iGEM 2019 — Team BSC_United

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# Team:BSC United/Notebook

iGEM BSC\_United

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## MINILOSS](https://2019.igem.org/Team%3ABSC_United)

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* Team
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  + [Special Thanks](https://2019.igem.org/Team%3ABSC_United/Thanks)
* Project
  + [Description](https://2019.igem.org/Team%3ABSC_United/Description)
  + [Experiments](https://2019.igem.org/Team%3ABSC_United/Experiments)
  + [Notebook](https://2019.igem.org/Team%3ABSC_United/Notebook)
  + [Results](https://2019.igem.org/Team%3ABSC_United/Results)
  + [Demonstrate](https://2019.igem.org/Team%3ABSC_United/Demonstrate)
* Design
  + [Overview](https://2019.igem.org/Team%3ABSC_United/Design)
  + [Biological Part](https://2019.igem.org/Team%3ABSC_United/Bio)
  + [Sensor](https://2019.igem.org/Team%3ABSC_United/Sensor)
  + [Organ Chip](https://2019.igem.org/Team%3ABSC_United/Organ_Chip)
  + [Hardware](https://2019.igem.org/Team%3ABSC_United/Hardware)
  + [Software](https://2019.igem.org/Team%3ABSC_United/Software)
  + [Model](https://2019.igem.org/Team%3ABSC_United/Model)
* [Parts](https://2019.igem.org/Team%3ABSC_United/Parts)
* [Safety](https://2019.igem.org/Team%3ABSC_United/Safety)
* HP
  + [Human Practices](https://2019.igem.org/Team%3ABSC_United/Human_Practices)
  + [Education&Engagement](https://2019.igem.org/Team%3ABSC_United/Public_Engagement)
* [Judging Form](https://igem.org/2019_Judging_Form?team=BSC_United)

# PROJECT

![](https://static.igem.org/mediawiki/2019/4/41/T--BSC_United--Notebook.svg)

## NOTEBOOK

* [◆ April](#p1)
* [◆ May](#p2)
* [◆ June](#p3)
* [◆ July](#p4)
* [◆ August](#p5)
* [◆ September](#p6)
* [◆ October](#p7)

### April

##### Week1 Apr 1st-7th

Recruit BSC\_United members. 14 people from 10 cities worldwide joined the team for the iGEM 2019 competition.

##### Week2&3 Apr 8th-21st

Brainstorming. Through the discussion, team decided to build an in vitro model as the first step for exploration of novel diabetic treatments, and our ultimate goal is to cure diabetes by gut microbiota therapy.

##### Week4&5 Apr 22nd-May 3rd

Discussing details about the design. Between fish and rats, we choose fish as the experimental subject.

### May

##### Week6 May 4th-12th

The basic plan was established, and the equipment used has been chosen. Electrochemical sensor is chosen for its high sensitivity, reliable reproducibility, and simple operation.
Through market investigation, we found out that most blood glucose level detectors employ the one-time test paper slips and could not fulfill our need to continuously monitor blood glucose level; thus, we decided to create our own electrochemical sensors.
Considering the size of sensor, we rejected the previous plan to employ fish as the experimental subject.

##### Week7 May 13th-19th

Consult documents and design the electrochemical sensor. We design our electrochemical sensor with CAD software and later simulate and verify the design with comsol software. After the result indicates that our electrochemical sensor is functionally operational, we finally produce home-made electrochemical sensors with screen printing. Organ-on-a-chip is planned to be the test object.

##### Week8 May 20th-26th

Consult documents about organ-on-a-chip; learn basics in Synbio and determine the synthetic microbial chassis. Conduct an interview with diabetes expert Dr. Pingsheng Liu.

##### Week9 May 27th-Jun 2th

Work suspended for exams.

### June

##### Week10&11 Jun 3rd-16th

Design the biological experiment and organ-on-a-chip.
For the experiment, our team consulted documentation, prepare E. coli and B. subtilis strains for the experiment, and design the experimental procedures.
For organ-on-a-chip, our team studied the mechanism of insulin synthesis and release. Knowing that liver is essential in glucose reduction, our team investigated basic functional unit of the liver and design organ-on-a-chip.

##### Week12&13 Jun 17th-30th

Our team analyzed the test requirement, designed analog and digital modules, determined the selection of modules, and started the hardware design documents and hardware schematic framing.

### July

##### Week14&15 Jul 1st-14th

Study laboratory safety protocols and conduct safety training, prepare needed materials. Electric circuit scheme and PCB design are finalized.

##### Week16 Jul 15th-21st

Produce the first batch of electrochemical sensors. Conduct basic biological experiments, and familiarize ourselves with experiment tools. Let factory produce circuit boards and weld chips and modules, and start the development of device debugging software. Have the first communication with UCAS-China. Learn to use laser engraving machine and micro processing technology to prepare for organ chip.

