igem.wiki/teams/5740/result/11.webp)

Figure 11. A. Fermentation test of strain 123 (three replicates), B. Sample preparation, C. Reaction, D. End of
reaction, E. Absorbance test

We first developed a standard curve, as shown in Figure 12A. We then calculated the L-DOPA content in the sample
based on the standard curve and absorbance values. It can be seen that strain 1 had the highest L-DOPA production,
as shown in Figure 12B. That is, TyrA derived from K12 showed better enzyme activity in the modified strain. For
this modified strain, genes derived from similar species have better adaptability, so this gene was selected for
subsequent RBS screening.

![](https://static.igem.wiki/teams/5740/result/12.webp)

Figure 12. A. Standard curve of the ELISA test kit; B. L-DOPA production by three strains, where Strain 1 contains
TyrA from K12, Strain 2 contains TyrA from SO, and Strain 3 contains TyrA from HI.

',
},
]
},
{
"title": '3. Second round of recombinant plasmid construction',
"content": '',
"children": [
{
"title": '3.1 pETDuet-TyrA(K12)-RRS2-hpaB-hpaC',
"content": '',
},
{
"title": '3.2 pETDuet-TyrA(K12)-RRS3-hpaB-hpaC',
"content": '

After confirming that TyrA from K12 was the most effective, we needed to further optimize the RBS sequence of this
fragment. Since this gene is a key enzyme in L-dopa synthesis, we only replaced the RBS at the front end of this
fragment, from the default RBS1: AAGGAG to RBS2: ATTAAAGAGGAGAA and RBS3: TCACACAGG, as shown in Figure 13.

![](https://static.igem.wiki/teams/5740/result/13.webp)

Figure 13. Schematic diagram for replacing the RBS

To replace the RBS at the beginning of the TyrA gene in pETDuet-TyrA(K12)-hpaB-hpaC, we used inverse PCR. We
designed the two RBS sequences to be replaced into two pairs of primers and then amplified them using the original
pETDuet-TyrA(K12)-hpaB-hpaC plasmid as a template. Compared to the original plasmid, only the RBS sequence changed
in the amplified plasmid. Since the length of the newly amplified plasmid was almost the same as the original, we
did not perform agarose gel electrophoresis. Instead, we transformed the plasmid after amplification and extracted
it for sequencing. The sequencing results in Figure 14 demonstrate that the new RBS sequence has been successfully
replaced. The sequence on the left shows the replacement of RBS2, while the sequence on the right shows the
replacement of RBS3.

![](https://static.igem.wiki/teams/5740/result/14.webp)

Figure 14. Sequencing results after replacing the RBS sequence

After the RBS sequence replacement was completed, fermentation testing was repeated to determine which RBS
sequence performed best. Three strains were inoculated into LBG (20 g/L glucose) medium at an initial OD of 0.05.
After fermentation, product testing was performed to identify the optimal RBS sequence for the TyrA gene and
improve expression of the key enzyme.

The fermentation testing steps were similar to those described above. Three replicates of different RBS strains
were tested, as shown in Figure 15A. The assays were performed using a kit, with the reaction progressing and
reaction completion times (Figure 15BC). Finally, the fermentation yields were calculated using a standard curve.

![](https://static.igem.wiki/teams/5740/result/15.webp)

Figure 15. A Fermentation test of different RBS strains (three replicates), BC is divided into reaction progress
and reaction completion

By substituting the absorbance value into the standard curve for calculation, we can obtain the amount of L-dopa
produced by the strain under different RBS sequences. According to the bar graph in Figure 16, the three different
RBS sequences do have a certain effect on gene expression. Among the RBS sequences we screened, the optimal
expression level increased the strain\'s yield by 16.4%, reaching 500.3 mg/L.

![](https://static.igem.wiki/teams/5740/result/16.webp)

Figure 16. The yield of levodopa corresponding to different RBS sequences

',
},
]
},
{
"title": '4. Second round of fermentation testing',
"content": '',
"children": [
{
"title": '4.1 Detection of L-dopa production using an ELISA test kit. (Temperature, time, inducer concentration, glucose
concentration）',
"content": '

At this point, we have confirmed the optimal TyrA source (K12) and RBS sequence TCACACAGG. We then initially
tested the effect of different glucose concentrations on L-DOPA production by fermentation in the BL21 (DE3) host
strain. We set initial glucose concentrations of 10 g/L, 20 g/L, 30 g/L, 40 g/L, and 50 g/L, and the fermentation
conditions were the same as before, After this, we will add some new parameters to explore the factors affecting
levodopa production, such as inducer concentration and time.

Figure 17 shows that the yield of the strain increased with increasing glucose concentration, reaching a peak of
689.3 mg/L. However, as the glucose concentration continued to increase to 50 g/L, the yield began to decline. We
speculate that the high glucose concentration affected the strain\'s metabolism, resulting in a decrease in yield.

![](https://static.igem.wiki/teams/5740/result/17.webp)

Figure 17. Analysis of results at different initial sugar concentrations (optimal RBS sequence strain)

To further explore the factors affecting levodopa, we simultaneously investigated the inducer concentration,
glucose concentration, and fermentation time. The glucose concentration was still set at 10 g/L to 50 g/L, with 5
gradients, and the inducer concentration was 0, 0.2, 0.4, 0.6, 0.8, 1.0 mM, with 6 gradients, and the fermentation
time was 36 h and 48 h. The strain was inoculated into LBG medium with an initial OD of 0.05. After the
fermentation was completed, the product was tested.

