iGEM 2025 — Team bioplus-shanghai

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
{% block page\_content %}

![Project Banner](https://static.igem.wiki/teams/5762/description/slice6.webp)

# Notebook

![Title Line](https://static.igem.wiki/teams/5762/description/frame445.webp)

![Icon](https://static.igem.wiki/teams/5762/description/slice2.webp)

## Notebook

![Heartbeat]({{ url_for('static', filename='images/xintiao.gif') }})

### Notebook

#### 7.8

1. The PCR system was formulated according to the table below (YFP)

| Reagent | Volume(μL) |
| --- | --- |
| DNA | 1 |
| KpnI-YFP-F | 0.5 |
| BamH I-YFP-R | 0.5 |
| 2X Master Mix | 5 |
| ddH2O | 3 |

2. Put in PCR instrument, PCR program

| Temperature | Time |
| --- | --- |
| 98℃ | 30s |
| 98℃ | 10s |
| 65℃ | 30s |
| 72℃ | 21s |
| 72℃ | 10min |
| Cycle: 35 | |

3. Agarose gel electrophoresis

1. Weigh 0.5g agarose and add to 50ml 1X TAE
2. Microwave until completely melted
3. Cool to 50 °C to 60 °C and add 3μL DNA stain
4. Inverted board
5. After cooling, sample in sequence
6. 130V 30min
7. After the process is completed, place the gel under a UV lamp, excise the portion of the gel containing the target fragment, and place the excised gel into a 1.5 mL EP tube.

#### 7.9

1. Gel recovery

1. Add 200 μL of PN to the colloid and incubate in a water bath at 50°C for 10 minutes.
2. Add 500 μL of BL to the chromatography column and place it in a centrifuge at 12,000 rpm for 1 minute. Discard the eluate.
3. Transfer the liquid in the EP tube to the chromatography column and let it stand at room temperature for 2 minutes. Then place it in the centrifuge at 12,000 rpm for 1 minute and discard the eluate.
4. Add 600 μL of PW to the column and place it in the centrifuge at 12,000 rpm for 1 minute. Discard the eluate.
5. Repeat the previous step once.
6. Place the tube in the centrifuge at 12,000 rpm for 2 minutes without any liquid, then open the lid and let it stand for 5 minutes.
7. Place the chromatography column in a new EP tube and add 30 μL of ddH2O to the column. Let it stand for 2 minutes and then place it in the centrifuge at 12,000 rpm for 2 minutes. Collect the eluate.
8. Add the eluate back to the chromatography column and place the system in the centrifuge at 12,000 rpm for 2 minutes. Discard the chromatography column and keep the EP tube.

2. Enzymatic digestion

1) Prepare the enzyme digestion system

| Regant | Volume(μL) |
| --- | --- |
| pRESTa | 3 |
| Kpn I | 0.2 |
| BamH I | 0.2 |
| rcut smart buffer | 2 |
| ddH2O | 13μL |

2) 37℃ 40min

#### 3. Agarose gel electrophoresis + Gel recovery

1. Weigh 0.5g agarose and add to 50ml 1X TAE
2. Microwave until completely melted
3. Cool to 50 °C to 60 °C and add 3μL DNA stain
4. Inverted board
5. After cooling, sample in sequence
6. 130V 30min
7. After the process is completed, place the gel under a UV lamp, excise the portion of the gel containing the target fragment, and place the excised gel into a 1.5 mL EP tube.
8. Add 200 μL of PN to the colloid and incubate in a water bath at 50°C for 10 minutes.
9. Add 500 μL of BL to the chromatography column and place it in a centrifuge at 12,000 rpm for 1 minute. Discard the eluate.
10. Transfer the liquid in the EP tube to the chromatography column and let it stand at room temperature for 2 minutes. Then place it in the centrifuge at 12,000 rpm for 1 minute and discard the eluate.
11. Add 600 μL of PW to the column and place it in the centrifuge at 12,000 rpm for 1 minute. Discard the eluate.
12. Repeat the previous step once.
13. Place the tube in the centrifuge at 12,000 rpm for 2 minutes without any liquid, then open the lid and let it stand for 5 minutes.
14. Place the chromatography column in a new EP tube and add 30 μL of ddH2O to the column. Let it stand for 2 minutes and then place it in the centrifuge at 12,000 rpm for 2 minutes. Collect the eluate.
15. Add the eluate back to the chromatography column and place the system in the centrifuge at 12,000 rpm for 2 minutes. Discard the chromatography column and keep the EP tube.

#### 4. The connection of the YFP and pRSETa vectors

1）Prepare the connection system（10μL）

| Regant | Volume(μL) |
| --- | --- |
| pRESTa | 1.5 |
| YFP | 3.5 |
| T4 DNA Ligase | 0.5 |
| T4 DNA Ligase buffer | 1 |
| ddH2O | 3.5 |

2）4℃ for the whole night

#### 7.10

#### 1． Transformation of the connected product

1. Add 10 μL of plasmid to the EP tube.
2. Add 20 μL of competent cells to the EP tube.
3. Place the system on ice and let it stand for 30 minutes.
4. After completion, quickly transfer the system to a water bath and perform a heat shock at 42°C for 35 seconds.
5. Add 500 μL of LB culture medium to the system and place the entire system in a shaker for 1 hour.
6. After the previous section is completed, place the entire reaction system on the centrifuge and centrifuge it at 4000 rpm for 5 minutes.
7. After completion, discard the supernatant of the system, 450 μL, and then use the remaining liquid to resuspend and mix the precipitate before coating the plate.

#### 2. Amplification of the mCherry fragment

1） The PCR system was formulated according to the table below（mCherry）

| Reagent | Volume(μL) |
| --- | --- |
| DNA | 1 |
| KpnI-mCherry-F | 0.5 |
| BamH I-mCherry-R | 0.5 |
| 2X Master Mix | 5 |
| ddH2O | 3 |

2）．Put in PCR instrument, PCR program

| Temperature | Time |
| --- | --- |
| 98℃ | 30s |
| 98℃ | 10s |
| 65℃ | 30s |
| 72℃ | 45s |
| 72℃ | 10min |

Cycle：35

#### 7.11

#### 1. Perform single-cell cloning culture and identification-YFP

1. Add 500 μL LB culture medium to a 2 mL centrifuge tube
2. Pick a single colony into the tube
3. Place the system in a shaking incubator at 37°C and 220 rpm for culture until the OD value reaches 0.5
4. Perform PCR identification

#### 2. Send the sequenced strains for further sequencing and identification.

#### 7.14

#### 1. Gel recovery(mCherry)

1. Add 200 μL of PN to the colloid and incubate in a water bath at 50°C for 10 minutes.
2. Add 500 μL of BL to the chromatography column and place it in a centrifuge at 12,000 rpm for 1 minute. Discard the eluate.
3. Transfer the liquid in the EP tube to the chromatography column and let it stand at room temperature for 2 minutes. Then place it in the centrifuge at 12,000 rpm for 1 minute and discard the eluate.
4. Add 600 μL of PW to the column and place it in the centrifuge at 12,000 rpm for 1 minute. Discard the eluate.
5. Repeat the previous step once.
6. Place the tube in the centrifuge at 12,000 rpm for 2 minutes without any liquid, then open the lid and let it stand for 5 minutes.
7. Place the chromatography column in a new EP tube and add 30 μL of ddH2O to the column. Let it stand for 2 minutes and then place it in the centrifuge at 12,000 rpm for 2 minutes. Collect the eluate.
8. Add the eluate back to the chromatography column and place the system in the centrifuge at 12,000 rpm for 2 minutes. Discard the chromatography column and keep the EP tube.

#### 2. Isolation and cultivation of positive clones of E. coli (YFP)

1. Add 5mL LB and 5 μL Amp to a 50 mL centrifuge tube, and then add the positive cloning bacterial solution that was saved last week.
2. Shake at 37℃ and 220 rpm for over night.

