iGEM 2024 — Team termosz-selye-hun

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Notebook

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Engineering

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# PCR reactions

---

# 1.

We set up our first gradient PCR reaction to determine the optimal annealing temperature for each of our genes.

* We centrifuged and deluted 10 μL of our 100μM reverse and forward primer solution to 100μL with TE Buffer.
* We dissolved our 500ng of otimized HNMT in 1000μL TE Buffer then we vortexed and centrifuged the solution.
* We dissolved our 500ng of wild type HNMT in 1000μL TE Buffer then we vortexed and centrifuged the solution.
* We dissolved our 1000ng of otimized DAO in 1000μL TE Buffer then we vortexed and centrifuged the solution.
* We dissolved our 1000ng of wild type DAO in 1000μL TE Buffer then we vortexed and centrifuged the solution.

We added the following substrates in order:

| Component | Final concentration | 50-μL rxn |
| --- | --- | --- |
| 2X Platinum TM SuperFi TM II PCR Master Mix | 1X | 25 μL |
| Forward primer | 0.5 μM | 3 μL |
| Reverse primer | 0.5 μM | 3 μL |
| Template DNA | 0.1–10 ng plasmid (5-100 ng genomic DNA) | 2 μL |
| Water, nuclease free | - | 17 μL |

We ran the following PCR program:
![](https://static.igem.wiki/teams/5492/engineering/k-p1.webp)
![](https://static.igem.wiki/teams/5492/engineering/k-p2.webp)
We placed our PCR tube into the maschine in the following order:
A: O1 and W1
B: O1 and W1
C: -
D: O2 and W2
E: O2 and W2
F: -
G: O3 and W3
H: O3 and W3

We added the 6X DNA Loading dye to each PCR tubes marked in red above. We loaded the wells of the gels with 12μL of HNMT solution in the following order:

1. O1
2. O2
3. O3
4. Ladder (8 μL)
5. W1
6. W3

![](https://static.igem.wiki/teams/5492/engineering/k-p3.webp)
We loaded the wells of the gels with 12μL of DAO solution in the following order:

1. O1
2. O2
3. O3
4. Ladder (8 μL)
5. W1
6. W3

![](https://static.igem.wiki/teams/5492/engineering/k-p4.webp)

---

# 2.

Problem: few PCR products were generated
Solution: We decided to change the volume of the primer - and the DNA solutions added to the PCR reaction, to produce more DNA.
We added the following substrates in order:

| Component | Final concentration | 50-μL rxn |
| --- | --- | --- |
| 2X PlatinumTM SuperFiTM II PCR Master Mix | 1X | 25 μL |
| Forward primer | 0.5 μM | 2 μL |
| Reverse primer | 0.5 μM | 2 μL |
| Template DNA | 0.1–10 ng plasmid (5-100 ng genomic DNA) | 3 μL |
| Water, nuclease free | - | 18 μL |

![](https://static.igem.wiki/teams/5492/engineering/k-p5.webp)
We added the 6X DNA Loading dye to each PCR tubes.
We loaded the wells of the gels with 12μL of HNMT solution in the following order:

1. TO1
2. TO2
3. TO3
4. Ladder
5. GO1
6. GO2
7. GO3
8. GW1
9. GW2

![](https://static.igem.wiki/teams/5492/engineering/k-p6.webp)
We loaded the wells of the gels with 12μL of DAO solution in the following order:

1. -
2. TW1
3. TW2
4. TW4
5. Ladder
6. TO1
7. TO2
8. TO3
9. -

![](https://static.igem.wiki/teams/5492/engineering/k-p7.webp)

---

# 3. HNMT

Problem: few PCR products were generated
Solution: We raised the MgCl2 concentration
We ran more cycles and set them longer
We used 3 different Master Mix, containing different Taq polimerases.
We added the following substrates in order: (each optimized and wild type)

|  | S1(O/W) | S2(O/W) |
| --- | --- | --- |
| SuperFi Master Mix (2X) | 14 μL | 14 μL |
| Forward primer | 2 μL | 2 μL |
| Reverse primer | 2 μL | 2 μL |
| HNMT gene | 8 μL | 8 μL |
| Water, nuclease free | 16 μL | 20 μL |
| 10X Dream Taq Buffer | 8 μL | 4 μL |
| Total | 50 μL | 50 μL |

