iGEM 2021 — Team NCKU_Tainan

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# Team:NCKU Tainan/Notebook

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* Project

  [Description](https://2021.igem.org/Team%3ANCKU_Tainan/Description)
  [Design](https://2021.igem.org/Team%3ANCKU_Tainan/Design)
  [Experiments](https://2021.igem.org/Team%3ANCKU_Tainan/Experiments)
  [Measurement](https://2021.igem.org/Team%3ANCKU_Tainan/Measurement)
  [Model](https://2021.igem.org/Team%3ANCKU_Tainan/Model)
  [Hardware](https://2021.igem.org/Team%3ANCKU_Tainan/Hardware)
  [Safety](https://2021.igem.org/Team%3ANCKU_Tainan/Safety)
* Results

  [Results](https://2021.igem.org/Team%3ANCKU_Tainan/Results)
  [Proof of Concept](https://2021.igem.org/Team%3ANCKU_Tainan/Proof_Of_Concept)
  [Implementation](https://2021.igem.org/Team%3ANCKU_Tainan/Implementation)
  [Engineering](https://2021.igem.org/Team%3ANCKU_Tainan/Engineering)
  [Contribution](https://2021.igem.org/Team%3ANCKU_Tainan/Contribution)
  [Future](https://2021.igem.org/Team%3ANCKU_Tainan/Future)
* HP

  [Human Practice](https://2021.igem.org/Team%3ANCKU_Tainan/Human_Practices)
  [Communication](https://2021.igem.org/Team%3ANCKU_Tainan/Communication)
  [Collaboration](https://2021.igem.org/Team%3ANCKU_Tainan/Collaborations)
  [Entrepreneurship](https://2021.igem.org/Team%3ANCKU_Tainan/Entrepreneurship)
  [Partnership](https://2021.igem.org/Team%3ANCKU_Tainan/Partnership)
  [Inclusivity](https://2021.igem.org/Team%3ANCKU_Tainan/Inclusivity)
  [Sustainable Development](https://2021.igem.org/Team%3ANCKU_Tainan/Sustainable)
  [Excellence in Another Area](https://2021.igem.org/Team%3ANCKU_Tainan/Excellence)
* Parts

  [Parts](https://2021.igem.org/Team%3ANCKU_Tainan/Parts)
  [Improvement](https://2021.igem.org/Team%3ANCKU_Tainan/Improvement)
* Team

  [Team](https://2021.igem.org/Team%3ANCKU_Tainan/Team)
  [Attributions](https://2021.igem.org/Team%3ANCKU_Tainan/Attributions)
* Notebook

  [Notebook](https://2021.igem.org/Team%3ANCKU_Tainan/Notebook)
* Awards

  [Awards](https://2021.igem.org/Team%3ANCKU_Tainan/Awards)
  [Criteria](https://2021.igem.org/Team%3ANCKU_Tainan/Criteria)

Collab

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e-mail : igem.ncku.tainan@gmail.com

phone : 06-2757575

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# Team:NCKU Tainan/Engineering

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* Project

  [Description](https://2021.igem.org/Team%3ANCKU_Tainan/Description)
  [Design](https://2021.igem.org/Team%3ANCKU_Tainan/Design)
  [Experiments](https://2021.igem.org/Team%3ANCKU_Tainan/Experiments)
  [Measurement](https://2021.igem.org/Team%3ANCKU_Tainan/Measurement)
  [Model](https://2021.igem.org/Team%3ANCKU_Tainan/Model)
  [Hardware](https://2021.igem.org/Team%3ANCKU_Tainan/Hardware)
  [Safety](https://2021.igem.org/Team%3ANCKU_Tainan/Safety)
* Results

  [Results](https://2021.igem.org/Team%3ANCKU_Tainan/Results)
  [Proof of Concept](https://2021.igem.org/Team%3ANCKU_Tainan/Proof_Of_Concept)
  [Implementation](https://2021.igem.org/Team%3ANCKU_Tainan/Implementation)
  [Engineering](https://2021.igem.org/Team%3ANCKU_Tainan/Engineering)
  [Contribution](https://2021.igem.org/Team%3ANCKU_Tainan/Contribution)
  [Future](https://2021.igem.org/Team%3ANCKU_Tainan/Future)
* HP

  [Human Practice](https://2021.igem.org/Team%3ANCKU_Tainan/Human_Practices)
  [Communication](https://2021.igem.org/Team%3ANCKU_Tainan/Communication)
  [Collaboration](https://2021.igem.org/Team%3ANCKU_Tainan/Collaborations)
  [Entrepreneurship](https://2021.igem.org/Team%3ANCKU_Tainan/Entrepreneurship)
  [Partnership](https://2021.igem.org/Team%3ANCKU_Tainan/Partnership)
  [Inclusivity](https://2021.igem.org/Team%3ANCKU_Tainan/Inclusivity)
  [Sustainable Development](https://2021.igem.org/Team%3ANCKU_Tainan/Sustainable)
  [Excellence in Another Area](https://2021.igem.org/Team%3ANCKU_Tainan/Excellence)
* Parts

  [Parts](https://2021.igem.org/Team%3ANCKU_Tainan/Parts)
  [Improvement](https://2021.igem.org/Team%3ANCKU_Tainan/Improvement)
* Team

  [Team](https://2021.igem.org/Team%3ANCKU_Tainan/Team)
  [Attributions](https://2021.igem.org/Team%3ANCKU_Tainan/Attributions)
* Notebook

  [Notebook](https://2021.igem.org/Team%3ANCKU_Tainan/Notebook)
* Awards

  [Awards](https://2021.igem.org/Team%3ANCKU_Tainan/Awards)
  [Criteria](https://2021.igem.org/Team%3ANCKU_Tainan/Criteria)

* [Design](#Subtitle1)
* [Build](#Subtitle2)
* [Test](#Subtitle3)
* [Learn](#Subtitle4)

![](https://static.igem.org/mediawiki/2021/5/53/T--NCKU_Tainan--Engineering_header.png)

## Design

  The double knockout strain was built for the IFN-γ induced system and the total taurine production. The two knockout genes are wild type *ompA* gene and *tauD* (taurine dioxygenase) gene.

  First, without conducting wild type *ompA* gene knockout, the mutant OmpA protein that can sense the IFN-γ cannot be well expressed on the *E. coli* cytoplasmic membrane. Second, *tauD* (taurine dioxygenase) gene knockout was conducted to decrease the taurine degradation for in vivo studies. The *tauD* mutant affects bacteria’s ability to use taurine as its sulfur source without affecting other physiological functions[[1](#ref1)].

---

## Build

  The mutant strain construction was finished in three procedures. The three procedures are PCR gene replacement method, P1 bacteriophage transduction and the deletion of antibiotic resistance genes.

  First, the mutant strain construction was done in *E. coli* W3110 by PCR gene replacement method according to the research[[2](#ref2)]. Second, the P1 bacteriophage was prepared to carry the mutant gene (with kanamycin resistance gene and two *FRT* genes) into *E. coli* MG1655 by P1 bacteriophage transduction. Last but not least, the kanamycin resistance gene was deleted by doing electroporation with pCP20 plasmid (with FLP recombinase gene and ampicillin resistance gene). The pCP20 plasmid can remove the gene between two *FRT* genes and it is simply cured by culturing at a temperature higher than 30°C because of its temperature sensitivity[[3](#ref3)](Fig. 1).

[![](https://static.igem.org/mediawiki/2021/7/78/T--NCKU_Tainan--engineering.gif)](https://static.igem.org/mediawiki/2021/7/78/T--NCKU_Tainan--engineering.gif)

Fig. 1. The mutation and deletion methods.

---

## Test

  The plate selection was used to test the deletion of antibiotic resistance genes by picking the colonies and culturing them on three LB agar plates, one directly on the plate, another with ampicillin, and the other with kanamycin. The colonies that can only survive on the LB agar plate were selected (Fig. 2) to make sure there is no biosafety issue once the bacteria leak out into the environment.

[![](https://static.igem.org/mediawiki/2021/8/8a/T--NCKU_Tainan--plate_selection-1.png)](https://static.igem.org/mediawiki/2021/8/8a/T--NCKU_Tainan--plate_selection-1.png)

Fig. 2. The colonies that can only grow on the LB plate are those that the antibiotic resistance gene was deleted and the pCP20 plasmid was cured.

---

## Learn

  To confirm whether the deletion of the *ompA* gene succeeded, PCR was conducted to amplify the *ompA* gene with *ompA* primer. The DNA sequence lengths of our sample was shorter than that of the control *ompA* gene, which shows that the *ompA* gene was successfully deleted(Fig. 3).