#### 7.15

#### 1. Plasmid extraction

1. Take 5ml overnight culture bacterial solution, centrifuge at 9000 rpm for 2 minutes, and collect all bacterial bodies after multiple centrifugation
2. The supernatant was removed and 250μL buffer P1 was added to the precipitation
3. Blow the precipitation repeatedly with the pipette gun until the bacteria are completely suspended
4. Add 250μL Buffer P2, and immediately gently reverse the centrifugal tube for 5-10 times and mix well. Let stand at room temperature for 2-4 minutes. You'll see the liquid clear and clear
5. Add 300μL Buffer P3 and immediately gently reverse the centrifuge tube for 5-10 times to mix. At this point you can see the white precipitate forming
6. Centrifuge at 12000rpm for 5-10 min. Transfer the supernatant into the adsorption column, centrifuge at 12000rpm for 30 seconds, and drain the liquid in the collection tube. If the supernatant liquid is too much, it can be centrifuged
7. Add 600μL Wash Solution, centrifuge at 9000rpm for 30 seconds, and drain the liquid in the collection tube. Repeat twice
8. The empty adsorption column is placed in the collection tube at 9000rpm for 2min
9. Place the adsorption column in a new centrifuge tube and place it in the fume hood for 10min to remove residual ethanol
10. Add 30μL Elution Buffer into the adsorption column, leave at room temperature for 2min, and then centrifuge at 9000rpm for 2min to collect the liquid in the centrifugal tube
11. In order to ensure the concentration of glue recovery, you can repeat the step j)

#### 2. Send the purified plasmid for sequencing

#### 3. Enzymatic digestion

Prepare the enzyme digestion system

| Regant | Volume(μL) |
| --- | --- |
| mCherry/pRSETa | 3 |
| Kpn I | 0.2 |
| BamH I | 0.2 |
| rcut smart buffer | 2 |
| ddH2O | 13μL |

37℃ 40min

#### 3. Agarose gel electrophoresis+ Gel recovery

1. Weigh 0.5g agarose and add to 50ml 1X TAE
2. Microwave until completely melted
3. Cool to 50 ° C to 60 ° C and add 3μL DNA stain
4. Inverted board
5. After cooling, sample in sequence
6. 130V 30min
7. After the process is completed, place the gel under a UV lamp, excise the portion of the gel containing the target fragment, and place the excised gel into a 1.5 mL EP tube.
8. Add 200 μL of PN to the colloid and incubate in a water bath at 50°C for 10 minutes.
9. Add 500 μL of BL to the chromatography column and place it in a centrifuge at 12,000 rpm for 1 minute. Discard the eluate.
10. Transfer the liquid in the EP tube to the chromatography column and let it stand at room temperature for 2 minutes. Then place it in the centrifuge at 12,000 rpm for 1 minute and discard the eluate.
11. Add 600 μL of PW to the column and place it in the centrifuge at 12,000 rpm for 1 minute. Discard the eluate.
12. Repeat the previous step once.
13. Place the tube in the centrifuge at 12,000 rpm for 2 minutes without any liquid, then open the lid and let it stand for 5 minutes.
14. Place the chromatography column in a new EP tube and add 30 μL of ddH2O to the column. Let it stand for 2 minutes and then place it in the centrifuge at 12,000 rpm for 2 minutes. Collect the eluate.
15. Add the eluate back to the chromatography column and place the system in the centrifuge at 12,000 rpm for 2 minutes. Discard the chromatography column and keep the EP tube.

#### 4. The connection of the mCherry and pRSETa vectors

1）Prepare the connection system（10μL）

| Regant | Volume(μL) |
| --- | --- |
| pRESTa | 1.5 |
| mCherry | 3.5 |
| T4 DNA Ligase | 0.5 |
| T4 DNA Ligase buffer | 1 |
| ddH2O | 3.5 |

2）4℃ for the whole night

#### 7.16

#### 1． Transformation of the connected product

1. Add 10 μL of plasmid to the EP tube.
2. Add 20 μL of competent cells to the EP tube.
3. Place the system on ice and let it stand for 30 minutes.
4. After completion, quickly transfer the system to a water bath and perform a heat shock at 42°C for 35 seconds.
5. Add 500 μL of LB culture medium to the system and place the entire system in a shaker for 1 hour.
6. After the previous section is completed, place the entire reaction system on the centrifuge and centrifuge it at 4000 rpm for 5 minutes.
7. After completion, discard the supernatant of the system, 450 μL, and then use the remaining liquid to resuspend and mix the precipitate before coating the plate.

#### 7.17

#### 1. Monoclonal culture

1. Add 1 mL of LB medium and 1 μL of Amp (100 mg/mL) into a 1.5 mL EP tube.
2. Pick a single colony from the overnight grown solid medium of Escherichia coli and add it to the above 1.5 mL EP tube.
3. Shake at 37°C and 220 rpm until the bacterial solution becomes turbid.

#### 2. Bacterial liquid PCR identification

The clarified bacterial solution obtained above was used as the template for PCR analysis. The PCR system was based on the PCR amplification of mCherry.

#### 7.18

#### 1. Isolation and cultivation of positive clones of E. coli (mCherry)

1. Add 5mL LB and 5 μL Amp to a 50 mL centrifuge tube, and then add the positive cloning bacterial solution that was saved last week.
2. Shake at 37℃ and 220 rpm for over night.

#### 2. Plasmid extraction

The process refers to the plasmid extraction method of YFP.

#### 7.21

#### 1. PCR

Using different primers to perform PCR amplification on the sequence of YFP, different lengths of Linker were constructed. The PCR system followed the amplification of YFP, and the primer sequences are as follows.

| Name | Sequences |
| --- | --- |
| NO-N1-F | GCAGGCTACAACAGCGACAAC |
| NO-N1-R | AAAAGGAGTAGCATCTCCAGGTAG |
| NO-N2-F | GGCTACAACAGCGACAACGTC |
| NO-N3-F | TACAACAGCGACAACGTCTATATC |

#### 2. Send the extracted mCherry plasmid for sequence analysis

#### 3. Gel recovery （sequences of N1-N3 PCR）

#### 7.22

1. Enzymatic digestion: DpnI enzyme digests the gel recovery product and the vector at 37°C for 40 minutes.
2. Ligation: T4 DNA ligase ligates the above-mentioned ligated products at 4°C for 2 hours.
3. Transformation: The above-mentioned ligated products are transformed into Escherichia coli competent cells. The experimental procedure follows the previous one.

#### 7.23

#### 1. PCR

Using different primers to perform PCR amplification on the sequence of YFP, different lengths of Linker were constructed. The PCR system followed the amplification of YFP, and the primer sequences are as follows.

| Name | Sequences |
| --- | --- |
| NO-C1-F | GAAGCTGTAAAACAAACACAAATGA |
| NO-C1-R | GGTACCGTTGTACTCCAGCTTGT |
| NO-C2-R | ACCGTTGTACTCCAGCTTGTGC |
| NO-C3-R | GTTGTACTCCAGCTTGTGCCC |

#### 2. Monoclonal culture: Select the monoclonal cells grown on the solid medium after transformation on July 22nd and cultivate them at 37°C with a speed of 220 rpm.

#### 7.24

#### 1. Plasmid extraction

The process refers to the plasmid extraction method of YFP.

#### 2. Send the extracted plasmid for sequencing

#### 3. Gel recovery：Recycle the PCR product with a concentration of 7.23

#### 7.25

1. Enzymatic digestion: DpnI enzyme digests the gel recovery product and the vector at 37°C for 40 minutes.
2. Ligation: T4 DNA ligase ligates the above-mentioned ligated products at 4°C for 2 hours.
3. Transformation: The above-mentioned ligated products are transformed into Escherichia coli competent cells. The experimental procedure follows the previous one.

#### 7.28

1. transformation: Transform the sequenced correct YFP plasmid into BL21 competent cells.
2. Pick and cultivate the cloned colonies obtained from the 7.25 transformation of E. coli.