|  | T1(O/W) | T2(O/W) |
| --- | --- | --- |
| DreamTaq Master Mix (2X) | 14 μL | 14 μL |
| Forward primer | 2 μL | 2 μL |
| Reverse primer | 2 μL | 2 μL |
| HNMT gene | 8 μL | 8 μL |
| Water, nuclease free | 16 μL | 20 μL |
| 10X Dream Taq Buffer | 8 μL | 4 μL |
| Total | 50 μL | 50 μL |

|  | N1(O/W) | N2(O/W) |
| --- | --- | --- |
| NED Master Mix (2X) | 14 μL | 14 μL |
| Forward primer | 2 μL | 2 μL |
| Reverse primer | 2 μL | 2 μL |
| HNMT gene | 8 μL | 8 μL |
| Water, nuclease free | 16 μL | 20 μL |
| 10X Dream Taq Buffer | 8 μL | 4 μL |
| Total | 50 μL | 50 μL |

We ran our PCR reaction for HNMT:
![](https://static.igem.wiki/teams/5492/engineering/k-p8.webp)
We added the 6X DNA Loading dye to each PCR tubes.
We loaded the wells of the gels with 12L of optimized HNMT solution in the following order:

1. -
2. S1O
3. S2O
4. T1O
5. Ladder
6. T2O
7. N1O
ű8. N2O
9. -

![](https://static.igem.wiki/teams/5492/engineering/k-p9.webp)
We loaded the wells of the gels with 12μL of wild type HNMT solution in the following order:

1. -
2. S1W
3. S2W
4. T1W
5. Ladder
6. T2W
7. N1W
ű8. N2W
9. -

![](https://static.igem.wiki/teams/5492/engineering/k-p10.webp)

---

# 4. DAO

Problem: unnecessary large fragments in PCR products
Solution: We decreased the MgCl2 concentration
We annealing time for 20s.
We added more EDTA.
We added the following substrates in order: (each optimized and wild type)

|  | D31 (O/W) | D32 (O/W) | D33 (O/W) | D34 (O/W) |
| --- | --- | --- | --- | --- |
| MM (1X) | 25 μL | 25 μL | 25 μL | 25 μL |
| Forward primer | 1 μL | 1 μL | 1 μL | 1 μL |
| Reverse primer | 1 μL | 1 μL | 2 μL | 1 μL |
| DAO gene | 4 μL | 4 μL | 4 μL | 4 μL |
| Water, nuclease free | 14 μL | 17 μL | 12 μL | 9 μL |
| EDTA | 5 μL | 2 μL | 5 μL | 10 μL |
| Total | 50 μL | 50 μL | 50 μL | 50 μL |

We ran our PCR reaction for DAO:
![](https://static.igem.wiki/teams/5492/engineering/k-p11.webp)
We added the 6X DNA Loading dye to each PCR tubes.
We loaded the wells of the gels with 12μL of DAO solution in the following order:

1. D31O
2. D32O
3. D33O
4. D34O
5. Ladder
6. D31W
7. D32W
8. D33W
9. D34W

![](https://static.igem.wiki/teams/5492/engineering/k-p12.webp)

---

# 5. and 6. HNMT

According to the previous PCR reaction we decided to use S1 and N1.
We added the following substrates in order: (each optimized and wild type)

|  | S1(O/W) |
| --- | --- |
| SuperFi Master Mix (2X) | 14 μL |
| Forward primer | 2 μL |
| Reverse primer | 2 μL |
| HNMT gene | 8 μL |
| Water, nuclease free | 16 μL |
| 10X Dream Taq Buffer | 8 μL |
| Total | 50 μL |

|  | N1(O/W) |
| --- | --- |
| NED Master Mix (2X) | 14 μL |
| Forward primer | 2 μL |
| Reverse primer | 2 μL |
| HNMT gene | 8 μL |
| Water, nuclease free | 16 μL |
| 10X Dream Taq Buffer | 8 μL |
| Total | 50 μL |

We set up previously used PCR reaction

# DAO

According to the previous PCR reaction we decided to use D31 and D32.

|  | D41(O/W) | D42(O/W) |
| --- | --- | --- |
| MM (1x) | 25 μL | 25 μL |
| Forward primer | 1 μL | 1 μL |
| Reverse primer | 1 μL | 1 μL |
| DAO gene | 4 μL | 4 μL |
| Water, nuclease free | 14 μL | 17 μL |
| EDTA | 5 μL | 2 μL |
| Total | 50 μL | 50 μL |

We set up previously used PCR reaction.
We added the 6X DNA Loading dye to each PCR tubes. We loaded the wells of the gels with 12μL of DNA solution in the following order:

1. S1O
2. S1W
3. N1O
4. N1W
5. Ladder
6. D310
7. D31W
8. D32O
9. D32W

![](https://static.igem.wiki/teams/5492/engineering/k-p13.webp)