[![](https://static.igem.org/mediawiki/2021/c/c5/T--NCKU_Tainan--ompA-mutation-confirm.png)](https://static.igem.org/mediawiki/2021/c/c5/T--NCKU_Tainan--ompA-mutation-confirm.png)

Fig. 3. The electrophoresis result of *ompA* fragment from the chromosome PCR.
M: Marker; Lane 1: wild-type *E.coli* MG1655 (1638 bp); Lane 2: *∆ompA E.coli* MG1655 (763 bp).

  Due to time constraints, we could not confirm the deletion of the *tauD* gene. In our future plan, we will use *tauD* primers to compare *tauD* DNA sequence lengths of our deleted strain with that of the control.

## References

1. van der Ploeg JR, Weiss MA, Saller E, et al. Identification of sulfate starvation-regulated genes in Escherichia coli: a gene cluster involved in the utilization of taurine as a sulfur source. *Journal of Bacteriology*. 1996;178(18):5438-5446. doi:10.1128/jb.178.18.5438-5446.1996.
2. Baba T, Ara T, Hasegawa M, et al. Construction of Escherichia coli K‐12 in‐frame, single‐gene knockout mutants: the Keio collection. *Molecular Systems Biology*. 2006;2(1). doi:10.1038/msb4100050.
3. Datsenko KA, Wanner BL. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. *Proceedings of the National Academy of Sciences*. 2000;97(12):6640-6645. doi:10.1073/pnas.120163297.

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e-mail : igem.ncku.tainan@gmail.com

phone : 06-2757575

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# Team:NCKU Tainan/Experiments

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* Project

  [Description](https://2021.igem.org/Team%3ANCKU_Tainan/Description)
  [Design](https://2021.igem.org/Team%3ANCKU_Tainan/Design)
  [Experiments](https://2021.igem.org/Team%3ANCKU_Tainan/Experiments)
  [Measurement](https://2021.igem.org/Team%3ANCKU_Tainan/Measurement)
  [Model](https://2021.igem.org/Team%3ANCKU_Tainan/Model)
  [Hardware](https://2021.igem.org/Team%3ANCKU_Tainan/Hardware)
  [Safety](https://2021.igem.org/Team%3ANCKU_Tainan/Safety)
* Results

  [Results](https://2021.igem.org/Team%3ANCKU_Tainan/Results)
  [Proof of Concept](https://2021.igem.org/Team%3ANCKU_Tainan/Proof_Of_Concept)
  [Implementation](https://2021.igem.org/Team%3ANCKU_Tainan/Implementation)
  [Engineering](https://2021.igem.org/Team%3ANCKU_Tainan/Engineering)
  [Contribution](https://2021.igem.org/Team%3ANCKU_Tainan/Contribution)
  [Future](https://2021.igem.org/Team%3ANCKU_Tainan/Future)
* HP

  [Human Practice](https://2021.igem.org/Team%3ANCKU_Tainan/Human_Practices)
  [Communication](https://2021.igem.org/Team%3ANCKU_Tainan/Communication)
  [Collaboration](https://2021.igem.org/Team%3ANCKU_Tainan/Collaborations)
  [Entrepreneurship](https://2021.igem.org/Team%3ANCKU_Tainan/Entrepreneurship)
  [Partnership](https://2021.igem.org/Team%3ANCKU_Tainan/Partnership)
  [Inclusivity](https://2021.igem.org/Team%3ANCKU_Tainan/Inclusivity)
  [Sustainable Development](https://2021.igem.org/Team%3ANCKU_Tainan/Sustainable)
  [Excellence in Another Area](https://2021.igem.org/Team%3ANCKU_Tainan/Excellence)
* Parts

  [Parts](https://2021.igem.org/Team%3ANCKU_Tainan/Parts)
  [Improvement](https://2021.igem.org/Team%3ANCKU_Tainan/Improvement)
* Team

  [Team](https://2021.igem.org/Team%3ANCKU_Tainan/Team)
  [Attributions](https://2021.igem.org/Team%3ANCKU_Tainan/Attributions)
* Notebook

  [Notebook](https://2021.igem.org/Team%3ANCKU_Tainan/Notebook)
* Awards

  [Awards](https://2021.igem.org/Team%3ANCKU_Tainan/Awards)
  [Criteria](https://2021.igem.org/Team%3ANCKU_Tainan/Criteria)

![](https://static.igem.org/mediawiki/2021/f/f1/T--NCKU_Tainan--Exp_header.png)

Protocol:

1. On ice, add all components in a PCR tube, making up to 50 µl volume reaction.

| Components | Volume (μl) |
| --- | --- |
| MQ or ddH2O 1.1 | |
| 10x PCR buffer 1 | |
| dNTP 2.5 mM 0.8 | |
| Template 1 | |
| Forward primer 10 μM 1 | |
| Reverse primer 10 μM 0.1 | |
| Taq polymerase + vent 0.1 | |
| Total Volume | 10 |

2. Gently mix the PCR reactions and centrifuge briefly.

3. Transfer the PCR tubes to a thermocycler.

| Step | Temperature | Time |
| --- | --- | --- |
| Initial denaturalization 94°C | 5 mins | |
| 25 - 35 cycles 94°C (denaturation) | 30 secs | |
| 55°C (annealing) | 30 secs |
| 72°C (extension) | 2 mins (depend on sequence size 2 kbp/min.) |
| Final extension 72°C | 5 mins | |
| Hold 16°C (holding for a short time) or 4°C (holding for a long time) | ∞ | |

## Electroporation *Escherichia coli* for homologous

Materials:

#### Equipment:

1. Pipette and pipette tips

2. Eppendorf tubes

3. Centrifuge

4. Flasks

5. Electroporation cuvettes (2 mm gap)

6. Electroporator

#### Consumables:

1. SOB

2. Chilled 10% glycerol

3. SOC

4. Selection plates

5. DNA fragment

6. 10% L-arabinose

7. Ampicillin

Protocol:

##### Competent cell making:

1. Inoculate 500 μl of overnight culture (*Escherichia coli* with pKD46) to 50 ml of fresh SOB-Mg medium with 50 μl of ampicillin at 30°C.

2. Grow cells with shaking at 30°C for 1 hour and 50 mins.

3. Add 1 ml of 10% L-arabinose and culture for 1 hour.

4. Transfer the culture into 50 ml centrifuge tube, and centrifuge it for 5 min at 4°C and 6,000 rpm to harvest cells. (After this stage, the cells should be kept cool throughout the cell preparation)

5. Remove the supernatant and suspend the cells with 1 ml of chilled 10% glycerol. Transfer the cell suspension into a 1.5 ml tube and centrifuge again for 1 min at 4°C and 12,000 rpm. (first wash)

6. Remove the supernatant using a pipette and suspend the cells with 1 ml of chilled 10% glycerol. Centrifuge again for 1 min at 4°C and 12,000 rpm. (second wash)

7. Remove the supernatant using a pipette and suspend the cells with 1 ml of chilled 10% glycerol. Centrifuge again for 1 min at 4°C and 12,000 rpm. (third wash)

8. Remove the supernatant using a pipette and suspend the cells with 250 μl (depends on the amount of the cell) of chilled 10% glycerol.

##### Electroporation:

1. Mix 50 μl of the competent cell and 5 μl DNA solution in a fresh 1.5 ml tube.

2. Transfer the mixture into chilled electroporation cuvette (2 mm gap), and pulse (Ec 2).

3. Add 1 ml of SOC with 20 μl 10% arabinose immediately after the pulse.

4. Incubate it for 1 hour at 37°C.

5. Plate 100 μl of the culture on a selection plate, and incubate at 30°C.

## Electroporation *Escherichia coli* with plasmid

Materials:

#### Equipment:

1. Pipette and pipette tips

2. Eppendorf tubes

3. Centrifuge

4. Flasks

5. Electroporation cuvettes (2 mm gap)

6. Eletroporator

#### Consumables:

1. SOB

2. Chilled 10% glycerol

3. SOC

4. Selection plates

5. DNA plasmid

Protocol:

##### Competent cell making:

1. Inoculate 1 ml of overnight culture to 50 ml of fresh SOB-Mg medium.

2. Grow cells with shaking at 37°C for 1.5 hours (recA+ strain) or 1.75 hours (recA- strain).

3. Chill the flask on ice for 5 min, transfer the culture into 50 ml centrifuge tube, and centrifuge it for 5 min at 4°C and 6,000 rpm to harvest cells. (After this stage, the cells should be kept cool throughout the cell preparation)

4. Remove the supernatant and suspend the cells with 1 ml of chilled 10% glycerol. Transfer the cell suspension into a 1.5 ml tube and centrifuge again for 1 min at 4°C and 12,000 rpm.