#### 7.29

1. Protein induction: Induce the BL21 strain containing the YFP plasmid with IPTG. The induction conditions are 18°C, 220 rpm, and 24 hours.
2. Plasmid extraction
3. The process refers to the plasmid extraction method of YFP.
4. Send the extracted plasmid for sequencing

#### 7.30

#### 1. The cultivation of E. coli

1. Add 20 microliters of Amp
2. Add 20 milliliters of LB
3. Add 20 microliters of bacterial solution
4. Shaking culture at 37℃ and 220 rpm on a shaker

#### 2. transformation

1. Take the competent cells out from the -80°C refrigerator.
2. Add 1 μL of plasmid and 4-5 μL of competent cells to the EP tube.
3. Incubate on ice for 30 minutes, then in a water bath at 42°C for 35 seconds.
4. Add 500 μL of LB in the ultrapure bench, incubate at 37°C, on a 220 rpm shaker for 1 hour. During this period, sterilize the inoculation rod with an alcohol lamp.
5. Centrifuge at 4000 rpm for 5 minutes, discard 450 μL of the supernatant, retain 100 μL of the precipitate and the supernatant. Take 100 μL and evenly drop onto the petri dish, spread, and finish by placing upside down and sealing.
6. Cultivate in an inverted 37°C incubator overnight.

#### 7.31 Purification of NorR-YFP probe protein

1. Check whether there is fluorescence in the overnight-induced E. coli culture medium.
2. Centrifuge E. coli for 5 minutes at 5000 rpm
3. Discard the upper layer of water
4. Add 1 ml of PBS and vortex to mix evenly
5. Transfer to a 2 ml tube
6. Use diaphragm meter + magnetic beads
7. Centrifuge for 10 minutes at 12,000 rpm
8. Discard the upper clear liquid.
9. Add the egg white to the magnetic beads.
10. Place it in the protein purification apparatus for 3 hours.
11. Discard the upper clear liquid.
12. Add 500ml of 50kpi and shake to discard the upper clear liquid.
13. Add 500ml of 100kpi and shake to retain the upper clear liquid.
14. Add 500ml of 300kpi and shake to retain the upper clear liquid.

#### 8.1

#### 1. Fluorescent protein spectroscopy - Analysis of the full spectrum (300 - 800)

1. Wash the quartz glass dish.
2. Add 200 μL of YFP fluorescent protein.
3. Place the sample in the instrument.
4. Set the corresponding program and start the test.
5. Record the data.
6. Test completed and output results

#### 2. Detect the specificity of fluorescent proteins

#### 8.4

#### The influence of temperature and pH on the probe

1. Prepare solutions of different pH using NaOH solution and HCI solution.
2. Add 90 μL of solutions with pH values of 7, 7.4, 7.5, and 7.6 to a 96-well plate (7 groups for each treatment, totaling 28 wells). Add 10 μL of protein probe solution to each well. After adding the protein, place the plate in the microplate reader for detection at 37°C for 20 times. After testing 5 groups, add 10 μL of furazolidone to each well.

#### 8.5

#### mCherry E. coli cloning culture

Pick and transfer 500 μL of LB medium + 0.5 μL Amp + the monoclonal bacteria

#### 8.6

#### 1. PCR（GFP）

| Reagent | Volume(μL) |
| --- | --- |
| DNA | 1 |
| KpnI-GFP-F | 0.5 |
| BamH I-GFP-R | 0.5 |
| 2X Master Mix | 5 |
| ddH2O | 3 |

Tm：55℃，cycle：35

#### 2. Agarose gel electrophoresis and gel recovery

1. Run the gel: Inject 5 μL of the marker into the first well of the gel, and all the amplified products into the remaining wells of the gel.
2. Gel recovery: #PN actually adds 200 μL per tube, PW actually adds 500 μL per tube. Place for 5 minutes in the seventh step, and do not use EB but use 30 μL of ddH2O in the eighth step.

#### 8.7

#### 1. Induction of mCherry protein expression

The bacterial culture containing mCherry was induced with IPTG at low temperature (18℃, 220 rpm) for 24 hours, then left at room temperature for 30 minutes, and then centrifuged at 4000 rpm for 5 minutes to break the bacteria and release the protein.

#### 2. Protein extraction and detection of protein's response to NO

Discard the supernatant, add 1 mL of HEPES buffer, vortex to detach the protein from the tube wall, and add 2 mL into the EP tube

#### 3. Protein disruption

(instrument needs repair, will be done next week)

#### 4. Gel recovery

Gel recovery: #PN actually adds 200 μL per tube, PW actually adds 500 μL per tube. Place for 5 minutes in the seventh step, and do not use EB but use 30 μL of ddH2O in the eighth step.

#### 5. The connection of the GFP and pRSETa vectors

1）Prepare the connection system（10μL）

| Reagent | Volume(μL) |
| --- | --- |
| pRESTa | 1.5 |
| GFP | 3.5 |
| T4 DNA Ligase | 0.5 |
| T4 DNA Ligase buffer | 1 |
| ddH2O | 3.5 |

2）4℃ for the whole night

#### 8.8

#### Transformation of the connected product

1. Add 10 μL of connected product to the EP tube.
2. Add 20 μL of competent cells to the EP tube.
3. Place the system on ice and let it stand for 30 minutes.
4. After completion, quickly transfer the system to a water bath and perform a heat shock at 42°C for 35 seconds.
5. Add 500 μL of LB culture medium to the system and place the entire system in a shaker for 1 hour.
6. After the previous section is completed, place the entire reaction system on the centrifuge and centrifuge it at 4000 rpm for 5 minutes.
7. After completion, discard the supernatant of the system, 450 μL, and then use the remaining liquid to resuspend and mix the precipitate before coating the plate.

#### 8.11

#### 1. Pick a single clone [Expand the bacterial quantity]

500 μL LB + 0.5 μL Amp + single clone

#### 2. Serum detection (test probe)

Artificial mouse plasma was divided into four portions (50 μL each), and 0, 300 μmol, 500 μmol, 1 mmol, and 1.5 mmol of furazan were added respectively. Then, 50 μL of probe solution was added to each portion, and the mixtures were observed under a blue light instrument. The final volumes of the five groups were 100, 121.4, 150, 105.6, and 108.8 μL, respectively.

#### 3. Recovery of 293T engineering cells:

Remove the cells from the -80℃ refrigerator and quickly place them in a water bath at 37℃.

2 mL of DMEM medium (containing 10% fetal bovine serum) + one tube of frozen cells, 1000 rpm (but in actual operation, it was set to 1000 \* g) for 3 minutes, discard the supernatant (the cryopreservation solution is toxic to the cells), add 2 mL of DMEM and vortex, add to the petri dish, then add 6 mL of DMEM.

![冻存的细胞](https://static.igem.wiki/teams/5762/note/note1.webp)

冻存的细胞

#### 8.12

1. Plasmid extraction: Take 2 mL of bacterial solution and transfer it to a 2 mL ELP tube. Centrifuge at 12000 rpm for 1 min \* 3
2. 293T cell passage

![Parental cells before passage](https://static.igem.wiki/teams/5762/note/note2.webp)

Parental cells before passage

Place 6 cell cards into the six-well plate respectively, add 2 mL of DMEM to each well, add 8 mL of DMEM to the empty wells, discard all the supernatant from the previous culture plates, and add 1 mL of trypsin to cause the cells to detach.

Rinse off the remaining cells with pancreatic enzyme solution, resuspend, and transfer to a 15 mL tube. Centrifuge at 1000 rpm for 3 minutes, discard the supernatant, add 2 ml of new medium, resuspend. Add 500 microliters to a petri dish and mix thoroughly in a cross pattern [for preservation]. Add 150 microliters per well to a six-well plate.

#### 8.13

1. Transfection

   The final shape of the cell

   The optimal density of cells

   Add 80 microliters of DMEM (without serum) to each of the 6 pear-shaped tubes, and add 2 microliters of PIvX-NO {cyto,mito,nuclear} (with volumes of {2.53, 2.31, 2.61 microliters}) to {2x3} of the tubes. Add 36 microliters of EZtrans 480 microliters of DMEM (without serum) to tube 7, and distribute it into tubes 1-6 in six portions and vortex. Let it stand for 10 minutes.