##### Week17 Jul 22nd-28th

Test the first batch of electrochemical sensors at the electrochemical workstation; the result shows that these sensors cannot meet our requirement, so we decided to refine them and make another batch of sensors. In laboratory, we constructed plasmids and conducted subcloning experiments, including purification, restriction digestion, gel extraction, ligation, transformation, and colony PCR.

##### Week18 Jul 29th-Aug 4

Produce the second batch of electrochemical sensors; our team added potassium ferrocyanide. The CV curve of the second batch of sensors is better than that of the first batch. We also began to produce the third batch.
The vectors were successfully constructed. It was verified at the DNA level that the plasmid is transformed into the B. subtilis.
An issue in organ-on-a-chip: the liver cells didn’t attach to the PET membrane. We consulted documents and conducted surface specialization in order to increase cell adhesion.
Interview doctors and patients at hospital, disseminate knowledge in Synbio and iGEM, as well as our own project.

### August

##### Week19 Aug 5th-11th

Specialization of team members. Discuss logo design. Cultivate liver cancer cells, fermentation of engineering bacteria, extract protein and conduct SDS-PAGE electrophorosis to verify expression of HPI gene at protein level. The third batch of electrochemical sensors meets the requirement of our design.

##### Week20 Aug 12th-18th

Cultivate liver cancer cells from different sources and select strong ones for primary experiments. Construct the organ-on-a-chip.

##### Week21 Aug 19th-25th

Electric circuit was produced and we started software debugging. Test conditions of planting liver cancer cells into organ chip.

##### Week22 Aug 26th-31st

Continue cultivating liver cancer cells in organ chip. Keep debugging the device, finish debugging BT module, continue debugging ADC module

### September

##### Week23 Sep 1st-8th

Work suspended due to beginning of school.

##### Week24 Sep 9th-15th

Liver cancer cells are successfully cultivated in organ-on-a-chip. Software debugging of electronic circuit equipment is completed.

##### Week25 Sep 16th-22nd

Start composing wiki materials.Assemble sensors on organ-on-a-chip. The liver cells are implanted into organ-on-a-chip. After 24-hours cultivation, our team observe the attachment of cells, and finally add engineering bacteria and sample. Then we would test the complete version of organ-on-a-chip at electrochemical workstation. Test connection between device and app.

##### Week26 Sep 23rd-30th

Assemble sensors on organ-on-a-chip , and test the equipment.

### October

##### Week27 Oct 1st-7th

Days for rest

##### Week28 Oct 8th-14th

Compose and revise the wiki documents, evaluate and submit Judging Form.

##### Week29 Oct 15th-21st

Revise the wiki documents.

![](https://static.igem.org/mediawiki/2019/3/39/T--BSC_United--arrow.svg)

Ⓒ 2019 iGEM BSC\_United


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# Team:BSC United/Experiments

iGEM BSC\_United

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## MINILOSS](https://2019.igem.org/Team%3ABSC_United)

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* Project
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* Design
  + [Overview](https://2019.igem.org/Team%3ABSC_United/Design)
  + [Biological Part](https://2019.igem.org/Team%3ABSC_United/Bio)
  + [Sensor](https://2019.igem.org/Team%3ABSC_United/Sensor)
  + [Organ Chip](https://2019.igem.org/Team%3ABSC_United/Organ_Chip)
  + [Hardware](https://2019.igem.org/Team%3ABSC_United/Hardware)
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* HP
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  + [Education&Engagement](https://2019.igem.org/Team%3ABSC_United/Public_Engagement)
* [Judging Form](https://igem.org/2019_Judging_Form?team=BSC_United)

# PROJECT

![](https://static.igem.org/mediawiki/2019/c/c3/T--BSC_United--Experiments.svg)

## EXPERIMENTS

### Gene Fragment PCR Amplification

The PCR is an effective procedure for generating large quantities of specific DNA sequence in vitro. This amplification is achieved by a three-step cycling process.

|  |  |
| --- | --- |
| **Reaction Mixture** | **20µL** |
| 2x Mix (Taq DNA polymerase (0.05 U/µL), reaction buffer, 4 mM MgCl2, and 0.4 mM of each dNTP) | 10µL |
| ddH2O | 7µL |
| Template DNA | 2µL |
| Primer R | 0.5µL |
| Primer F | 0.5µL |

1. Make the reaction mixture with the above recipe in 1.5 mL Eppendorf tubes
2. Use microcentrifuge to make the liquid in Eppendorf tubes homogenous
3. Put the Eppendorf tubes into the PCR machine and start the thermocycling process with the parameters below:

1. 94˚C for 3min
2. b.94˚C for 30s
3. 48˚C for 30s
4. 72˚C for 30s
5. Cycle for 30 times
6. 72˚C for 10min
7. 4˚C storage

### Colony PCR

|  |  |
| --- | --- |
| **Reaction Mixture** | **20µL** |
| 2x Mix (Taq DNA polymerase (0.05 U/µL), reaction buffer, 4 mM MgCl2, and 0.4 mM of each dNTP) | 10µL |
| ddH2O | 7µL |
| DNA Dilution | 2µL |
| Primer R | 0.5µL |
| Primer F | 0.5µL |

1. Pick a single colony with pipette tip from the solid growth on the plate
2. Dilute them with 5µl ddH2O in an Eppendorf tube
3. Make the master mix with 2µL of the diluted DNA
4. Put the whole mixture in Eppendorf tubes into the PCR machine and start the thermocycling process with the parameters below:

1. 94˚C for 3min
2. b.94˚C for 30s
3. 48˚C for 30s
4. 72˚C for 30s
5. Cycle for 30 times
6. 72˚C for 10min
7. 4˚C storage

### Gel Preparation

|  |  |
| --- | --- |
| Agarose | 1.2g |
| 0.5x TAE | 100mL |
| Goldview I (Nuclear Staining Dye) | 3µL |

1. Weigh the agarose
2. Add TAE and agarose together
3. Heat the mixture in the microwave
4. Cool for 30min
5. Add Goldview I
6. Pour it into the mold
7. Cool for 30min

### Gel Electrophoresis

**Material:** TAE buffer

1. Immerse the gel into the chamber
2. Pour the TAE buffer until it completely covers the gel
3. Add 2µL of DNA into each well
4. Put the marker into the leftmost lane
5. Connect the electrodes
6. Turn on the power supply to 90v for 30 minutes
7. Put the gel under UV light and observe

### Gel Extraction

1. Cut the desired gel fragment and place it into a tube
2. Add equal weight of Binding Buffer as the gel (XP2)
3. Vortex
4. Place the tube into 55˚C water bath for 10 minutes
5. Vortex
6. Add 300µL of Binding Buffer into the tube and centrifuge it for 1 minute at 12000rpm
7. Discard the filtrate
8. Add 700µL of SPW Wash Buffer into the tube and centrifuge it for 1 minute at 12000rpm
9. Discard the filtrate
10. Repeat steps 9~10 once
11. Centrifuge for 3 minutes at 12000rpm
12. Use 25µL of ddH2O to wash down the pellet
13. Let it stand for 2 minutes
14. Centrifuge it for 1 minute at 12000rpm

### Restriction Digest

|  |  |
| --- | --- |
| **Reaction Mixture** | **20µL** |
| 10x Buffer | 2µL |
| BamH I | 1µL |
| EcoR I | 1µL |
| ddH2O | ~up to 20µL |
| DNA Fragments | 1µg |

1. Make the mixture in a 1.5 mL Eppendorf tube
2. Put it into the PCR machine for 15 minutes at 37˚C

### Nanodrop UV Spectrophotometer

1. Use distilled water and filter paper to clean the nanodrop UV spectrophotometer
2. Calibrate it using distilled water
3. Use the wipes to dry it completely
4. Pipette 1µL of DNA sample onto the platform
5. Record the measurements

### Ligation

|  |  |
| --- | --- |
| **Reaction Mixture** | **10µL** |
| ddH2O | ~up to 10µL |
| Insert DNA | 50ng |
| 10x Buffer | 1µL |
| T4 Ligase | 0.5µL |
| Vector | 10ng |

1. Make the mixture in a 1.5 mL Eppendorf tube
2. Put it into the PCR at 16˚C for 3 hours

### Revival of Bacteria

|  |  |
| --- | --- |
| GM I | 10mL |
| ddH2O | 8.604mL |
| 10% conc. Yeast Extract | 1mL |
| 10x C-Salts | 956µL |
| 20% conc. Glucose Solution | 250µL |
| 10mg/mL Amino Acid Solution | 50µL |
| 5% conc. Casein Hydrolysate | 40µL |

1. inoculate bacteria into the culture dishes that contain antibiotic and grow overnight at 37 ˚C
2. Make GMI
3. Transfer the cells from the culture dishes onto the GMI
4. Incubate at 30˚C 150rpm overnight

### Transformation of E. Coli

1. Mix 10µL of plasmid with 100µL of E. Coli in a 1.5mL Eppendorf tube
2. Ice bath it for 30 minutes
3. Put it into hot water bath for 45 seconds at 42˚C
4. Ice bath it for 2 minutes
5. Mix with 500µL LB medium
6. Shake the bacteria culture for 30 minutes at 37˚C with 200rpm
7. Spread the bacteria culture onto plates