5. Remove the supernatant using a pipette and suspend the cells with 1 ml of chilled 10% glycerol. Centrifuge again for 1 min at 4°C and 12,000 rpm.

6. Remove the supernatant using a pipette and suspend the cells with 250 μl (depends on the amount of the cell) of chilled 10% glycerol.

##### Electroporation:

1. Mix 50 μl of the competent cell and DNA solution (the amount depends on the DNA plasmid) in a fresh 1.5 ml tube.

2. Transfer the mixture into chilled electroporation cuvette (2 mm gap), and pulse (Ec 2).

3. Add 1 ml of SOC immediately after the pulse.

4. Incubate it for 1 hour at 37°C.

5. Plate the culture on a selection plate, and incubate.

## Gel Electrophoresis

Materials:

#### Equipment:

1. Microwave

2. Electrophoresis comb

3. Electrophoresis trays

4. Pipette and pipette tips

5. Buffer tank

6. Voltage source

7. Glass bottle or Erlenmeyer flask

#### Consumables:

1. Agarose powder

2. TBE or TAE buffer

3. λ EH (λ DNA/EcoRI+HindIII) marker (2X) - Promega

4. 2X SLB or 10X SLB (sample loading buffer)

5. DNA sample

6. EtBr (Ethidium bromide)

7. RO (reverse osmosis)

Protocol:

##### Agarose gel preparation:

1. Adjust the amount of agarose to get the desired gel concentration (in this protocol, we’ll be using 0.7% agarose).

2. Weight 0.7 grams of agarose powder and add it to a glass bottle or Erlenmeyer flask.

3. Measure 100 ml of TBE buffer or TAE buffer and add it to the glass bottle or Erlenmeyer.

4. Put the mix into the microwave and heat it until the agarose is completely dissolved.

5. Let the mix cool down and pour it to the electrophoresis tray.

6. Set the electrophoresis comb and wait for the gel to solidify.

##### Sample preparation:

1. Samples are prepared in various ways depends on its proposes.

| Purpose | DNA Solution | SLB | Total Volume |
| --- | --- | --- | --- |
| Size checking 2 μl | 10 μl (2X) | 12 μl | |
| Gel extraction depends on how much DNA solution to be extracted | 5 μl (10X) |  | |

##### Agarose gel running:

1. When the agarose gel solidified, take out the electrophoresis comb.

2. Pour the TBE buffer or TAE buffer to the buffer tank. Note: buffer used for running has to be the same as the buffer used to prepare the gel solution.

3. Put the gel in the buffer tank.

4. Load 5 μl λ EH (λ DNA/EcoRI+HindIII) marker (2X) in a well and load the DNA sample mix with SLB in the remaining wells.

5. Run for 20 mins at 100V. Note: voltage and time varies.

##### Agarose gel visualization:

1. Take the gel out of the buffer tank.

2. Soak the gel in the EtBr solution for 8 mins and in RO for 30 secs.

3. Take the gel out of RO and visualize the DNA under UV transilluminator.

## Gel Extraction and Purification

Materials:

#### Equipment:

1. Eppendorf tubes

2. Collection tubes

3. Spin column

4. Pipette and pipette tips

5. Centrifuge

6. Scalpel

7. Dry Thermounit

#### Consumables:

1. Isopropanol

2. Binding buffer

3. Washing buffer

4. Elution buffer

Protocol:

##### Gel excision, solubilization and DNA binding:

1. Excise band with scalpel and transfer to a new eppendorf tube.

2. Weigh the gel slice in a tube (by measuring the weight difference of an empty eppendorf and the eppendorf with gel slice in the tube).

3. Add 3 volumes of binding buffer to 1 volume of gel (100 mg = 100 μl).

4. Incubate at 60°C for 2-10 mins (or until the gel has completely dissolved).

5. Add 1.5 volume of isopropanol and invert the eppendorf 10 times.

6. Place a spin column in a provided collection tube. Transfer 700 μl sample to the spin column and centrifuge for 30 secs, 12,000 rpm.

7. Discard flow-through and place the spin column back into the collection tube.

##### 2. Wash:

1. Add 500 μl of washing buffer to the spin column and centrifuge for 30 secs, 12,000 rpm.

2. Discard flow-through and place the spin column back into the collection tube.

3. Add 200 μl of washing buffer to the spin column and centrifuge for 5 mins, 12,000 rpm.

4. Discard flow-through and place the spin column back into the collection tube.

##### 3. DNA elution:

1. Transfer spin column to clean eppendorf. Add 50 μl of elution buffer to the spin column. Centrifuge for 2 mins, 12,000 rpm.

2. Collect the pure sample in the eppendorf and discard the spin column.

Keep the DNA solution in -20°C freezer.

## Ligation

Materials:

#### Equipment:

1. Eppendorf tubes

2. Pipette and pipette tips

3. Centrifuge

#### Consumables:

1. T4 DNA ligase (NEB)

2. T4 DNA ligase buffer (NEB)

Protocol:

1. Set up the following reaction on ice:

| Vector | 12 μl |
| --- | --- |
| Insert 40 μl | |
| T4 DNA ligase buffer (10x) 5.8 μl | |
| T4 DNA ligase 0.5 μl | |
| Total Volume | 58.3 μl |

2. Gently mix the reaction and centrifuge.

3. Incubate at 16°C overnight.

4. Keep the DNA solution in -20°C freezer.

## PCR Clean-up

Materials:

#### Equipment:

1. Eppendorf tubes

2. Collection tubes

3. Spin column

4. Pipette and pipette tips

5. Centrifuge

#### Consumables:

1. Isopropanol

2. Binding buffer

3. Washing buffer

4. Elution buffer

5. MQ or ddH2O

Protocol:

##### DNA binding:

1. Add MQ or ddH2O to DNA solution until the volume reaches 100 μl.

2. Add 300 μl of binding buffer to the solution.

3. Add 150 μl of isopropanol and invert the eppendorf 10 times.

4. Place a spin column in a provided collection tube. Transfer 550 μl sample to the spin column and centrifuge for 30 secs, 12,000 rpm.

5. Discard flow-through and place the spin column back into the collection tube.

##### Wash

1. Add 500 μl of washing buffer to the spin column and centrifuge for 30 secs, 12,000 rpm.

2. Discard flow-through and place the spin column back into the collection tube.

3. Add 200 μl of washing buffer to the spin column and centrifuge for 5 mins, 12,000 rpm.

4. Discard flow-through and place the spin column back into the collection tube.

##### DNA elution

1. Transfer spin column to clean eppendorf. Add 50 μl of elution buffer to the spin column. Centrifuge for 2 mins, 12,000 rpm.

2. Collect the pure sample in the eppendorf and discard the spin column.

3. Keep the DNA solution in -20°C freezer.

## Plasmid Isolation: Miniprep

Materials:

#### Equipment:

1. Eppendorf tubes

2. Collection tubes

3. Spin column

4. Pipette and pipette tips

5. Centrifuge

6. Ice bucket

#### Consumables:

1. Suspension buffer

2. Lysis buffer

3. Binding buffer

4. Washing buffer I

5. Washing buffer II

6. Elution buffer

7. LB broth

8. Antibiotic as needed

Protocol:

1. Prepare 2-5 ml overnight culture in LB medium with the antibiotic (depends on the plasmid to be extracted).

2. Collect cells from the overnight culture with a centrifuge (12,000 rpm 30 sec) and discard the supernatant.

3. Add 250 μl of suspension buffer and suspend bacterial cells by vortex.

4. Add 250 μl of lysis buffer and mix gently by inverting the tube 10 times.

5. Add 350 μl of binding buffer and mix gently by inverting the tube 10 times.

6. Incubate on ice for 5 mins.

7. Centrifuge at 4°C, 12,000 rpm for 10 mins.

8. Place a spin column in a provided collection tube. Transfer 750 μl of supernatant to spin column.

9. Centrifuge at room temperature, 12,000 rpm for 30 sec and discard the flow-through.

10. Add 500 μl of washing buffer I to the spin column and centrifuge for 30 sec. Discard the flow-through.

## Restriction Enzyme Digestion

| Restriction Enzymes | Buffer  (Buffer will depend on restriction enzyme being used) |
| --- | --- |
| EcoRI SuR E/Cut Buffer H (10X) - Roche | |
| XbaI CutSmart Reaction Buffer (10x) - NEB or 3.1 - NEB | |
| SpeI CutSmart Reaction Buffer (10x) - NEB | |
| PstI 3.1 (10X) - NEB | |

Materials:

#### Equipment:

1. Eppendorf tubes

2. Pipette and pipette tips

3. Ice bucket

4. Incubator (37°C)

#### Consumables:

1. Restriction enzymes

2. Buffer 10x (depends on its restriction enzymes)

3. DNA sample

4. MQ od ddH2O (for single digestion)

Protocol:

1. On the ice, add all the components.

Double Digestion:

| DNA | 43 μl |
| --- | --- |
| Buffer 10x | 5 μl |
| Restriction enzyme 1 | 1 μl |
| Restriction enzyme 2 | 1 μl |
| Total Volume | 50 μl |

Single Digestion (Structure Check):

| Plasmid DNA 2 μl | |
| --- | --- |
| Buffer 10x 1.5 μl | |
| Restriction enzyme 0.5 μl | |
| MQ or ddH2O 11 μl | |
| Total Volume | 15 μl |

2.Mix gently and incubate for 1-2 hours for double digestion or 30 mins - 1 hour for single digestion at 37°C. Note: Incubation time varies along the total volume of the reaction.