   Replace the liquid in the six-well culture medium with 2 mL of DMEM (without serum)

   Add the solutions after standing to the six-well plates respectively

   The transfected cells
2. Transformation(mCherry-BL21)

#### 8.14

1. Single-cell cloning (mCherry-BL21)
2. Passage of 293T cells
3. Observation of fluorescence
4. Bacterial inoculation: Add bacterial culture solution (20 mL), LB medium, and 20 microliters of Amp to a 50 mL centrifuge tube

#### 8.15

1. 293T cell passage
2. Confocal imaging: Attach the thin slices containing 293T cells that have been transfected for 48 hours onto the confocal carrier, and rinse with 500 μL of sugar-free HBSS.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
{% block page\_content %}

![Engineering Banner](https://static.igem.wiki/teams/5762/description/slice6.webp)

# ENGINEERING

![Title Line](https://static.igem.wiki/teams/5762/description/frame445.webp)

![Heart](https://static.igem.wiki/teams/5762/description/slice1.webp)

* Background
* Cycle1
* Cycle2
* References

![Background Icon](https://static.igem.wiki/teams/5762/description/slice2.webp)

## Background

![Title Line]({{ url_for('static', filename='images/xintiao.gif') }})

Cardiovascular diseases (CVDs) remain the leading cause of death worldwide. Nitric oxide (NO), a key biomarker closely associated with vascular function and the progression of CVDs, exhibits dynamic changes that hold significant pathophysiological implications [1]. As an endogenous gaseous signaling molecule, NO is involved in various physiological processes such as regulating vasodilation, maintaining blood pressure homeostasis, and modulating inflammatory responses [2-4].

Clinical studies have demonstrated that abnormal expression levels of NO are significantly correlated with the development and progression of major diseases including hypertension, atherosclerosis, and heart failure. However, direct and dynamic monitoring of NO in blood remains challenging due to its short half-life, low concentration in vivo, and high reactivity with substances such as oxygen radicals.

Current commonly used NO detection methods—such as the Griess assay, electrochemical sensing, and electron spin resonance (ESR)—are often labor-intensive, rely on bulky instruments, and are unsuitable for real-time in situ detection, limiting their application in rapid clinical diagnostics and point-of-care testing (POCT). Therefore, there is an urgent need in the biomedical detection field to develop a highly sensitive, specific, and user-friendly NO detection tool for quantitative analysis in complex biological fluids such as blood.

![Design Icon](https://static.igem.wiki/teams/5762/description/slice2.webp)

## Construction and Validation of the Core NorR-based Biosensor

![Title Line]({{ url_for('static', filename='images/xintiao.gif') }})

### Design

In this study, the NO-binding domain of NorR [5] was selected as the recognition module and fused to yellow fluorescent protein (YFP), red fluorescent protein (mCherry), and green fluorescent protein (GFP) via a flexible linker. Upon NO binding to the NorR domain, the induced conformational change alters the optical properties of the fluorescent protein through an allosteric effect, thereby converting NO concentration into a quantifiable fluorescent signal.

This strategy provides an effective means for highly specific and real-time detection of NO in complex biological environments. We propose to produce the fluorescent probes using a prokaryotic expression system in E. coli. The anticipated experimental workflow is shown in Fig.1.

![Expected experimental flowchart](https://static.igem.wiki/teams/5762/test/eng1.webp)

Fig.1 Gene circuit for prokaryotic expression

### Build

We constructed the prokaryotic expression vectors using a double restriction enzyme digestion strategy, as outlined in Fig. 2. First, the DNA fragments encoding YFP, mCherry, and GFP were amplified by PCR. Both the PCR products and the pRSETa-NorR vector were digested with the restriction enzymes BamH I and KpnI. The digested DNA fragments were purified and ligated into the linearized vector using T4 DNA ligase, resulting in the recombinant plasmids NorR-YFP\_pRSETa, NorR-mCherry\_pRSETa, and NorR-GFP\_pRSETa. These constructs were then transformed into E. coli DH5α competent cells. Positive clones were identified by colony PCR (Fig. 4), and selected clones were cultured for plasmid extraction and subsequent sequencing.

![Workflow diagram of plasmid construction](https://static.igem.wiki/teams/5762/test/engbuild1.webp)

Fig.2 Workflow diagram of plasmid construction

![Vector construction](https://static.igem.wiki/teams/5762/test/engbuild2.webp)

Fig.3 Vector construction of NorR-YFP\_pRSETa, NorR-mCherry\_pRSETa and NorR-GFP\_pRSETa

A: PCR of YFP sequences; B: PCR of mCherry sequence; C: PCR of GFP sequences; D: The pRSETa vector after double enzyme digestion.

![PCR Verification of the recombinant plasmid](https://static.igem.wiki/teams/5762/test/engbuild3.webp)

Fig.4 PCR Verification of the recombinant plasmid

### Test

### 1. Protein expression and purification

Based on the successfully constructed recombinant plasmids, the NorR-fluorescent protein fusion was expressed in E. coli BL21(DE3) cells. Soluble expression was achieved through low-temperature induction with 0.42 mM IPTG. After cell lysis, the fusion protein was purified by nickel affinity chromatography via its His-tag, and high-purity protein fractions were obtained using gradient imidazole elution (Fig. 5). These purified samples were subsequently used for further studies.

![Purification results](https://static.igem.wiki/teams/5762/test/test1.webp)

Fig.5 The purification results of recombinant proteins NorR-YFP, NorR-GFP and NorR-mCherry

### 2. The influence of different fluorescent protein carriers on the sensitivity of nitric oxide (NO) probes

To evaluate the NO-binding capabilities of YFP, GFP, and mCherry, each probe was incubated with varying concentrations of the NO donor Furoxan, and their specific fluorescence signals were detected. YFP and GFP were measured in dual-excitation mode (excitation at 420 nm and 485 nm, emission at 515 nm) to determine the fluorescence ratio, while mCherry was detected under 590 nm excitation and 645 nm emission. The binding performance was quantitatively assessed by calculating the ratio of fluorescence intensity after NO binding to that before binding. As shown in Fig. 6, with increasing Furoxan concentration, the fluorescence ratios of YFP and GFP changed significantly and gradually saturated, demonstrating a concentration-dependent response. In contrast, the fluorescence ratio of mCherry showed no notable change across the concentration range, indicating its inability to effectively bind NO. Further comparison revealed that YFP exhibited a significantly higher response amplitude than GFP, indicating superior sensitivity. Therefore, YFP was selected as the NO-sensing probe for subsequent experiments.

![Fluorescence response curves](https://static.igem.wiki/teams/5762/test/test2.webp)

Fig.6 Fluorescence response curves of the YFP-, GFP- and mCherry-NO probes

### 3. Verification of the NO sensing function of NorR-YFP

#### 3.1 pH value and temperature stability

We further evaluated the stability of the protein under different temperatures and pH conditions. Each 100 μL protein sample was treated with 100 μM Furoxan (NO donor) in the experimental group, while the control group received an equal volume of buffer. As shown in Fig. 7, after NO release induced by Furoxan, the fluorescence ratio of NorR-YFP at 420/480 nm increased significantly. The protein exhibited a stable fluorescence response within the pH range of 7.2–8.0 and the temperature range of 20–40°C, demonstrating excellent environmental adaptability and detection reliability, making it suitable for NO detection in complex physiological systems.

![pH value and temperature stability](https://static.igem.wiki/teams/5762/test/test3.webp)

Fig.7 pH value and temperature stability of NorR-YFP

#### 3.2 Specific detection of NO

To assess the specificity of NorR-YFP, we examined its fluorescence response in the presence of various reactive molecules. The system was treated with 100 μM of Furoxan (NO donor), CO, H₂O₂, NaNO₂, or NaHS, and changes in the fluorescence ratio were monitored. The results (Fig. 8) showed that only Furoxan treatment induced a rapid and significant increase in the fluorescence ratio, while other reactive molecules caused no notable signal changes. This confirms that NorR-YFP possesses highly selective recognition capability for NO and can effectively distinguish NO from other similar reactive small molecules.