### Transformation of Bacillus subtilis

|  |  |
| --- | --- |
| GM II | 100 mL |
| ddH2O | 83.95 mL |
| 10× C-Salts | 9.328mL |
| 20% conc. Glucose Solution | 2.5mL |
| 1M MgCl2 | 2mL |
| 1M CaCl2 | 2mL |
| 10 mg/mL Amino Acid Solution | 0.1mL |
| 5% conc. Casein Hydrolysate | 0.08mL |
| 10% conc. Yeast Extract | 0.04mL |

1. Make GMII
2. Add 5 mL of B. subtilis into GM II
3. Shake the mixture for 1.5 hours at 120 rpm
4. Centrifuge the mixture for 10 minutes at 500g
5. Discard the supernatant
6. Resuspend pellet in 5 mL fresh medium
7. Discard the other supernatant
8. Add the plasmid into the bacterial culture medium
9. Shake the mixture for 1 hour at 150 rpm
10. Centrifuge the mixture for 5 minutes at 5000 rpm
11. Resuspend the pellet in 100 µL fresh medium
12. Discard the other supernatant
13. Spread the growth medium onto plates and incubate overnight at 37 °C

### Protein Gel Electrophoresis

|  |  |  |
| --- | --- | --- |
|  | Running Gel | Stacking Gel |
| 30% Acrylamide | 5mL | 0.33mL |
| Tris-HCl | 2.5mL (1.5M, pH 8.8) | 0.25mL (1M, pH 6.8) |
| ddH2O | 2.3mL | 1.4mL |
| 10% SDS | 0.1mL | 0.02mL |
| 10% APS | 0.1mL | 0.02mL |
| TEMED | 0.004mL | 0.002mL |

1. Make the gel
2. Mix 4 µL of 5x SDS loading buffer and 16 µL culture medium
3. Water bath for 10 minutes at 100°C- Put the gel into the device
   - Load the samples into the gel
   - Turn on the power supply at 60V until the samples reach the running gel
   - Turn up the voltage to 120V for 90 minutes
   - Dye the gel with G-250 solution (100mg G-250, 50 ml 95% ethanol, 100 ml 85% phosphoric acid)
   - Shake and wash with destaining solution (50ml methanol, 75ml ethanoic acid, 875ml ddH2O

### Protein Extraction (lysozyme)

1. Centrifuge the bacteria in 40 ml of cell culture at 12000 rpm for 1 minute
2. Discard the supernatant
3. Add 10 ml ddH2O
4. Distribute into 9 test-tubes
5. Centrifuge at 12000 rm for 1 minute
6. Discard the supernatant
7. Add 100 µL lysozyme
8. Water bath at 37 °C for 60 min

### Protein Extraction (extraction kit)

Material: Invent Biotech Minute™ Total Protein Extraction Kit

1. Centrifuge cell culture
2. Add 1 ml water
3. Centrifuge at top speed in a microcentrifuge for 2 min
4. Remove supernatant completely
5. Resuspend the pellet in 50 µl buffer A
6. Weigh out 80-90 mg protein extraction powder and add to the bottom of the tube
7. Grind the tube repeatedly with pestle for about 2 min with twisting force
8. Add 50 µl buffer A and 100 µl buffer B to the same tube and continue to grind for about thirty seconds
9. Vortex vigorously for 10 seconds
10. Centrifuge the tube at top speed for 3-4 min
11. Proteins will be in the supernatant

### Bradford Assay

Materials:

* Bradford solution

|  |  |
| --- | --- |
| **Bradford Solution** | **1000mL** |
| Coomassie Brilliant Blue G-250 | 100mg |
| 95% ethanol | 50 mL |
| 85% phosphoric acid | 100 mL |
| ddH2O | ~up to 1000 mL |

* BSA standard solution 0.1 µg/µl
* 0.1% NaCl Solution

1. Prepare the following protein solution

|  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Test-tube | 0 | 1 | 2 | 3 | 4 | 5 | 6 |
| BSA standard solution /mL | 0.0 | 0.1 | 0.2 | 0.4 | 0.6 | 0.8 | 1.0 |
| 0.1% NaCl Solution | 1 | 0.9 | 0.8 | 0.6 | 0.4 | 0.4 | 0.0 |
| Bradford solution /mL | 5 | 5 | 5 | 5 | 5 | 5 | 5 |

2. Use the microcentrifuge for the solution to be thoroughly mixed
3. Let them rest for 5 minutes
4. Measure absorbance at 595 nm
5. Prepare a calibration cure by plotting absorbance against protein amount in µg
6. Determine the amount of protein in our sample using the equation of the curve