## SDS-PAGE

Materials:

#### Equipment:

1. Eppendorf tubes

2. Pipette and pipette tips

3. Centrifuge

4. Vortex

5. Ice bucket

6. Empty box

7. Dry Thermounit

8. Glass plates

9. 10-well comb

10. Spacer

11. Clamp

12. Casting stand

13. Buffer tank

14. Voltage source

15. Shaker

#### Consumables:

1. H2O

2. 30% acrylamide

3. 1.5M tris pH 8.8

4. 1M tris pH 6.8

5. 10% SDS

6. 10% APS

7. TEMED

8. Prestained Protein Marker - Bioman

9. Protein dye

10. Overnight culture (sample)

11. CBB (Coomassie Brilliant Blue) staining solution

12. Destaining buffer

13. Tank buffer (1X)

14. Iso-propanol

Protocol:

##### Resolving gel preparation

1. Adjust the amount of agarose to get the desired gel concentration

2. In a 50 ml eppendorf tube add all the components 15% gel concentration

| Total Volume | 5 ml | 10 ml | 15 ml |
| --- | --- | --- | --- |
| H2O (ml) 1.15 | 2.3 | 3.4 | |
| 30% acrylamide mix (ml) 2.5 | 5 | 7.5 | |
| 1.5M Tris pH 8.8 (ml) 1.25 | 2.5 | 3.3 | |
| 10% SDS (ml) 0.05 | 0.1 | 0.15 | |
| 10% APS (ml) 0.10 | 0.2 | 0.3 | |
| TEMED (ml) 0.002 | 0.004 | 0.006 | |

3. Pour the mixture in between the glasses and add iso-propanol afterward.

4. Wait for 10-15 mins or until the gel solidifies.

5. Pour out the iso-propanol.

##### Stacking gel preparation

1. In a 50 ml eppendorf tube add all the components

| Total Volume | 1.5 ml | 3 ml | 5 ml |
| --- | --- | --- | --- |
| H2O (ml) 1.05 | 2.1 | 3.4 | |
| 30% acrylamide mix (ml) 0.25 | 0.5 | 0.83 | |
| 1M Tris pH 6.8 (ml) 0.19 | 0.38 | 0.63 | |
| 10% SDS (ml) 0.015 | 0.03 | 0.05 | |
| 10% APS (ml) 0.03 | 0.06 | 0.05 | |
| TEMED (ml) 0.0015 | 0.003 | 0.005 | |

2. Pour the mixture in between the glass plates and add the 10-well comb.

3. Wait for 10-15 mins or until the gel solidifies.

##### Sample preparation

1. Take overnight cultures with the desired volume and centrifuge.

2. Take 12 μl of pellet or supernatant (depends on necessity) and move to a new fresh eppendorf.

3. Add 3 μl dye into the eppendorf. Vortex and centrifuge briefly.

4. Heat the eppendorf at 100°C for 10 mins and centrifuge briefly.

5. Put samples in the ice bucket.

##### Gel running

1. When the gel solidified, set up the running equipment and pour 1X tank buffer.

2. Take out the comb and load 3 μl of the marker into a well and load the samples into the remaining wells.

3. Run the first 30 mins at 120V then continue to run for 1 hour at 150 V. Note: voltage and time may vary.

##### Staining

1. Take out the glass plates out of the buffer tank and split up the glass plates to take out the gel.

2. the stacking gel and put the resolving gel inside an empty box, then add the CBB staining solution until it covers the gel.

Put the box on a shaker and shake for 30 mins or until the gel turns blue.

##### Destaining

1. Pour the CBB staining solution back to the bottle and add the destaining buffer into the box until it covers the gel.

2. Put the box on a shaker and shake until the protein bands are visible or until the gel turns white.

3. Pour out the destaining buffer.

## Standard PCR

Materials:

#### Equipment:

1. PCR tubes

2. Ice bucket

3. Thermocycler

4. Pipette and pipette tips

#### Consumables:

1. MQ or ddH2O

2. 2x PCR buffer for KOD FX

3. dNTPs 2 mM

4. Forward and reverse primer (10 μM)

5. KOD FX polymerase

Protocol:

1. On the ice, add all components in a PCR tube, making up to 50 µl volume reaction.

| Components | Volume (μl) |
| --- | --- |
| MQ or ddH2O 1.1 | |
| 10x PCR buffer 1 | |
| dNTP 2.5 mM 0.8 | |
| Template 1 | |
| Forward primer 10 μM 1 | |
| Reverse primer 10 μM 0.1 | |
| Taq polymerase + vent 0.1 | |
| Total Volume | 10 |

2. Gently mix the PCR reactions and centrifuge briefly.

3. Transfer the PCR tubes to a thermocycler.

| Step | Temperature | Time |
| --- | --- | --- |
| Initial denaturalization 94°C | 2 mins | |
| 25 - 35 cycles 98°C (denaturation) | 30 secs | |
| 55°C (annealing) | 30 secs |
| 68°C (extension) | 2 mins |
| Final extension 68°C | 2 mins | |
| Hold 16°C (holding for a short time) or 4°C (holding for a long time) | ∞ | |

## Media Preparation

### 1.LB (Lysogeny Broth)

---

Materials:

#### Equipment:

1. Glass bottle (for LB broth) or Erlenmeyer flask (for agar medium)

2. Magnetic stirrer

3. Magnetic stirring bar

4. Plastic measure jug

5. Measure cylinder

6. Spoon

7. Petri dish (for agar medium)

8. Aluminum foil

9. Electronic balance

10. Weighing paper or weighing bowl

11. Pipette and pipette tips

#### Consumables:

1. DW (distilled water)

2. Tryptone

3. Yeast extract

4. NaCl

5. NaOH (10N)

6. Agar (for LB agar)

7. Antibiotics (for selection plate)

Protocol:

1. Prepare mixture as the following inside a jar with a magnetic stirring bar inside and place it on the magnetic stirrer.

| DW 98 ml | |
| --- | --- |
| Tryptone 1 g | |
| Yeast Extract 0.5 g | |
| NaCl 1 g | |
| NaOH (10N) 20 μl | |
| Total Volume | 100 ml |
| Agar (for LB agar) 1.5% | |

2. Mix well, pour the mixture into the bottle or flask and autoclave. Notes: For agar medium, pour the mixture to the petri dish and dry the plates.

For selection plates, add antibiotics as the following before pouring the mixture to the petri dish:

- Kanamycin: 100 μl/100 ml media

- Chloramphenicol: 50 μl/100 ml media

- Ampicillin: 100 μl/100 μl media

### 2.SOB (Super Optimal Broth)

---

Materials:

#### Equipment:

1. Flask

2. Magnetic stirrer

3. Magnetic stirring bar

4. Plastic measure jug

5. Measure cylinder

6. Spoon

7. Aluminum foil

8. Electronic balance

9. Weighing paper or weighing bowl

10. Pipette and pipette tips

#### Consumables:

1. DW (distilled water)

2. Tryptone

3. Yeast extract

4. 5M NaCl

5. 3M KCl

Protocol:

1. Prepare mixture as the following inside a jar with a magnetic stirring bar inside and place it on the magnetic stirrer.

| DW 96 ml | |
| --- | --- |
| Tryptone 2 g | |
| Yeast Extract 0.5 g | |
| 5M NaCl 0.2 ml | |
| 3M KCL 83 μl | |
| Total Volume | 100 ml |

2. Mix well, pour the mixture into the flask and autoclave.

### 3.SOC

---

Materials:

#### Equipment:

1. Pipette and pipette tips

#### Consumables:

1. SOB medium in a glass bottle

2. 1M MgCl2

3. 1M MgSO4

4. 20% glucose

Protocol:

1. Add chemicals as the following to a bottle of 100 ml SOB (autoclaved)

| 1M MgCl2 96 ml | |
| --- | --- |
| 1M MgSO4 2 g | |
| 20% glucose 0.5 g | |

### 4. M9 Medium

---

Materials:

#### Equipment:

1. Pipette and pipette tips

#### Consumables:

1. M9 salts (5x)

2. 20% glucose

3. 1M MgSO4

4. 1M CaCl2

5. H2O

Protocol:

1. Add chemicals as the following to a bottle

| M9 salts (5X) 20 ml | |
| --- | --- |
| 20% glucose 2 ml | |
| 1M MgSO4 200 μl | |
| 1M CaCl2 10 μl | |
| H2O 78 ml | |
| Total Volume | 100 ml |

2. Mix well, pour the mixture into the flask and autoclave.

## Bubble Production

Materials:

#### Equipment:

1. Beaker

2. Hot plate magnetic heater

3. Weighing scale

4. Pipette and pipette tips

5. Centrifuge tubes

#### Consumables:

1. 1 g sodium alginate (mixed with 100 ml ddH2O, heated and mixed using a magnetic stirrer until no powder precipitate is left)

2. MQ or ddH2O

3. 1.5 g CaCl2 (mixed with 100 ml ddH2O)

4. 15.6 g NaH2PO4 (mixed with 200 ml ddH2O, then divided into 16 tubes each containing 12.5 ml)

5. 2 ml freshly cultured bacteria (OD600= 1)

Protocol:

1. Prepare freshly cultured bacteria.

2. Adjust the OD600 of the bacteria to 1.

3. Pour 2 ml of the bacteria into 50 ml sodium alginate, and then mix well.

4. Using a pipette, slowly drip 2.5 ml of the sodium alginate solution drop by drop into 100 mL CaCl2.

5. Each experiment is supposed to accomodate around 7.7 x 107 cells of bacteria.

6. Separate the bubbles from the CaCl2 solution using filtration or decantation.

7. Pour the bubbles into 12.525 ml of buffer, and incubate for 2 hours before plating in plate medium.

## Oxidative Assay

Materials:

#### Equipment:

1. 96 wells

2. Pipette and pipette tips

3. Plate reader

#### Consumables:

1. Overnight culture (sample)

2. Paraquat (PQ)

3. Hydrogen peroxide (H2O2)

4. MQ or ddH2O

Protocol:

1. Apply 180 μl culture in 96 wells and measure OD600 and sfGFP expression as control value.

2. After 1.5 hour, dilute PQ and H2O2 with ddH2O and add it into the well as inducer.

3. Measure the OD600 and sfGFP expression through plate reader every hour.

4. Repeat Step 3 six times.

## Functional Test(*in vitro*)

Materials:

#### Equipment:

1. Eppendorf tubes

2. Pipette and pipette tips

3. High power press cell disruptor

4. 37°C incubator

#### Consumables:

1. 12 h cultured bacteria (JJU, CoaBC)

2. 50 mM L-Cysteine

3. 20 mM PLP

4. PBS (pH 7.4 buffer)

Protocol:

1. Wash bacteria twice.

2. Adjust the OD600 of bacteria to 10 and retrieve the supernatant after lysing the bacteria.

3. Run SDS-PAGE on the supernatant and whole cell samples.

4. Add a premix solution to eppendorf tubes, the contents of the premix is shown in the table below.

| Components | Volume(μl) | | |  |
| --- | --- | --- | --- | --- |
| 50mM L-Cysteiene | 20 | | |  |
| JJU | 80 | 107 | 53 | respectively 1:1/2:1/1:2 |
| CoaBC | 80 | 53 | 107 |
| 20mM PLP | 5 | | |  |
| PBS (pH 7.4 buffer) | 15 | | |  |
| Total Volume | 200 | | |  |

5. Eppendorf tubes are incubated at 37oC for 1 hour.

6. After incubation, eppendorf tubes is centrifuged at 12500 rpm for 5 minutes.

7. Rtrieve the supernatant to conduct HPLC, to measure taurine concentration.

## Functional Test(*in vivo*)

Materials:

#### Equipment:

1. Eppendorf tubes

2. Pipette and pipette tips

3. High power press cell disruptor

4. 37°C incubator

#### Consumables:

1. LB

2. 50 mM L-Cysteine

3. 20 mM PLP

4. IPTG

Protocol:

1. Preculture 4 ml LB with sample at 37°C overnight.

2. From preculture, take 2% of the bacterial culture to a fresh 20 ml LB culture.

3. Test OD600 per 2 h to determine the growth curve of the bacteria.

4. When OD600 is around 0.6, add in 20 μl IPTG as an inducer and then add L-Cystine and PLP for chemical reaction.

5. Collect samples at set hours.

6. To determine extracellular taurine concentration, centrifuge the sample and take supernatant for HPLC.

7. To determine intracellular taurine concentration, lysis the bacteria, centrifuge it then take the supernatant to conduct HPLC.

8. Run SDS-PAGE to confirm the protein expression.

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:NCKU Tainan/Results

[![Logo](https://static.igem.org/mediawiki/2021/d/df/T--NCKU_Tainan--final.png)
![logo-mobile](https://static.igem.org/mediawiki/2021/d/df/T--NCKU_Tainan--final.png)](https://2021.igem.org/Team%3ANCKU_Tainan)

* Project

  [Description](https://2021.igem.org/Team%3ANCKU_Tainan/Description)
  [Design](https://2021.igem.org/Team%3ANCKU_Tainan/Design)
  [Experiments](https://2021.igem.org/Team%3ANCKU_Tainan/Experiments)
  [Measurement](https://2021.igem.org/Team%3ANCKU_Tainan/Measurement)
  [Model](https://2021.igem.org/Team%3ANCKU_Tainan/Model)
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* Results

  [Results](https://2021.igem.org/Team%3ANCKU_Tainan/Results)
  [Proof of Concept](https://2021.igem.org/Team%3ANCKU_Tainan/Proof_Of_Concept)
  [Implementation](https://2021.igem.org/Team%3ANCKU_Tainan/Implementation)
  [Engineering](https://2021.igem.org/Team%3ANCKU_Tainan/Engineering)
  [Contribution](https://2021.igem.org/Team%3ANCKU_Tainan/Contribution)
  [Future](https://2021.igem.org/Team%3ANCKU_Tainan/Future)
* HP

  [Human Practice](https://2021.igem.org/Team%3ANCKU_Tainan/Human_Practices)
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  [Collaboration](https://2021.igem.org/Team%3ANCKU_Tainan/Collaborations)
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  [Partnership](https://2021.igem.org/Team%3ANCKU_Tainan/Partnership)
  [Inclusivity](https://2021.igem.org/Team%3ANCKU_Tainan/Inclusivity)
  [Sustainable Development](https://2021.igem.org/Team%3ANCKU_Tainan/Sustainable)
  [Excellence in Another Area](https://2021.igem.org/Team%3ANCKU_Tainan/Excellence)
* Parts

  [Parts](https://2021.igem.org/Team%3ANCKU_Tainan/Parts)
  [Improvement](https://2021.igem.org/Team%3ANCKU_Tainan/Improvement)
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  [Team](https://2021.igem.org/Team%3ANCKU_Tainan/Team)
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  [Notebook](https://2021.igem.org/Team%3ANCKU_Tainan/Notebook)
* Awards

  [Awards](https://2021.igem.org/Team%3ANCKU_Tainan/Awards)
  [Criteria](https://2021.igem.org/Team%3ANCKU_Tainan/Criteria)

* [Menbles](#Subtitle1)
* [Oxidative Stress Sensing](#Subtitle2)
* [Interferon-gamma Sensing](#Subtitle3)
* [Taurine Production](#Subtitle4)

![](https://static.igem.org/mediawiki/2021/e/e7/T--NCKU_Tainan--result.png)

## Menbles Production Experiments

### Goal

  To test whether Menbles final product can effectively protect *E. coli* Nissle 1917 under different pH values of milk tea, and to compare which bubble size is the most capable to protect our bacteria.

### Achievements

1. Proved that the bubble is effective to protect the bacteria inside by minimizing loss of bacteria, in the pH conditions of milk tea.

2. Identified which bubble size has the best structure based on recovery rates.

### Process

  After we decided on sodium alginate and calcium chloride as the raw material for Menbles, we conducted several experiments to test the number of bacteria recovered from the bubble at different pH levels of milk tea (4.5, 5.0, 5.5, 7.0). Menbles is produced by mixing 2 ml of freshly cultured *E. coli* Nissle 1917 (OD600 = 1) with 50 ml of alginate solution, then using different sizes of laboratory pipettes we dripped 2.5 ml of the solution drop by drop into calcium chloride solution. As the result of different pipette sizes being used, there are three different sizes of the bubble, with the volumes approximately equal to 14.14 mm3 (Large), 4.19 mm3 (Medium), and 1.77 mm3 (Small).