![Specificity assessment of NorR-YFP](https://static.igem.wiki/teams/5762/test/test4.webp)

Fig.8 Specificity assessment of NorR-YFP toward various reactive species

#### 3.3 Concentration-dependent detection

To elucidate the dose-response relationship between the fluorescence signal of NorR-YFP and NO concentration, the protein was treated with gradient concentrations of the NO donor Furoxan. The experimental system contained 100 μL of 1 μmol/L protein sample, and changes in the fluorescence ratio were continuously monitored over 40 minutes. The results showed that as the Furoxan concentration increased, the fluorescence response of NorR-YFP enhanced in a clear concentration-dependent manner (Fig. 9), fully demonstrating that the fusion protein can produce a sensitive dose-dependent response to NO.

![Real-time Monitoring of NorR-YFP Fluorescence Kinetics](https://static.igem.wiki/teams/5762/test/test5.webp)

Fig.9 Real-time Monitoring of NorR-YFP Fluorescence Kinetics upon Furoxan Stimulation

#### 3.4 Dependence of Nitric Oxide Concentration on Fluorescence Detection in Artificial Plasma

In the plasma NO detection experiment, different concentrations (0–1000 μM) of the NO donor Furoxan were incubated with artificial plasma and the probe protein. After blue light excitation, the fluorescence ratio significantly increased with rising Furoxan concentration, showing a clear dose-dependent relationship (Fig. 10). The results indicate that the probe can sensitively and reliably detect NO in plasma, laying the foundation for quantitative analysis of NO in biological samples.

![Dose-dependent response in artificial plasma](https://static.igem.wiki/teams/5762/test/test6.webp)

Fig.10 The dose-dependent response of probe protein fluorescence intensity in artificial plasma to the concentration of NO donor furoxan

#### 3.5 Subcellular localization detection

To achieve specific monitoring of NO at the subcellular level, lentiviral vectors targeting different cellular regions were synthesized: mito-NorR-YFP\_pLVX (mitochondria), cyto-NorR-YFP\_pLVX (cytoplasm), and nuclear-NorR-YFP\_pLVX (nucleus). At 48 hours after transfection of these vectors into 293T cells, fluorescence microscopy observations (Fig. 11) revealed effective expression of the NorR-YFP fusion protein in the cells, with fluorescence distributions fully consistent with the expected localizations: a reticular or punctate pattern in the mitochondria group, uniform diffusion in the cytoplasm group, and concentration in the nuclear region in the nucleus group. These results indicate that the constructed probes possess accurate subcellular targeting ability, providing a reliable tool for real-time and specific monitoring of NO dynamics in specific organelles.

![Subcellular localization of NO sensor](https://static.igem.wiki/teams/5762/test/test7.webp)

Fig.11 The subcellular localization of the NO sensor NorR-YFP

### Learn

This study successfully developed a novel NO biosensor based on the NorR recognition module. The NorR-YFP fusion protein demonstrated high sensitivity, excellent environmental stability, and specificity, enabling real-time quantitative detection of NO under physiologically relevant conditions.

It performed well in artificial plasma and various subcellular structures. This sensor provides a powerful tool for mechanistic research and clinical diagnosis of cardiovascular diseases.

In the future, this study is expected to further advance the development of in vivo real-time imaging technology and portable NO detection devices, offering innovative solutions for precision medicine and point-of-care diagnostics.

![Learn Icon](https://static.igem.wiki/teams/5762/description/slice2.webp)

## Optimization of the Linker in a Direct Fusion Protein

![Title Line]({{ url_for('static', filename='images/xintiao.gif') }})

### Design

Based on the successful construction of the original NorR-YFP fusion protein in the first cycle, we conducted a detailed analysis of its potential limitations. We hypothesized that the length of the Linker connecting the NorR sensing domain and the YFP reporter domain is a key but yet unoptimized parameter that affects the performance of the sensor. An inappropriate Linker length may lead to spatial steric hindrance between the domains or a low efficiency in conformational change transmission.

Therefore, we have designed a series of Linker variants, aiming to systematically study the influence of Linker length on the performance of the sensor. The specific strategy is to construct a set of variants that are shorter and longer than the original Linker, in order to find the optimal length that can maximize the signal response amplitude. The detailed DNA sequences of all the designed Linkers are shown in the Tab.1.

Tab.1 DNA sequences of Linkers

| Linker | Sequences |
| --- | --- |
| cycle1 | GCGGGCTACAACAGCGACAACGTCTATATC |
| cycle2-1 | GCAGGC |
| cycle2-2 | GCAGGCTACAACAGCGAC |
| cycle2-3 | GGAGGAGGAGGATCCGGAGGAGGAGGATCCGGAGGAGGAGGATCC |

### Build

To construct the aforementioned Linker variants, we employed the double restriction enzyme digestion method. We used specific restriction endonucleases to digest the vector backbone and the insertion fragments containing different Linker sequences, then purified, ligated, and transformed the ligated products into competent cells. However, after screening colonies by colony PCR and DNA sequencing verification on a large number of transformants, the results indicated that no correct recombinant plasmids of any variant could be obtained. All sequencing results showed phenomena such as vector self-ligation, incorrect insertion, or non-specific recombination, indicating a systematic failure in the cloning process.

### Test - Not Completed

Due to the failure to obtain any correct plasmid constructs during the "construction" phase, the subsequent protein expression, purification, and functional testing of this project cannot be carried out.

### Learn

Although this cycle failed to obtain functional Linker variants due to the failure in constructing double-cutting vectors, the work at this stage verified at the design level the significant potential of optimizing the Linker system to enhance the performance of the NorR-YFP probe. The gradient variants designed based on the Linker length provided a clear experimental foundation for the precise regulation of the sensor's performance. This technical bottleneck also prompted us to recognize that adopting more efficient cloning methods (such as Golden Gate assembly) is crucial for advancing such optimization studies. In the future, after overcoming the construction obstacles, the targeted modification of the Linker region will remain a core strategy for enhancing the probe's sensitivity and stability, and it is worthy of being pursued as a key direction.

![References Icon](https://static.igem.wiki/teams/5762/description/slice2.webp)

## References

![Title Line]({{ url_for('static', filename='images/xintiao.gif') }})

[1] Farah, C., Michel, L. & Balligand, JL. Nitric oxide signalling in cardiovascular health and disease. Nat Rev Cardiol 15, 292–316 (2018).

[2] Garthwaite, J. Glutamate, nitric oxide and cell-cell signalling in the nervous system. TINS. 1991, 14: 60-67

[3] Moncada, S., Palmer, R.M., Higgs, E.A. Nitric oxide: physiology, pathophysiology and pharmacology. Pharmacol. Rev. 1991, 43:109-142.

[4] Forman, H.J., Fukuto, J.M., Torres, M. Redox signaling: thiol chemistry defines which reactive oxygen and nitrogen species can act as second messengers. Am. J. Physiol. Cell Physiol. 2004, 287:C246-C256.

[5] Lee, Y. Y., Shearer, N., and Spiro, S. (2006) Microbiology 152, 1461–1470

{% endblock %}

{% extends "layout.html" %}
{% block title %}Wetlab Experiments{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
{% block page\_content %}

![Project Banner](https://static.igem.wiki/teams/5762/description/slice6.webp)

# WETLAB EXPERIMENTS

![Title Line](https://static.igem.wiki/teams/5762/description/frame445.webp)

![Heart](https://static.igem.wiki/teams/5762/description/slice1.webp)

PCR

Agarose gel electrophoresis

Gel recovery

Enzymatic digestion

The connection of the YFP

Transformation

Protein expression

Protein Purification

Temperature Stability Assay

pH Stability Assay

Concentration-Dependent Response Assay

Specificity Test

Thawing

Passaging

Transfection

Artificial plasma test

![Icon](https://static.igem.wiki/teams/5762/description/slice2.webp)

## PCR

![Heartbeat]({{ url_for('static', filename='images/xintiao.gif') }})

The PCR system was formulated according to the table below

| Reagent | Volume(μL) |
| --- | --- |
| DNA | 1 |
| Primer-F | 0.5 |
| Primer-R | 0.5 |
| 2X Master Mix | 5 |
| ddH2O | 3 |

Put in PCR instrument, PCR program

| Temperature | Time |
| --- | --- |
| 98°C | 30s |
| 98°C | 10s |
| 65°C | 30s |
| 72°C | 21s |
| 72°C | 10min |

Cycle：35

![Icon](https://static.igem.wiki/teams/5762/description/slice2.webp)

## Agarose gel electrophoresis

![Heartbeat]({{ url_for('static', filename='images/xintiao.gif') }})

1) Weigh 0.5g agarose and add to 50ml 1X TAE.