### Preparation of Competent Cells

1. Inoculate the yeast into YPD broth, and shake for overnight at 30˚C and 200rpm
2. Transfer 5 mL from the broth into 100 mL YPD and shake at 30˚C and 200rpm
3. Collect the yeast when the OD value lies between 1.2 and 2.0.
4. Collect yeast after 3000rpm for 5min
5. Centrifuge at 1600g for 5min at 4˚C
6. Discard the supernatant
7. Resuspend yeast with 10 mL 1mol/L D-Sorbitol suspension
8. Centrifuged at 1600g for 5 min at 4˚C
9. Discard the supernantent
10. Repeat step 7~9 five more times
11. Water bath the yeast in cool water

### Electrotransformation of Yeast

1. Add 80 μl of competent yeast cells onto pre-chilled electroporation cuvette.
2. Mix 20 μl of concentrated linearized plasmid solution with the yeast cells
3. Place on ice for 5 min
4. Start the electroporation process
5. Immediately add 1 mL pre-cooled sterile 1 mol/L D-Sorbitol and rest for 60 minutes
6. Spread on MD plate
7. Place in 30℃ oven for 20-60 min
8. Incubate for 3-5 days

### Colometric Assay

Material: 100 mL of the product of ASN hydrolysis

1. Add 3400μl deionized water to 100ml solution
2. Add 500μl Nessler's reagent
3. Carry out the nanodrop to find out the absorbance value

### Ingredients of Medium

For Bacillus subtilis cultures:
**LB Medium**

|  |  |
| --- | --- |
| **LB** | **1L** |
| Tryptone | 10g |
| NaCl | 10g |
| Yeast Extract | 5g |
| Agar (for solid medium) | 15g |

For yeast culture:
**YPG Medium**

|  |  |
| --- | --- |
| **YPG** | **1L** |
| Tryptone | 20g |
| Yeast Extract | 10g |
| Glycerol | 20mL |
| Agar | 20g |

**YPD Medium**

|  |  |
| --- | --- |
| **YPD** | **1L** |
| Tryptone | 20g |
| Yeast Extract | 10g |
| Glucose | 20g |
| Agar | 20g |
| ddH2O | ~up to 1L |

For yeast fermentation:
**BMGY**

|  |  |
| --- | --- |
| **BMGY** | **1L** |
| Tryptone | 20g |
| Yeast Extract | 10g |
| 1.34% YNB | 100mL |
| 2% glycerol | 50mL |
| 10 mmol/L potassium phosphate (pH 6.0) | 100mL |
| Biotin 4×10-5% | 2mL |
| ddH2O | ~up to 1L |

**BMMY**

|  |  |
| --- | --- |
| **BMMY** | **1L** |
| Tryptone | 20g |
| Yeast Extract | 10g |
| 1.34% YNB | 100mL |
| 0.5% methanol | 100mL |
| 10 mmol/L potassium phosphate (pH 6.0) | 100mL |
| Biotin 4×10-5% | 2mL |

![](https://static.igem.org/mediawiki/2019/3/39/T--BSC_United--arrow.svg)

Ⓒ 2019 iGEM BSC\_United

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:BSC United/Results

iGEM BSC\_United

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## MINILOSS](https://2019.igem.org/Team%3ABSC_United)

* [Home](https://2019.igem.org/Team%3ABSC_United)
* Team
  + [Team Members](https://2019.igem.org/Team%3ABSC_United/Team)
  + [Attributions](https://2019.igem.org/Team%3ABSC_United/Attributions)
  + [Collaborations](https://2019.igem.org/Team%3ABSC_United/Collaborations)
  + [Special Thanks](https://2019.igem.org/Team%3ABSC_United/Thanks)
* Project
  + [Description](https://2019.igem.org/Team%3ABSC_United/Description)
  + [Experiments](https://2019.igem.org/Team%3ABSC_United/Experiments)
  + [Notebook](https://2019.igem.org/Team%3ABSC_United/Notebook)
  + [Results](https://2019.igem.org/Team%3ABSC_United/Results)
  + [Demonstrate](https://2019.igem.org/Team%3ABSC_United/Demonstrate)
* Design
  + [Overview](https://2019.igem.org/Team%3ABSC_United/Design)
  + [Biological Part](https://2019.igem.org/Team%3ABSC_United/Bio)
  + [Sensor](https://2019.igem.org/Team%3ABSC_United/Sensor)
  + [Organ Chip](https://2019.igem.org/Team%3ABSC_United/Organ_Chip)
  + [Hardware](https://2019.igem.org/Team%3ABSC_United/Hardware)
  + [Software](https://2019.igem.org/Team%3ABSC_United/Software)
  + [Model](https://2019.igem.org/Team%3ABSC_United/Model)
* [Parts](https://2019.igem.org/Team%3ABSC_United/Parts)
* [Safety](https://2019.igem.org/Team%3ABSC_United/Safety)
* HP
  + [Human Practices](https://2019.igem.org/Team%3ABSC_United/Human_Practices)
  + [Education&Engagement](https://2019.igem.org/Team%3ABSC_United/Public_Engagement)
* [Judging Form](https://igem.org/2019_Judging_Form?team=BSC_United)