[![](https://static.igem.org/mediawiki/2021/4/4b/T--NCKU_Tainan--menblespiccombined.png)](https://static.igem.org/mediawiki/2021/4/4b/T--NCKU_Tainan--menblespiccombined.png)

Fig. 1. (A) 14.14 mm3 (Large) bubble (B) 4.19 mm3 (Medium) bubble (C) 1.77 mm3 (Small) bubble

[![](https://static.igem.org/mediawiki/2021/4/45/T--NCKU_Tainan--menblesexperimentprocedure.jpeg)](https://static.igem.org/mediawiki/2021/4/45/T--NCKU_Tainan--menblesexperimentprocedure.jpeg)

Fig. 2. Menbles Experiment Procedure

  Then, the bubbles were extracted from calcium chloride solution through filtration, which would be equally distributed and submerged into 12.5 ml of disodium hydrogen diphosphate buffer with 4 different pH levels for 2 hours at room temperature.

  After the bubbles were submerged for 2 hours, 60 μl of supernatant from every pH sample was smeared culture into LB plate medium, and the rest of the bubbles were separated from the buffer than would be crushed with the compression of a syringe. The crushed bubble should be submerged back into its original buffer, then 60 μl of supernatant samples were smeared again to another LB medium. Negative controls were also smeared into LB medium which involves buffer without any bubble present to identify any possible contamination.

  After the plates were incubated for 12 hours at 37°C, CFU quantification was conducted to determine the bacteria recovery rate from the bubble. By calculating the amount of bacteria supposed to be inside the bubble in each pH buffer, we can approximate the percentage of bacteria recovered both in the supernatant and the crushed bubble. The results shown in Fig. 3. are the bacteria recovery rate of the large, medium, and small bubbles, with additional negative control plates did not grow any colonies, so the results obtained are devoid of any contamination.

[![](https://static.igem.org/mediawiki/2021/3/3f/T--NCKU_Tainan--menbleresultcombined.png)](https://static.igem.org/mediawiki/2021/3/3f/T--NCKU_Tainan--menbleresultcombined.png)

Fig. 3. The recovery percentage of *E. coli* Nissle 1917 in bubbles with volume 14.14 mm³ (Large), 4.19 mm³ (Medium), and 1.77 mm³ (Small).

  Based on the results shown in Fig. 3, we concluded that the bubble with a volume of 4.19 mm3 (Medium) delivers the best results due to its stable recovery rate and suits our hypothesis of a lower pH will lower down the bacteria loss.

---

## Oxidative Stress Sensing

#### **Plasmid Construction for Oxidative Stress Assay**

##### **Achievement**

1. Construction of the plasmid containing different promoters that can detect oxidative stress.

##### Process

**In *E. coli*, there are two main promoters that can detect oxidative stress, the *katG* promoter (*PkatG*) ([BBa\_K3771047](http://parts.igem.org/Part%3ABBa_K3771047)) and the *soxS* promoter (*PsoxS*) ([BBa\_K3771048](http://parts.igem.org/Part%3ABBa_K3771048))**. To determine which promoter to use in our final biobrick in order to express the taurine-producing enzyme, an oxidative stress assay is essential. Hence, we need to construct plasmids with different promoters and utilize the reporter, sfGFP, to determine the intensity and sensitivity of both promoters.

  First, we conducted PCR to amplify the fragment of *PkatG*, *PsoxS*, and *soxR* gene with *PsoxS* from the chromosome of *E. coli* MG1655. SoxR is a transcriptional activator of *PsoxS*. The fragments were used to replace J23100 promoter in pSAA-J23100-*sfgfp* ([BBa\_K3771050](http://parts.igem.org/Part%3ABBa_K3771050)). We obtained three new plasmids, pSAA-*PkatG-sfgfp* ([BBa\_K3771091](http://parts.igem.org/Part%3ABBa_K3771091)), pSAA-*PsoxS-sfgfp* ([BBa\_K3771092](http://parts.igem.org/Part%3ABBa_K3771092)), and pSAA-*soxR*-*PsoxS-sfgfp* ([BBa\_K3771093](http://parts.igem.org/Part%3ABBa_K3771093)). To ensure the correctness of the sequence, we conducted colony PCR after transformation and the result showed that the plasmid contains the correct sequence (Fig. 4).

[![](https://static.igem.org/mediawiki/2021/1/15/T--NCKU_Tainan--PkatG%2C_PsoxS%2C_soxR-PsoxS_Colony_PCR.png)](https://static.igem.org/mediawiki/2021/1/15/T--NCKU_Tainan--PkatG%2C_PsoxS%2C_soxR-PsoxS_Colony_PCR.png)

Fig. 4. The electrophoresis result of colony PCR of (A) pSAA-*PkatG-sfgfp* (BBa\_K3771091), (B) pSAA-*PsoxS-sfgfp*, (BBa\_K3771092), and (C) pSAA-*soxR*-*PsoxS-sfgfp* (BBa\_K3771093). (A) M: Marker; Lane 1, 2: pSAA-*PkatG-sfgfp* (2629 bp); Lane 3: pSAA-J23100-*sfgfp* (2537 bp).​ (B) M: Marker; Lane 1: pSAA-J23100-*sfgfp* (2537 bp); Lane 2, 3: pSAA-*PsoxS-sfgfp* (3122 bp). (C) M: Marker; Lane 1, 2: pSAA-*soxR*-*PsoxS-sfgfp* (3327 bp); Lane 3: pSAA-*PsoxS-sfgfp* (2691 bp).

#### **Oxidative Stress Assay**

##### **Achievement**

1. Compared the intensity and sensitivity of different promoters under the stimulation by oxidative stress.

2. Selected a suitable promoter for regulating the expression of L-cysteine sulfinic acid decarboxylase (CSAD).

##### **Process**

  We compared the relative fluorescence intensity of sfGFP expressed by different promoters when induced by different concentrations of paraquat (PQ). Paraquat can react with oxygen to produce various types of reactive oxygen species (ROS), such as superoxide radical (O2−•), hydrogen peroxide (H2O2), and hydroxyl radical (OH−•)[[5](#ref5)], which is used to simulate abnormal oxidative stress in the intestines of stressed individuals.

  Over a period of 4.5 hours after paraquat was added, the relative fluorescence intensity was measured. J23100, a constitutive promoter, being the positive control, shows no difference in sfGFP expression level between the samples with and without paraquat. The activity of *PsoxS* ([BBa\_K3771048](http://parts.igem.org/Part%3ABBa_K3771048)) is higher than that of *PkatG* ([BBa\_K3771047](http://parts.igem.org/Part%3ABBa_K3771047)), but the activity is greatest when the SoxR transcription factor ([BBa\_K3771100](http://parts.igem.org/Part%3ABBa_K3771100)) is added in the construction together with *PsoxS* (Fig. 5 and 6).

[![](https://static.igem.org/mediawiki/parts/c/c2/T--NCKU_Tainan--sfGFP_Expression_%28PQ_%404.5h%29.png)](https://static.igem.org/mediawiki/parts/c/c2/T--NCKU_Tainan--sfGFP_Expression_%28PQ_%404.5h%29.png)

Fig. 5. sfGFP expression under high concentration of PQ

[![](https://static.igem.org/mediawiki/parts/0/0d/T--NCKU_Tainan--Low_sfGFP_Expression_%28PQ_%404.5h%29.png)](https://static.igem.org/mediawiki/parts/0/0d/T--NCKU_Tainan--Low_sfGFP_Expression_%28PQ_%404.5h%29.png)

Fig. 6. sfGFP expression under lower concentration of PQ. The comparison of different promoter strengths by relative fluorescence intensity after 4.5-hour incubation with paraquat in various concentrations was shown in these two pictures.

  Another oxidative stress assay was performed only with SoxR and *PsoxS* promoter to determine the efficiency of this detection system. The sfGFP level was measured hourly during the 4.5 hours after paraquat induction, and results showed that even low concentrations of paraquat were able to activate the detection system (Fig. 7 and 8).

[![](https://static.igem.org/mediawiki/parts/3/33/T--NCKU_Tainan--Del_Low_sfGFP_Expression_%28soxR-PsoxS%29.png)](https://static.igem.org/mediawiki/parts/3/33/T--NCKU_Tainan--Del_Low_sfGFP_Expression_%28soxR-PsoxS%29.png)

Fig. 7. sfGFP expression under low concentration of PQ.

[![](https://static.igem.org/mediawiki/parts/0/0c/T--NCKU_Tainan--Del_High_sfGFP_Expression_%28soxR-PsoxS%29.png)](https://static.igem.org/mediawiki/parts/0/0c/T--NCKU_Tainan--Del_High_sfGFP_Expression_%28soxR-PsoxS%29.png)

Fig. 8. sfGFP expression under high concentration of PQ. Relative fluorescence intensity of soxR-*PsoxS* after 4.5-hour incubation with paraquat in various concentrations was shown in these two pictures.