---

# 7. and 8. HNMT

According to the previous PCR reaction we decided to use S1.

|  | S1(O/W) |
| --- | --- |
| SuperFi Master Mix (2X) | 14 μL |
| Forward primer | 2 μL |
| Reverse primer | 2 μL |
| HNMT gene | 8 μL |
| Water, nuclease free | 16 μL |
| 10X Dream Taq Buffer | 8 μL |
| Total | 50 μL |

# DAO

According to the previous PCR reaction we decided to use D31.

|  | D41(O/W) |
| --- | --- |
| MM (1x) | 25 μL |
| Forward primer | 1 μL |
| Reverse primer | 1 μL |
| DAO gene | 4 μL |
| Water, nuclease free | 14 μL |
| EDTA | 5 μL |
| Total | 50 μL |

We set up previously used PCR reaction
We digested our DNA inserts. We loaded the wells of the gel in the following order:
1-3. optimized HNMT
4. ladder
5-6. optimized DAO
7-8. wild type HNMT
![](https://static.igem.wiki/teams/5492/engineering/k-p14.webp)

---

# 9.

We digested our petDUET plasmid.

![](https://static.igem.wiki/teams/5492/engineering/k-p15.webp)

[**Download**](https://static.igem.wiki/teams/5492/engineering/pcr-reactions.pdf)

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Experiments

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![](https://static.igem.wiki/teams/5492/experiments/measurements-abra.webp)

# Transdermal transport of the enzymes and aptamers

# Producing human HNMT and DAO enzymes with bacteria

# Experiments with aptamers

# Our Devices

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Results

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# Results

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## Scientific experiments

* We successfully created the E. coli optimized and wild type HNMT és DAO genes from bioinformatics databases and successfully ordered them from gene synthesis providers.
* We could amplify these genes with conventional and slightly modified PCR methods.
* We cloned the genes to the petDUET plasmid successfully
* We transformed E.coli DH 10 beta bacteria with the modified plasmid.
* We could check the presence of our target proteins with an ELISA plate method
* We successfully designed two collections of aptamers: one for fluorescence investigations and one cohort of ssDNA for the "fishing" method.
* We successfully proved that fluorescence method is a relevant way of measuring aptamer-target concentration.
* We proved that aptamers efficiently attach the histamine when the concentration is below 10 uM.
* Using ex-vivo transdermal device we proved that HNMT is able to transferred into the skin in liposome / hydrogel media, but the amplification time is long (6 hours or more) This suggests that the use of HNMT enzyme is worth it to use it in topical treatment, but mainly in patients with chronic pruritus symptoms.
* Ex-vivo transdermal device didn't release the DAO enzyme nor in liposomes, neither without liposomes, which makes HNMT a better choice in topical use.
* Our aptamers didn't enter the skin in the used hydrogels, but still can be efficient ingerdients to decrease the histamine level in food.

## Education

* We've created a LEGO set to educate students about the DNA alpha helix and the walkthrough of the steps of the central dogma.
* We performed 5 times for 120 8th-grade students.
* We wrote a questionnaire for the 8th-grade students which showed us the effectiveness of our lectures.
* We successfully implemented a video game on the basics of synthetic biology.
* We delivered our video game to 42 schools to educate 334 students.

## Human Practices

* We collaborated with other iGEM teams and we managed to make a lipid delivery system handbook together.
* We managed to codon optimize our genes and choose the right plasmid with the help of multiple experts from HUN-REN Biological Research Centre and the Faculty of Pharmacy.
* We consulted with drug/supplement producers to enlighten us about product development and manufacturing.
* We developed a pruritus questionnaire to assess itching and provide help in diagnosis.

## Entrepreneurship

* We presented the idea to investors who were positive about our concept and said to invest.
* We created a comprehensive business plan showing the initiative's feasibility and authenticity.
* Consulted with at least 5 professionals from fields like research, finance, cream production, and construction.

## Sustainable Development

* We developed a Sustainability Profile Assessment Tool which is a comprehensive questionnaire to measure a company’s sustainability from this, we obtained two results which we then evaluated.
* We created a sustainable packaging with a 3D printer for ointments in which we can store the active ingredient which was developed by us.

## Model

* We have modeled enzyme diffusion through the skin.
* We have examined the velocity of enzyme reaction with fixed enzyme concentration and with [E](t)=C(t)

## Safety

* We have covered some aspects of safety from several points of view (environmental safety, personal safety, and data safety).

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