![63080ce321ff2d29e92777e1eaebd1a4](https://static.igem.wiki/teams/5740/result/18.webp)

Figure 18. Effects of inducer concentration and glucose concentration on levodopa production at different times

Figure 18 shows the effects of inducer concentration and glucose concentration on levodopa production at different
times. The overall trend is that when the inducer concentration is 0.8mM and the glucose concentration is 40g/L,
the levodopa production is higher and significantly higher. When the inducer concentration is 0.2mM, the
production is higher without adding the inducer concentration than after adding it. This may be due to
contamination during fermentation. In addition, by comparing the overall situation at 36h and 48h, we can see that
the levodopa production at 48h is higher.

The heat map in Figure 19 is actually a correlation graph. The response surface also tells us a trend. Finding the
optimal inducer concentration and glucose concentration at different time points is consistent with the conclusion
of the above bar graph. We can see that the optimal glucose concentration is 40g/L, while the inducer
concentration is 0.8mM.

![](https://static.igem.wiki/teams/5740/result/19.webp)

Figure 19. Optimal inducer concentration and glucose concentration at different time points

Finally, we analyzed the inducer concentration, glucose concentration and time together. In the three-dimensional
results of Figure 20, we can see that 0.8-1.0mM, 40g/L, 36-48h, the fermentation results are better. Finally, we
chose the conditions of 0.8mM, 40g/L, 48h for the fermentation of E. coli ECN.

![af171a22dfe2162d17b224bb71f5419a](https://static.igem.wiki/teams/5740/result/20.webp)

Figure 20. Effects of different inducer concentrations, glucose concentrations, and time on levodopa

',
},
{
"title": '4.2 HPLC determination of levodopa content in ECN',
"content": '

After we have explored the optimal inducer concentration, glucose concentration and time for L-dopa production in
E. coli BL21 (DE3), the next step is to transform plasmid 1 and the optimized plasmid 2 into E. coli ECN, because
our ultimate goal is to let probiotics produce L-dopa.

As shown in Figure 21, the transformation diagram on the left proves that plasmid 1 and plasmid 2 were
successfully transformed into ECN. In addition, in order to further verify that there is no problem with the
transformed plasmid, we used colony PCR for verification. The final amplified band length is 800bp, indicating
that the transformation is successful. The next step is to select the correct single clone, expand the culture,
and then induce fermentation to produce levodopa.

![](https://static.igem.wiki/teams/5740/result/21.webp)

Figure 21. Plasmid 1 and plasmid 2 were transformed into E. coli ECN (left), and the electrophoresis results of
colony PCR identification were shown (right).

We then fermented the successfully transformed ECN using the optimal conditions obtained in the BL21(DE3) host
strain. After fermentation, we analyzed the fermentation samples using HPLC in triplicate. Figure 22 shows the
peaks of the three replicates, revealing the concentrations of 1.064 g/L, 1.065 g/L, and 1.069 g/L,
respectively.

![](https://static.igem.wiki/teams/5740/result/22.webp)

Figure 22. Peak diagram of levodopa production after HPLC detection of ECN fermentation

',
}
]
},
{
"title": 'Future plans',
"content": '

1. 1: Weaken the TCA cycle to increase carbon flux in the production pathway.
2. 2: Optimize the strain\'s glucose uptake and introduce new transporters to reduce precursor consumption by the PTS
   transport system.
3. 3: Knockout or weakening of the shikimate pathway can enhance product production.
4. 4: Product production is only the first step; further research on the isolation and purification of the substrate
   is also necessary to enhance the overall integrity of the research project.

',
},
{
"title": 'References',
"content": '

1. BALESTRINO R, SCHAPIRA AHV. Parkinson disease[J]. European Journal of Neurology, 2020, 27(1): 27-42.
2. YANG DM, ZHAO DM, ALI SHAH SZ, WU W, LAI MY, ZHANG XX, LI J, GUAN ZL, ZHAO HF, LI W, GAO HL, ZHOU XM, YANG LF.
   The role of the gut microbiota in the pathogenesis of Parkinson’s disease[J]. Frontiers in Neurology, 2019, 10:
   1155.
3. Puranik, N.; Song, M. Therapeutic Role of Heterocyclic Compounds in Neurodegenerative Diseases: Insights from
   Alzheimer’s and Parkinson’s Diseases. Neurol. Int. 2025, 17, 26. https://doi.org/10.3390/neurolint17020026
4. Schapira A H V, Emre M, Jenner P, et al. Levodopa in the treatment of Parkinson’s disease[J]. European journal
   of neurology, 2009, 16(9): 982-989.
5. Livingston C, Monroe-Duprey L. A review of levodopa formulations for the treatment of Parkinson’s disease
   available in the United States[J]. Journal of Pharmacy Practice, 2024, 37(2): 485-494.

',
},
] %}
{% block title %}Results{% endblock %}
{% block lead %}Results{% endblock %}
{% block page\_content %}
{% endblock %}