### Plasmid Construction for CSAD Enzyme Expression

#### Achievements

1. Construction of the plasmid containing the *soxRS* regulon and the *csad* gene.

#### Process

  After confirming the function of *soxR*-P*soxS*, the coding sequence of sfGFP was replaced with the *csad gene* ([BBa\_K3771008](http://parts.igem.org/Part%3ABBa_K3771008)), resulting in a new plasmid - pSAA-*soxR*-P*soxS-csad-6xHis* ([BBa\_K3771097](http://parts.igem.org/Part%3ABBa_K3771097)). This plasmid is essential for producing taurine in our project, and the 6xHis-tag was designed for detecting the expressed protein through western blot. We conducted colony PCR after transformation to verify whether *E. coli* uptook the correct plasmid. The size of the PCR product was as expected (Fig. 9). The plasmid was also confirmed by sequencing and has no mutations.

[![](https://static.igem.org/mediawiki/2021/0/03/T--NCKU_Tainan--soxR-CSAD_Colony_PCR.png)](https://static.igem.org/mediawiki/2021/0/03/T--NCKU_Tainan--soxR-CSAD_Colony_PCR.png)

Fig. 9. The electrophoresis result of colony PCR of plasmid pSAA-*soxR*-P*soxS-csad-6xHis*. (BBa\_K3771097) M: Marker; Lane 1: pSAA-*soxR*-P*soxS-sfgfp* (1050 bp); Lane 2, 3: pSAA-*soxR*-P*soxS-csad-6xHis* (1829 bp).

### Enzyme (CSAD) Production

#### Achievements

1. Confirmation of CSAD ([BBa\_K3771008](http://parts.igem.org/Part%3ABBa_K3771008)) enzyme expression under oxidative stress by western blot.

#### Process

  To confirm that the CSAD enzyme is successfully expressed when induced by paraquat, the western blot was performed. When the OD600 of the bacterial culture reached 0.5, we added 0.1 mM paraquat into the culture and collected samples at 2, 4, 6 hours after paraquat was added. The result confirmed that CSAD was expressed under oxidative stress. The sample collected at 6 hours after paraquat was added had the greatest expression, and CSAD expression increases as the time allowed for paraquat to induce the *SoxS* promoter increases (Fig. 10).

[![](https://static.igem.org/mediawiki/2021/5/58/T--NCKU_Tainan--soxR-CSAD_WB.png)](https://static.igem.org/mediawiki/2021/5/58/T--NCKU_Tainan--soxR-CSAD_WB.png)

Fig. 10. The western blot result. CSAD (~55 kDa); –: control; PQ: 0.1 mM paraquat.

---

## Interferon-gamma Sensing System and Production

#### **Plasmid Construction for IFN-γ Induction Assay**

##### **Achievements**

1. Constructed *PpspA-ofp*-*PlacI-ompA/oprF* ([BBa\_K3771017](http://parts.igem.org/Part%3ABBa_K3771017)) and *PpspA-ofp*-*PompA-ompA/oprF* ([BBa\_K3771018](http://parts.igem.org/Part%3ABBa_K3771018)) plasmids for the IFN-γ induction assay

##### **Process**

  For the IFN-γ induction assay, OFP is used as the reporter to compare the strengths of the *lacI* promoter ([BBa\_R0010](http://parts.igem.org/Part%3ABBa_R0010)) and the *ompA* promoter ([BBa\_K3771011](http://parts.igem.org/Part%3ABBa_K3771011)). Therefore, two plasmids with the respective promoters were constructed, while also containing the *pspA* promoter ([BBa\_K3071013](http://parts.igem.org/Part%3ABBa_K3771013)) required for the activation of the IFN-γ sensing system.

  First, the *pspA* promoter synthesized by IDT was amplified by PCR and then ligated to the *ofp* gene ([BBa\_K156009](http://parts.igem.org/Part%3ABBa_K156009)), already present in our starting pUC cloning vector, resulting in *PpspA-ofp* ([BBa\_K3771014](http://parts.igem.org/Part%3ABBa_K3771014)). To construct *PlacI-ompA/oprF* ([BBa\_K3771015](http://parts.igem.org/Part%3ABBa_K3771015)), *lacI* promoter in our starting pUC cloning vector, was ligated with the *ompA/oprF* fragment ([BBa\_K3771009](http://parts.igem.org/Part%3ABBa_K3771009)) synthesized by IDT. We then cloned the *ompA* promoter sequence ([BBa\_K3771011](http://parts.igem.org/Part%3ABBa_K3771011)) from *E. coli* MG1655 and replaced *lacI* promoter with *ompA* promoter, resulting in *PompA-ompA/oprF* ([BBa\_K3771016](http://parts.igem.org/Part%3ABBa_K3771016)).

  Both the *PlacI-ompA/oprF* ([BBa\_K3771015](http://parts.igem.org/Part%3ABBa_K3771015)) and *PompA-ompA/oprF* ([BBa\_K3771016](http://parts.igem.org/Part%3ABBa_K3771016)) fragments were double digested and ligated to the *PpspA-ofp* vector ([BBa\_K3771014](http://parts.igem.org/Part%3ABBa_K3771014)), respectively, which formed the final plasmids required for the IFN-γ assay: *PpspA-ofp-PlacI-ompA/oprF* ([BBa\_K3771017](http://parts.igem.org/Part%3ABBa_K3771017)) and *PpspA-ofp-PompA-ompA/oprF* ([BBa\_K3771018](http://parts.igem.org/Part%3ABBa_K3771018)) plasmids.

[![](https://static.igem.org/mediawiki/2021/f/f8/T--NCKU_Tainan--pspAompA.jpg)](https://static.igem.org/mediawiki/2021/f/f8/T--NCKU_Tainan--pspAompA.jpg)

Fig. 11. Confirmation of construction by PCR. (Left) M: Marker; Lane 1: *pspA* promoter (BBa\_K3071013) (~218bp); Lane 2: *ompA/oprF* (BBa\_K3771009) (~1110bp)

[![](https://static.igem.org/mediawiki/2021/1/12/T--NCKU_Tainan--pspApompAompA.jpg)](https://static.igem.org/mediawiki/2021/1/12/T--NCKU_Tainan--pspApompAompA.jpg)

Fig. 12. M: Marker; Lane 1: *pspA* promoter (~218bp); Lane 2: *ompA* promoter (BBa\_K3771011) (~309 bp); Lane 3: *ompA/oprF* (~1110bp)

#### **IFN-γ Induction Assay**

##### **Achievements**

1. Selected a suitable promoter for regulating the expression of chimeric OmpA/OprF protein

2. Determined the minimum IFN-γ concentration required for activation of the sensing system

3. Determined the time required for the system to produce a significant difference in protein expression

##### **Process**

  The two plasmids *PpspA-ofp*-*PlacI-ompA/oprF* ([BBa\_K3771017](http://parts.igem.org/Part%3ABBa_K3771017)) and *PpspA-ofp*-*PompA-ompA/oprF* ([BBa\_K3771018](http://parts.igem.org/Part%3ABBa_K3771018)) were transformed into *ompA* gene knockout strain, JW0940, to prevent OmpA from interfering with IFN-γ binding to chimeric OmpA/OprF. Human IFN-γ of concentrations 300 pM and 600 pM were added to bacteria cultures to induce the system. OFP expression (RFU) was recorded using a microplate reader and normalized by OD600. As shown in Fig. 13 and 14, starting at around 12 hours after induction, OFP expression of both samples increased dramatically.

[![](https://static.igem.org/mediawiki/2021/a/ab/T--NCKU_Tainan--proof_of_concept_4.jpg)](https://static.igem.org/mediawiki/2021/a/ab/T--NCKU_Tainan--proof_of_concept_4.jpg)

Fig. 13. OFP expression of *PpspA-ofp-PlacI-ompA/oprF* (sample 1) (BBa\_K3771017)

[![](https://static.igem.org/mediawiki/2021/a/a3/T--NCKU_Tainan--proof_of_concept_5.jpg)](https://static.igem.org/mediawiki/2021/a/a3/T--NCKU_Tainan--proof_of_concept_5.jpg)

Fig. 14. OFP expression of *PpspA-ofp-PompA-ompA/oprF* (sample 2) (BBa\_K3771018)

  As presented in Fig. 15, at 12 hours after induction, sample 2 (*PpspA-ofp-PompA-ompA/oprF*) ([BBa\_K3771018](http://parts.igem.org/Part%3ABBa_K3771018)) with the addition of 600 pM IFN-γ had higher OFP expression than that of sample 1 (*PpspA-ofp*-P*lacI-ompA/oprF*) ([BBa\_K3771017](http://parts.igem.org/Part%3ABBa_K3771017)). Since *ompA* promoter stimulates higher protein expression than *lacI* promoter, we decided to incorporate the *ompA* promoter in our final biobrick to regulate the expression of CS.