# PROJECT

![](https://static.igem.org/mediawiki/2019/f/f2/T--BSC_United--Results.svg)

## RESULTS

* [◆ Biological Part](#p1)
* [◆ Sensor](#p2)
* [◆ Organ Chip](#p3)
* [◆ Conclusion](#p4)

### Biological Part

##### DNA PCR

As the starting step, human proinsulin coding gene (HPI) was synthesized. Specific primers were synthesized to ligate HPI gene into the plasmid pHY-P43 with the two restriction sites using BamHI and EcoRI . The gene in this plasmid is of the length of 345 bp after successful ligation, as shown in the gel electrophoresis image (Fig. 1).

![](https://static.igem.org/mediawiki/2019/6/60/T--BSC_United--results_fig1.png)

##### Fig.1 Gel electrophoresis image of the bands for HPI genes

##### Plasmid Construction

After the HPI gene was inserted into pHY-P43 plasmid, The length of the plasmid with HPI gene is 5.5 K bp. The electrophoresis image below (Fig. 2) confirmed that the recombinant plasmid was constructed successfully.

![](https://static.igem.org/mediawiki/2019/9/98/T--BSC_United--results_fig2.png)

##### Fig.2 Gel electrophoresis image of the bands for pHY-P43 after double digestion. 1, 3, 4, and 5 are positive results with bands in the region of 500 bp ~ 200 bp which is the length of HPI gene (345 bp) and bands in the region of 5000 bp which is the length of the plasmid. 2 is a negative result. There’s no band in the region of 500 bp ~ 200 bp.

##### Gene Transformation

The plasmid was inserted into E. coli for replication and storage. Colony PCR was implemented to confirm successful transformation of HPI gene into the host E. coli cells. Electrophoresis image (Fig. 3) demonstrates the presence of a gene of length 250~500 bp, which is the proinsulin gene, in the E. coli constructs.

![](https://static.igem.org/mediawiki/2019/6/60/T--BSC_United--results_fig3.png)

##### Fig.3 Gel electrophoresis image of the bands for E.coli’s. 1, 2, 3, 4, and 5 are all positive results with bands in the region of 500 bp ~ 200 bp which is the length of HPI gene (345 bp). Thus, they all contained the HPI gene.

To ensure that the gene between length 250~500 bp was HPI instead of some other gene, the band was extracted from the gel and sequenced. The results show that it matches the sequence of the HPI genes we originally used for synthesis.

Genetically engineered E. coli strains bearing HPI gene were cultured overnight. The recombinant plasmid was extracted from E. coli and transformed into B. subtilis WB800N. We presumed that the modified B. subtilis is able to produce proinsulin. Three different validations were performed.

##### 1. Colony PCR

Colony PCR for B. subtilis was carried out to ensure that it contains HPI gene from E. coli. The gel electrophoresis image (Fig. 4) confirms that B. subtilis did have the target gene.

![](https://static.igem.org/mediawiki/2019/8/89/T--BSC_United--results_fig4.png)

##### Fig.4 Gel image of bands for HPI gene within B. subtilis

##### 2. Protein Electrophoresis

B. subtilis was cultured for two days. Then all the proteins from the bacteria were extracted to test for proinsulin by protein electrophoresis. Human proinsulin has a molecular weight of about 12 kDa, so there should be an additional band between 14 and 10.5 kDa on the protein electrophoresis image, comparing to the control group that didn’t contain any HPI genes (1 and 4 on Fig.5). Despite numerous attempts, we couldn’t obtain a clear picture showing that particular band.

![](https://static.igem.org/mediawiki/2019/c/c3/T--BSC_United--results_fig5.png)

##### Fig.5 Results from protein electrophoresis before spacer peptide is added. 1 is the control group where the bacteria doesn’t contain HPI gene. 2 and 3 are the experimental group where the bacteria contain HPI gene. 4, 5, and 6 are 10x dilutions of 1, 2, and 3 respectively. There is no visible difference between the bands of the control group and experimental group, meaning that the engineered bacteria didn’t produce HPI as we have expected.