2) Microwave until completely melted.

3) Cool to 50 ° C to 60 ° C and add 3μL DNA stain.

4) Inverted board.

5) After cooling, sample in sequence.

6) 130V 30min.

7) After the process is completed, place the gel under a UV lamp, excise the portion of the gel containing the target fragment, and place the excised gel into a 1.5 mL EP tube.

![Icon](https://static.igem.wiki/teams/5762/description/slice2.webp)

## Gel recovery

![Heartbeat]({{ url_for('static', filename='images/xintiao.gif') }})

1) Add 200 μL of PN to the colloid and incubate in a water bath at 50°C for 10 minutes.

2) Add 500 μL of BL to the chromatography column and place it in a centrifuge at 12,000 rpm for 1 minute. Discard the eluate.

3) Transfer the liquid in the EP tube to the chromatography column and let it stand at room temperature for 2 minutes. Then place it in the centrifuge at 12,000 rpm for 1 minute and discard the eluate.

4) Add 600 μL of PW to the column and place it in the centrifuge at 12,000 rpm for 1 minute. Discard the eluate.

5) Repeat the previous step once.

6) Place the tube in the centrifuge at 12,000 rpm for 2 minutes without any liquid, then open the lid and let it stand for 5 minutes.

7) Place the chromatography column in a new EP tube and add 30 μL of ddH2O to the column. Let it stand for 2 minutes and then place it in the centrifuge at 12,000 rpm for 2 minutes. Collect the eluate.

8) Add the eluate back to the chromatography column and place the system in the centrifuge at 12,000 rpm for 2 minutes. Discard the chromatography column and keep the EP tube.

![Icon](https://static.igem.wiki/teams/5762/description/slice2.webp)

## Enzymatic digestion

![Heartbeat]({{ url_for('static', filename='images/xintiao.gif') }})

1) Prepare the enzyme digestion system

| Reagent | Volume(μL) |
| --- | --- |
| pRESTa | 3 |
| Kpn I | 0.2 |
| BamH I | 0.2 |
| rcut smart buffer | 2 |
| ddH2O | 13μL |

2） 37℃ 40min

![Icon](https://static.igem.wiki/teams/5762/description/slice2.webp)

## The connection of the YFP and pRSETa vectors

![Heartbeat]({{ url_for('static', filename='images/xintiao.gif') }})

1）Prepare the connection system（10μL）

| Reagent | Volume(μL) |
| --- | --- |
| pRESTa | 1.5 |
| YFP | 3.5 |
| T4 DNA Ligase | 0.5 |
| T4 DNA Ligase buffer | 1 |
| ddH2O | 3.5 |

2） 4℃ for the whole night

![Icon](https://static.igem.wiki/teams/5762/description/slice2.webp)

## Transformation

![Heartbeat]({{ url_for('static', filename='images/xintiao.gif') }})

1) Add 10 μL of plasmid to the EP tube.

2) Add 20 μL of competent cells to the EP tube.

3) Place the system on ice and let it stand for 30 minutes.

4) After completion, quickly transfer the system to a water bath and perform a heat shock at 42°C for 35 seconds.

5) Add 500 μL of LB culture medium to the system and place the entire system in a shaker for 1 hour.

6) After the previous section is completed, place the entire reaction system on the centrifuge and centrifuge it at 4000 rpm for 5 minutes.

7) After completion, discard the supernatant of the system, 450 μL, and then use the remaining liquid to resuspend and mix the precipitate before coating the plate.

![Icon](https://static.igem.wiki/teams/5762/description/slice2.webp)

## Protein expression

![Heartbeat]({{ url_for('static', filename='images/xintiao.gif') }})

1) Add 500 μL of medium and 0.5 μL of ampicillin to a 1.5 mL centrifuge tube, pick a single colony, and incubate at 37°C with shaking at 220 rpm for 2-3 hours.

2) Inoculate the bacteria from the 1.5 mL centrifuge tube into a 50 mL centrifuge tube containing 20 mL of medium, add 20 μL of ampicillin, and incubate at 37°C with shaking at 220 rpm. Measure the OD value using a microplate reader. When the OD value reaches 0.5-0.8, take out the bacterial culture and allow it to cool. Add 20 μL of IPTG (concentration 0.42 M), and induce at 18°C with shaking at 220 rpm for 24 hours at low temperature.

3) After 24 hours, centrifuge at 4000 rpm for 5 minutes, resuspend in 1 mL of HEPES Buffer to obtain a protein suspension, and store at -80°C.

![Icon](https://static.igem.wiki/teams/5762/description/slice2.webp)

## Protein Purification

![Heartbeat]({{ url_for('static', filename='images/xintiao.gif') }})

1) Lysate Preparation: The protein suspension is sonicated on ice at 20–30% power for 5 minutes, then centrifuged at 12,000 rpm for 10 minutes. The target protein remains in the supernatant.

2) Magnetic Bead Activation: Transfer His-Tag magnetic beads to a 1.5 mL tube, add 600 µL of HEPES Buffer, and mix thoroughly using a 1 mL pipette. Separate the beads from the buffer using a magnetic stand and discard the buffer. Repeat this washing process three times. After the final wash, remove all buffer.

3) Protein Binding: Add the protein supernatant to the washed magnetic beads and incubate at 4 °C for 3 hours with gentle mixing to facilitate binding.

4) Elution: Elute the bound protein using elution buffers containing imidazole at concentrations of 50 mM, 100 mM, 300 mM, and 500 mM.

5)Sample Collection: Retain the eluted fractions from the 100 mM and 300 mM imidazole elutions for further analysi

![Icon](https://static.igem.wiki/teams/5762/description/slice2.webp)

## Temperature Stability Assay

![Heartbeat]({{ url_for('static', filename='images/xintiao.gif') }})

1) Prepare a 1 μmol/L protein solution in HEPES buffer (or appropriate buffer).

2) Add 100 μL of the protein solution to each well of a 96-well black microplate.

3) Place the plate in a pre-equilibrated microplate reader and measure fluorescence intensities at 420 nm and 485 nm under different temperatures (20 °C, 25 °C, 30 °C, 35 °C, 40 °C).

4) Add Furoxan to each well to a final concentration of 100 μM.

5) Immediately monitor fluorescence changes at 420 nm and 485 nm under each temperature condition.

6) Calculate the fluorescence ratio (F420/F485) over time.

![Icon](https://static.igem.wiki/teams/5762/description/slice2.webp)

## pH Stability Assay

![Heartbeat]({{ url_for('static', filename='images/xintiao.gif') }})

1) Prepare 1 μmol/L protein solutions in buffers with different pH values (7.0, 7.2, 7.4, 7.6, 7.8, 8.0). Use appropriate buffers (e.g., HEPES, Tris, phosphate) adjusted to target pH.

2) Add 100 μL of each pH-adjusted protein solution to a 96-well black microplate.

3) Measure initial fluorescence at 420 nm and 485 nm.

4) Add Furoxan to a final concentration of 100 μM in each well.

5) Record fluorescence changes at both wavelengths and calculate the F420/F485 ratio.

![Icon](https://static.igem.wiki/teams/5762/description/slice2.webp)

## Concentration-Dependent Response Assay

![Heartbeat]({{ url_for('static', filename='images/xintiao.gif') }})

1) Add 100 μL of 1 μmol/L protein solution to each well of a 96-well black microplate.

2) Measure baseline fluorescence at 420 nm and 485 nm.

3) Add Furoxan to final concentrations of 0, 1, 10, and 100 μM (using serial dilutions if needed).

4) Incubate for 5–10 minutes and record fluorescence intensities.

5) Calculate the fluorescence ratio (F420/F485) for each Furoxan concentration.

![Icon](https://static.igem.wiki/teams/5762/description/slice2.webp)

## Specificity Test

![Heartbeat]({{ url_for('static', filename='images/xintiao.gif') }})

1) Add 100 μL of 1 μmol/L protein solution to each well of a 96-well black microplate.

2) Record initial fluorescence at 420 nm and 485 nm.

3) Add different analytes (Furoxan, CO, H₂O₂, NaNO₂, NaHS) to a final concentration of 100 μM in separate wells.

4) Incubate for 10–15 minutes at room temperature.

5) Measure fluorescence intensities at both wavelengths and calculate F420/F485 ratios.

6) Include a negative control (buffer only) and positive control (Furoxan) in each experiment.

![Icon](https://static.igem.wiki/teams/5762/description/slice2.webp)

## Thawing

![Heartbeat]({{ url_for('static', filename='images/xintiao.gif') }})

Quickly remove the cryovial from liquid nitrogen and thaw it in a 37°C water bath. Transfer the cell suspension to a 15 mL centrifuge tube containing 5 mL of pre-warmed complete medium. Centrifuge at 1000 rpm for 5 minutes. Discard the supernatant, resuspend the cell pellet in fresh medium, and seed it into a T25 culture flask. Gently mix to ensure even distribution.

![Icon](https://static.igem.wiki/teams/5762/description/slice2.webp)

## Passaging

![Heartbeat]({{ url_for('static', filename='images/xintiao.gif') }})

a) Passage the cells when they reach 80-90% confluency. Avoid overgrowth.

b) Aspirate and discard the old medium.

c) Gently rinse the cells with 3-5 mL of DPBS (Dulbecco's Phosphate-Buffered Saline) to remove residual serum and dead cells.

d) Aspirate the DPBS and add 1-2 mL of 0.25% Trypsin-EDTA (for a T25 flask). Gently swirl to ensure even coverage of the cell layer.

e) Incubate at 37°C for 1-2 minutes, or observe under a microscope. Proceed immediately when the cells round up and gaps appear between them.

f) Add 2-3 volumes of complete medium (containing FBS to inhibit trypsin activity). Gently pipette the solution to dislodge all cells and form a single-cell suspension.

g) Transfer the cell suspension to a centrifuge tube and centrifuge at 1000 rpm for 5 minutes.

h) Discard the supernatant and resuspend the cell pellet in fresh medium.

i) Passage the cells at a 1:5 to 1:10 split ratio (e.g., from a T25 flask to a T75 flask, or from one T75 flask to three T75 flasks).

j) Add fresh medium, gently swirl to mix, and return the flask to the incubator.

![Icon](https://static.igem.wiki/teams/5762/description/slice2.webp)

## Transfection

![Heartbeat]({{ url_for('static', filename='images/xintiao.gif') }})

1) Take a sterile 1.5 mL centrifuge tube and sequentially add the following:

• Ultrapure water: to a final volume of 125 μL

• Plasmid DNA mixture: 3.0 μg (diluted with ultrapure water)

• 2.5M CaCl₂: 12.5 μL

Gently mix.

2) Take another sterile 1.5 mL centrifuge tube and add 125 μL of 2× HBS solution.
Using a pipette, slowly add the DNA/CaCl₂ mixture dropwise into the 2× HBS solution while gently blowing to mix (add slowly, drop by drop).

3) After adding, immediately pipette up and down gently 3-5 times to mix. The solution should appear slightly turbid without visible large aggregates.

4) Incubate at room temperature for 15-20 minutes. During this time, fine DNA-calcium phosphate complexes will form, and the solution will become slightly clearer.

5) Evenly add the 250 μL transfection complex mixture dropwise into the cell culture medium in the 6-well plate, gently rocking the plate while adding.

6) Return the cell culture plate to the 37°C, 5% CO₂ incubator.

7) After 6-8 hours (or following overnight incubation), observe under a microscope. The cells should be covered with fine black particles. At this point, replace the medium with fresh complete medium (2 mL/well). This step reduces toxicity and improves cell viability.

8) Continue culturing for 48-72 hours, then collect the supernatant containing viral particles (if performing virus packaging).

![Icon](https://static.igem.wiki/teams/5762/description/slice2.webp)

## Artificial plasma test

![Heartbeat]({{ url_for('static', filename='images/xintiao.gif') }})

1) Solution Preparation:

• Using artificial plasma, dilute the Furoxan stock solution to 2× the target final concentration. Specifically:

- 0 μM group: Artificial plasma + corresponding volume of solvent (e.g., DMSO).

- 200 μM group: Dilute to 400 μM (2× the final concentration of 100 μM).

- 1000 μM group: Dilute to 2000 μM (2× the final concentration of 500 μM).

- 2000 μM group: Dilute to 4000 μM (2× the final concentration of 1000 μM).

• Using artificial plasma, dilute the NO fluorescent probe protein to its working concentration (as recommended in the probe manual).

2) Sample Loading and Reaction:

• Take four 1.5 mL centrifuge tubes and label them as 0, 100, 500, and 1000 μM.

• To each tube, add 50 μL of the corresponding 2× Furoxan solution.

• Then, add 50 μL of the diluted probe protein working solution to each tube.

• Vortex immediately to mix thoroughly and start the timer.

3) Incubation and Detection

• Transfer the entire reaction mixture to a black 96-well plate (or cuvette).

• Incubate the reaction system at 37°C in the dark for a specific duration (e.g., 30 minutes, or as required by the experimental design).

• Place the plate into the blue light excitation instrument.

• Set the excitation/emission wavelengths.

• Detect and record the fluorescence intensity of each well.

{% endblock %}

{% extends "layout.html" %}
{% block title %}HUMAN Result{% endblock %}
{% block lead %}Results{% endblock %}
{% block page\_content %}

![Project Banner](https://static.igem.wiki/teams/5762/description/slice6.webp)

# RESULTS

![Title Line](https://static.igem.wiki/teams/5762/description/frame445.webp)

![Heart](https://static.igem.wiki/teams/5762/description/slice1.webp)

Overview

Successful Construction

Heterologous Expression

Functional Validation of the Protein

![Icon](https://static.igem.wiki/teams/5762/description/slice2.webp)

## Overview

![Heartbeat]({{ url_for('static', filename='images/xintiao.gif') }})

This project aims to develop a novel, high-performance genetically encoded fluorescent probe for nitric oxide (NO) using protein engineering strategies. The probe is designed based on the regulatory domain of the bacterial transcription factor NorR, which will be rationally engineered and evolutionarily optimized through directed evolution before being fused with a fluorescent protein.

This approach enables highly sensitive and specific fluorescent responses to changes in NO concentration. Furthermore, using molecular biology techniques, the probe will be precisely targeted to specific subcellular locations—such as the cytoplasm and mitochondria—to achieve in situ, high spatiotemporal resolution imaging of NO dynamics in living cells.

The successful development of this probe will provide a crucial tool for elucidating the central role of NO in physiological and pathological processes. It also holds promise for applications in early diagnosis of suboptimal health conditions, evaluation of therapeutic efficacy, and the development of targeted drugs, thereby advancing the field of precision medicine.

![Icon](https://static.igem.wiki/teams/5762/description/slice2.webp)

## Successful Construction

![Heartbeat]({{ url_for('static', filename='images/xintiao.gif') }})

To develop novel nitric oxide (NO) biosensors, we inserted the YFP, GFP, and mCherry sequences into the pRSETa-NorR vector using BamHI and KpnI restriction cloning (see Fig. 1 for construction workflow). This engineered vector provides an N-terminal 6×His tag to facilitate subsequent protein purification and contains a T7 promoter for high-level expression in host cells.

![Vector construction workflow](https://static.igem.wiki/teams/5762/result/result1.webp)

Fig.1 Vector construction of NorR-YFP\_pRSETa, NorR-mCherry\_pRSETa and NorR-GFP\_pRSETa

A: PCR of YFP sequences; B: PCR of mCherry sequence; C: PCR of GFP sequences; D: The pRSETa vector after double enzyme digestion.