After rigorously analyzing our previous experiments for any mistakes, we found that the inability to produce the band on the gel image was because low expression of proinsulin. Although a strong promoter was used, the quantity of proinsulin obtained was still low. It is evident that the expression efficiency has to be increased. After a careful consideration, we decided to add a spacer peptide on the upstream region of the HPI gene. The results indicate a visible additional band between 14 kDa and 10.5 kDa, comparing to the control group (1 and 4 on Fig. 6). It is seen that the spacer peptide has actually enhance the gene expression, and the bacteria synthesized our desired protein, proinsulin.

![](https://static.igem.org/mediawiki/2019/c/c9/T--BSC_United--results_fig6.png)

##### Fig.6 Protein electrophoresis after spacer peptide is added. 1 is the control group where the bacteria doesn’t contain HPI gene. 2 and 3 are the experimental group where the bacteria contain HPI gene. 4, 5, and 6 are 10x dilutions of 1, 2, and 3 respectively. There are bands of 5 and 6 with deep color between 14kDa band (grey) and 10.5 kDa band (dark grey) of marker which 4 lacks. This shows that the bacteria do produce proinsulin.

##### 3. Microfluidic Analysis

The last approach is based on our microfluidics model (Fig. 7), composed of an organ-on-a-chip, dual channel electrochemical sensors, home-made data acquisition board, and our biological parts. If B. subtilis produce sufficient proinsulin, the liver cells grown on the organ chip should absorb glucose, and lower down the glucose concentration. As a result, the sensors should detect a decrease in current, which is directly proportional to glucose level. More details can be seen in the following organ-on-a-chip section.

![](https://static.igem.org/mediawiki/2019/5/56/T--BSC_United--results_fig7.png)

##### Fig.7 Data acquisition board with the biochemical sensors inside the organ-on-a-chip

### Electrochemical Sensor

Dual channel electrochemical sensors were made to monitor blood glucose level. (Fig.8)

![](https://static.igem.org/mediawiki/2019/5/52/T--BSC_United--results_fig8.png)

##### Fig.8 Our finished sensors connected to wires

To test the performance of the sensors, electric potential was altered between the range -0.6V to 0.6V on the working electrode of the sensors. A current was produced against the reference electrode which was 0V all the time. The current was measured continuously, and the electric potential against current was plotted below in Fig. 9. The change of current, 0.8 mA to -1.8 mA, on the graph with respect to electric potential shows that the electrodes on the sensors were functional, and the medium on the sensors were conductive.

![](https://static.igem.org/mediawiki/2019/f/f7/T--BSC_United--results_fig9.png)

##### Fig.9 Cyclic voltammetry graph of the electrodes on the sensor. Current changes with respect to electric potential

After function of the electrodes was confirmed, testing on the sensor was done to demonstrate its overall operationality. The sensor was inserted into a glucose solution of concentration 0.1 mM/L. After an interval of 50s, 5 µL of glucose is added into the solution. The sensor measured the current output. Fig. 10 showed a clear trend between current and time. As time increases, more glucose was added into the solution, so the current increased, from 0 µA to 195 µA. Thus, the sensors have the capability to detect glucose concentration in the whole range since there is clearly a relationship between the glucose concentration and the current measured by the sensor.

![](https://static.igem.org/mediawiki/2019/5/52/T--BSC_United--results_fig10.png)

##### Fig.10 Graph showing the overall rise of current measured by the sensor after a series of nine addition of glucose

Correlation between glucose concentration and current was then made. 12 readings were taken for 12 different standard solutions. The glucose concentration of each of the solutions is shown below on the graph (Fig. 11). We applied linear regression to the data and made the line of best-fit which is I=75.43c+1.31. The correlation coefficient is 0.976. This means that the equation constructed should be very close to the actual value according to mathematical calculations. Also, it indicted that the sensors were precise.

![](https://static.igem.org/mediawiki/2019/8/88/T--BSC_United--results_fig11.png)

##### Fig.11 Relationship between current and glucose concentration

To confirm that it’s accurate to deduce the unknow glucose concentration just from the current data by using the equation, c = ( I - 1.31 ) / 75.43, four test of known concentration of glucose solution (0.2 mmol/L, 0.4 mmol/L, 0.8 mmol/L, 1.6 mmol/L) were implemented. The graph below was obtained (Fig. 12).

![](https://static.igem.org/mediawiki/2019/f/fd/T--BSC_United--results_fig12.png)

##### Fig.12 A validation of our equation. The sensor is dipped into known concentration of glucose. The current is plotted out.

In order to make sure that two glucose concentration sensors can work side by side, two sensors were dipped into two solutions (a 2mM glucose solution and a 1x PBS solution) (Fig. 13). The current outputs (Fig. 14) from the two sensors were different. The reading for the sensor to the glucose solution was in the range of 3838~3508 mV, while the other one was in the range of 2732~2683 mV. The difference indicates that our sensors are capable of working together at the same time and still obtaining accurate values.