Following transformation of the ligation product into DH5α competent cells, overnight cultivation yielded abundant transformant colonies (Fig.2). Preliminary screening of the clones was performed by colony PCR using vector-specific primers (T7 promoter and terminator primers). As shown in Fig.3, all randomly selected clones produced a single DNA band of the expected size, indicating preliminary success in the insertion of the target fragment into the vector.

![Screening plate for transformation](https://static.igem.wiki/teams/5762/result/result2.webp)

Fig.2 The screening plate for the transformation of recombinant plasmid by DH5α

![PCR verification of recombinant plasmid](https://static.igem.wiki/teams/5762/result/result3.webp)

Fig.3 PCR Verification of the recombinant plasmid

To obtain final confirmation, the purified plasmid constructs were subjected to Sanger sequencing. The sequencing results indicated that no mutations were introduced into the recombinant vectors, confirming the successful construction of the plasmids.

![Icon](https://static.igem.wiki/teams/5762/description/slice2.webp)

## Protein Fusion

![Heartbeat]({{ url_for('static', filename='images/xintiao.gif') }})

Based on the successfully constructed plasmids, the NorR-fluorescent protein fusion was expressed in E. coli BL21(DE3) cells. The bacterial culture was induced with 0.42 mM IPTG and incubated at 18°C with shaking at 220 rpm for 24 hours. After centrifugation, the cell pellets were resuspended in HEPES buffer and stored at –80°C. For purification, the resuspended cells were disrupted by sonication followed by centrifugation. The supernatant was incubated with nickel magnetic beads at 4°C for 3 hours to allow binding of the His-tagged fusion protein. Stepwise elution was performed using imidazole concentrations ranging from 50 to 500 mM. Fractions eluted with 100 mM and 300 mM imidazole were collected for further analysis. The purified proteins are shown in Fig. 4.

![Purification results of recombinant proteins](https://static.igem.wiki/teams/5762/result/result5.webp)

Fig.4 The purification results of recombinant proteins NorR-YFP, NorR-GFP and NorR-mCherry

![Icon](https://static.igem.wiki/teams/5762/description/slice2.webp)

## Functional Validation of the Protein

![Heartbeat]({{ url_for('static', filename='images/xintiao.gif') }})

## 4.1Functional Comparison of Different Fluorescent Probes

After obtaining the three fluorescent probes—YFP, GFP, and mCherry—we systematically evaluated their nitric oxide (NO) binding capabilities to identify the optimal performer. YFP and GFP were analyzed using a dual-excitation ratiometric method, measuring fluorescence intensity at 515 nm under excitations of 420 nm and 485 nm, followed by ratio calculation. mCherry was assessed by direct fluorescence intensity measurement under 590 nm excitation and 645 nm emission. Each probe was incubated with varying concentrations of the NO donor Furoxan. The ratio of fluorescence intensity after adding the NO donor to that under baseline (zero NO) conditions was used as the evaluation metric (also applied in subsequent experiments). As shown in Fig.5, with increasing Furoxan concentration, both YFP and GFP exhibited significant ratiometric changes in a dose-dependent manner, eventually reaching saturation. In contrast, mCherry showed no notable change in fluorescence ratio across the concentration range, indicating its inability to effectively respond to NO. Further comparison revealed that YFP demonstrated a markedly greater response amplitude than GFP, suggesting higher sensitivity. Therefore, YFP was selected as the NO-sensing probe for subsequent studies.

![Fluorescence response curves](https://static.igem.wiki/teams/5762/result/result6.webp)

Fig.5 Fluorescence response curves of the YFP-, GFP- and mCherry-NO probes

## 4.2Functional Validation of the NorR-YFP Fluorescent Probe

After identifying YFP as the optimal probe, we conducted systematic functional validation of the NorR-YFP fusion protein to comprehensively evaluate its performance under practical application conditions. Key parameters including pH stability, thermal stability, concentration-dependent response, binding specificity, detection reliability in plasma, and subcellular localization capability were assessed to ensure the probe's accuracy and reliability in complex biological environments.

First, the thermal and pH stability of NorR-YFP were examined. Each reaction system contained 100 μL of 1 μmol/L NorR-YFP protein. The experimental group was treated with 100 μM Furoxan (NO donor), while the control group received an equal volume of buffer. As illustrated in Fig.6, a significant increase in the fluorescence ratio was observed after Furoxan-induced NO release. The probe exhibited consistent responsiveness within the pH range of 7.2–8.0 and the temperature range of 20–40°C, demonstrating robust environmental adaptability and detection stability.

![pH value and temperature stability of NorR-YFP](https://static.igem.wiki/teams/5762/result/result7.webp)

Fig.6 pH value and temperature stability of NorR-YFP

To assess the binding specificity of NorR-YFP, we introduced 100 μM of Furoxan, CO, H₂O₂, NaNO₂, and NaHS, respectively, and measured corresponding changes in the fluorescence ratio. As shown in Fig. 7, a rapid and significant increase in the fluorescence ratio occurred only in the Furoxan group, with no notable changes observed in other groups, confirming highly specific responsiveness of the probe to nitric oxide (NO).

![Specificity assessment of NorR-YFP](https://static.igem.wiki/teams/5762/result/result8.webp)

Fig.7 Specificity assessment of NorR-YFP toward various reactive species

To establish the quantitative relationship between fluorescence intensity of NorR-YFP and NO concentration, the protein was treated with different concentrations of the NO donor Furoxan. A solution of 100 μL of 1 μmol/L protein was used, and changes in the fluorescence ratio were dynamically monitored over 40 minutes. A positive correlation was observed between Furoxan concentration and the fluorescence signal of the NorR-YFP sensor (Fig. 8), indicating a clear dose-response relationship to NO.

![Real-time Monitoring of NorR-YFP Fluorescence Kinetics](https://static.igem.wiki/teams/5762/result/result9.webp)

Fig.8 Real-time Monitoring of NorR-YFP Fluorescence Kinetics upon Furoxan Stimulation

In plasma NO detection experiments, 50 μL of artificial plasma samples were mixed with an equal volume of NO donor Furoxan at varying concentrations (0–1000 μM), followed by the addition of a quantified amount of probe protein (Fig.9A). Fluorescence intensity increased in a dose-dependent manner with rising Furoxan concentration (Fig.9B), demonstrating the probe's sensitive and reliable response to changes in NO concentration within a complex matrix and validating its feasibility for quantitative detection in such environments.

![Dose-dependent response of probe protein fluorescence intensity](https://static.igem.wiki/teams/5762/result/result10.webp)

Fig.9 The dose-dependent response of probe protein fluorescence intensity in artificial plasma to the concentration of NO donor furoxan

To investigate the functionality of the NO-sensitive probe NorR-YFP across different cellular compartments, we targeted it to the cytoplasm, mitochondria, and nucleus, respectively. Expression and validation were carried out in human embryonic kidney 293T cells (cell culture process shown in Fig. 10).

![293T cells at different stages](https://static.igem.wiki/teams/5762/result/result11.webp)

Fig.10 293T cells at different stages observed under a microscope

A: Resuscitated 293T cells; B: 293T cells after 1-day culture; C:The detached 293T cells; D:293T cells grown in large petri dishes (at low density)；E: 293T cells at optimal density；F: 293T cells after transfection treatment.

At 48 hours post-transfection, strong YFP fluorescence signals were observed in all three groups under fluorescence microscopy (Fig. 11), indicating successful expression of the NorR-YFP fusion protein in 293T cells. More importantly, the fluorescence signals exhibited distinct distribution patterns consistent with the intended subcellular targeting strategies. These results confirm the successful construction of NorR-YFP probes specifically localized to the cytoplasm, mitochondria, and nucleus. These tool probes demonstrated efficient expression and accurate localization in 293T cells, providing a solid foundation for real-time and specific monitoring of NO dynamics within specific subcellular compartments in subsequent studies.

![Subcellular localization of the NO sensor NorR-YFP](https://static.igem.wiki/teams/5762/result/result12.webp)

Fig.11 The subcellular localization of the NO sensor NorR-YFP

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