![](https://static.igem.org/mediawiki/2019/0/00/T--BSC_United--results_fig13.png)

##### Fig.13 A demonstration of the test carried out. One sensor is dipped into 2mM glucose solution whereas the other is inserted into 1x PBS solution.

![](https://static.igem.org/mediawiki/2019/e/e7/T--BSC_United--results_fig14.png)

##### Fig.14 The current data from the two sensors of the above test. It’s easy to see that they are different.

### Organ-on-a-Chip

The final part of our experiment was performed. The organ chip was fabricated (Fig. 15) and incorporated with the functional biochemical sensors (Fig. 16). Several tests on the chip were carried out before the bacteria was injected.

![](https://static.igem.org/mediawiki/2019/0/00/T--BSC_United--results_fig15.png)

##### Fig.15 Our home-made organ-on-a-chip

![](https://static.igem.org/mediawiki/2019/6/6d/T--BSC_United--results_fig16.png)

##### Fig.16 Integration of organ-on-a-chip with sensors

##### Test of the blank chambers

First, diffusion rate of glucose inside the organ on chip was tested. The two chambers were filled with PBS. Glucose was injected into the upper chamber (simulating the blood stream). After the current readings of the top and bottom chamber stabilized, another batch of glucose was added. The two sensors on the upper and lower chamber continuously recorded current. From the graph (Fig. 17), it is seen that after 40-80 seconds, the glucose concentration became relatively equal in the two chambers. It can be inferred that little time was taken for glucose to achieve equilibrium between the top and bottom chamber, and glucose can diffuse very easily in between.

![](https://static.igem.org/mediawiki/2019/a/a3/T--BSC_United--results_fig17.png)

##### Fig.17 The current of the top and bottom chamber before and after glucose addition into the upper chamber

In order to mimic the liver, cancerous liver cell lines, hepatocytes, were transplanted onto the lower chamber of our organ chip (Fig. 18). Two fluorescent dyes, DAPI and CDFDA, were used to assess the liver cell growth. After 48 hours, several images (Fig. 19) were taken using fluorescence microscope. The abundance of blue-colored nucleus, stained by DAPI, in these pictures confirm that there were plenty of viable liver cells present in the chip. Here, the red color in the surroundings signifies MRP2 activity for the hepatocyte efflux transporter. This means that liver cells are functioning normally.

![](https://static.igem.org/mediawiki/2019/f/f2/T--BSC_United--results_fig18.png)

##### Fig.18 Liver cells injected into the organ chip

![](https://static.igem.org/mediawiki/2019/1/1d/T--BSC_United--results_fig19.png)

##### Fig.19 Fluorescent image of the liver cells in our organ-on-a-chip. The blue part represents cell nucleus while the red color indicates MRP2 activity.

With the assurance of liver cells growing finely, the same test of glucose diffusion was carried out again. It took much longer time for the stabilization of glucose concentration in the lower chamber, on average 80 seconds (Fig. 20). We concluded that the liver cell impeded the diffusion of glucose, thus the time for equilibrium increased.

![](https://static.igem.org/mediawiki/2019/b/bf/T--BSC_United--results_fig20.png)

##### Fig.20 The current of the top and bottom chamber containing liver cells before and after glucose addition

In our last experiment, proinsulin-bearing B. subtilis cells were injected into the organ chip. After 48 hours, lysozyme was added to release the proinsulin. The recorded time for glucose equilibrium was about 90 seconds (Fig. 21), longer than previous experiment without proinsulin-producing B. subtilis. Also, the glucose concentration (current) for the bottom chamber was significantly less than that of the top chamber. The above results provide convincing evidence that the release of proinsulin triggered the liver cells to take in glucose. The glucose level was lowered down within our in vitro model by the proinsulin from engineered B. subtilis, The objective of our project to simulate bacterial supply of insulin in vitro for the treatment of diabetes is achieved.

![](https://static.igem.org/mediawiki/2019/3/31/T--BSC_United--results_fig21.png)

##### Fig.21 Graph showing the current of the top chamber, containing bacteria, and the bottom chamber containing liver cells before and after glucose addition

### Conclusion

The completion of the in vitro modeling is a crucial step towards our ultimate goal of bacterial implantation in the human intestines to replace pancreas for the secretion of insulin. We have successfully mimicked the human liver in absorbing glucose in an in vitro environment. Novel combination of electrochemical sensors, organ-in-a-chip and use of lysozymes for fast insulin release is unique. In the near future, our research paradigm will be shifted to construction of in vivo system in human body for a brand new diabetic treatment.

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