[![](https://static.igem.org/mediawiki/2021/1/1a/T--NCKU_Tainan--ifn_12h.jpg)](https://static.igem.org/mediawiki/2021/1/1a/T--NCKU_Tainan--ifn_12h.jpg)

Fig. 15. OFP expression of sample 1 (*PpspA-ofp-PlacI-ompA/oprF*) (BBa\_K3771018) and sample 2 (*PpspA-ofp-PompA-ompA/oprF*) (BBa\_K3771018)

### Plasmid Construction for CS Enzyme Expression

#### Achievements

1. Completed construction of the plasmid P*pspA-cs*-6xHis-P*ompA-ompA/oprF* ([BBa\_K3771044](http://parts.igem.org/Part%3ABBa_K3771044)) for confirming expression of CS ([BBa\_K3771012](http://parts.igem.org/Part%3ABBa_K3771012)) when induced by IFN-γ

#### Process

  In our final biobrick, the production of CS is regulated by the IFN-γ sensing system. For CS expression to be confirmed by western blot, a 6xHis-tag sequence was attached to the 3’ end of the cs sequence.

  First, *ofp* ([BBa\_K156009](http://parts.igem.org/Part%3ABBa_K156009)) in P*pspA-ofp* ([BBa\_K3771014](http://parts.igem.org/Part%3ABBa_K3771014)) was replaced with the *cs* enzyme sequence, resulting in P*pspA-cs*-6xHis ([BBa\_K3771039](http://parts.igem.org/Part%3ABBa_K3771039)). Similar to the construction of plasmids for the IFN-γ assay, P*ompA-ompA/oprF* ([BBa\_K3771016](http://parts.igem.org/Part%3ABBa_K3771016)) was double digested and ligated to the P*pspA-cs*-6xHis vector. The final P*pspA-cs*-6xHis-P*ompA-ompA/oprF* ([BBa\_K3771044](http://parts.igem.org/Part%3ABBa_K3771044)) is induced by IFN-γ, and its expression was confirmed by western blot (Fig. 16).

[![](https://static.igem.org/mediawiki/2021/b/b7/T--NCKU_Tainan--pspAcspompAompA.jpg)](https://static.igem.org/mediawiki/2021/b/b7/T--NCKU_Tainan--pspAcspompAompA.jpg)

Fig. 16. Confirmation of construction by PCR. M: Marker; Lane 1: *pspA* promoter (BBa\_K3071013) (~218bp); Lane 2: *ompA* promoter (BBa\_K3771011) (~309 bp); Lane 3: *ompA/oprF* (BBa\_K3771009) (~1110bp); Lane 4: *cs*-6xHis (BBa\_K3771012) (~1284 bp)

### Chimeric OmpA/OprF Expression

#### Achievements

1. Confirmed the expression of chimeric OmpA/OprF protein ([BBa\_K3771009](http://parts.igem.org/Part%3ABBa_K3771009))[[1](#ref1)].

#### Process

  The two plasmids P*pspA-ofp*-P*lacI-ompA/oprF* ([BBa\_K3771017](http://parts.igem.org/Part%3ABBa_K3771017)) and P*pspA-ofp*-P*ompA-ompA/oprF* ([BBa\_K3771018](http://parts.igem.org/Part%3ABBa_K3771018)) were transformed into *ompA* gene knockout strain, JW0940, to prevent simultaneous expression of both OmpA ([BBa\_K3771010](http://parts.igem.org/Part%3ABBa_K3771010)) and chimeric OmpA/OprF in the same sample. Then, western blot was performed to confirm that the chimeric OmpA/OprF protein can be expressed in the *E. coli* membrane.

  As shown in Fig. 17, MG1655 strain in lane 1 expresses OmpA, the positive control, and serves as comparison to chimeric OmpA/OprF. In lane 2, the ompA gene knockout strain, JW0940, with no transformed plasmid is the negative control. In lanes 3 and 4, chimeric OmpA/OprF (~39 kDa) is successfully expressed in JW0940, albeit in lower amounts compared to that of OmpA (~35kDa).

[![](https://static.igem.org/mediawiki/2021/c/c2/T--NCKU_Tainan--ompA_wb.jpg)](https://static.igem.org/mediawiki/2021/c/c2/T--NCKU_Tainan--ompA_wb.jpg)

Fig. 17. Confirmation of protein expression by western blot. Lane 1: OmpA (BBa\_K3771010) (~35 kDa); Lane 2: N/A (no transformed plasmid) ; Lane 3&4: OmpA/OprF chimeric protein (BBa\_K3771009) (~39kDa)

### Enzyme (CS) Production

#### Achievements

  Confirmed CS expression by western blot when under regulation of the IFN-γ sensing system.

#### Process

  The plasmid with P*pspA-cs*-6xHis-P*ompA-ompA/oprF* ([BBa\_K3771044](http://parts.igem.org/Part%3ABBa_K3771044)) was transformed into *ompA* gene knockout strain JW0940 to prevent OmpA from interfering with IFN-γ binding to chimeric OmpA/OprF. Human IFN-γ concentrations of 600pM and 1200pM were added to bacteria cultures, which were then incubated at 37°C. Bacterial pellets of each sample were collected at 6 and 12 hours after IFN-γ induction.

  Western blot of the collected samples was performed to confirm the expression of CS enzyme after IFN-γ induction. Although CS enzyme is successfully expressed, the results are not as expected, since even without IFN-γ, CS enzyme is produced (Fig. 18).

[![](https://static.igem.org/mediawiki/2021/0/07/T--NCKU_Tainan--cs_wb.jpg)](https://static.igem.org/mediawiki/2021/0/07/T--NCKU_Tainan--cs_wb.jpg)

Fig. 18. Confirmation of CS (BBa\_K3771012)(~46 kDa) expression by western blot.

  In the future, we hope to improve the sensitivity of the *pspA* promoter ([BBa\_K3071013](http://parts.igem.org/Part%3ABBa_K3071013)) by adding an inhibitor or another transcriptional element to the biobrick so that this sensing system could function more properly according to the fluctuation of IFN-γ.

---

## Proof of Function: Taurine Production

### *In Vitro* vs *In Vivo* testing

*In vitro* testing refers to experiments conducted outside the living organism, while *in vivo* testing refers to research done in the living organism. To determine taurine production that most closely resembles that of the human intestine, we wanted to conduct *in vivo* testing of the function of the JJU-CoaBC pathway. However, transformation of both JJU and CoaBC plasmids into one bacteria failed and no colonies grew. Therefore, the JJU and CoaBC plasmids were transformed and cultured separately, and JJU and CoaBC supernatants were added to a reaction solution. In this case, since the substrates are added after incubation, this is considered as *in vitro* testing of taurine production by JJU and CoaBC.

  In our second attempt to conduct *in vivo* testing of taurine production, we used the enzymes of the other taurine production pathway, including CSAD, CS, and CDO1 enzymes, which are not naturally found in the human body. There was successful transformation of the three plasmids into one bacterial colony, so *in vivo* tests of taurine production of these enzymes were conducted.

  The step by step *in vitr*o and *in vivo* testing of the taurine production pathways are described in the following sections.

### Plasmid Construction

#### Achievements

Constructed the plasmids required for determining the taurine production yield:

1. For *in vitro* testing: P*T7-jju* ([BBa\_K3771079](http://parts.igem.org/Part%3ABBa_K3771079)) and P*T7-coaBC* ([BBa\_K3771080](http://parts.igem.org/Part%3ABBa_K3771080))

2. For *in vivo* testing: P*trc-csad* ([BBa\_K3771077](http://parts.igem.org/Part%3ABBa_K3771077)), P*trc-cdo1* ([BBa\_K3771075](http://parts.igem.org/Part%3ABBa_K3771075)) , *Ptrc-cs* ([BBa\_K3771078](http://parts.igem.org/Part%3ABBa_K3771078)), and P*lacI-csad* ([BBa\_K3771076](http://parts.igem.org/Part%3ABBa_K3771076))

#### Process

##### Plasmid construction for *In Vitro* Enzyme Testing

  To construct P*T7-jju* ([BBa\_K3771079](http://parts.igem.org/Part%3ABBa_K3771079)), the *jju* ([BBa\_K3771000](http://parts.igem.org/Part%3ABBa_K3771000)) sequence synthesized by IDT was amplified by PCR and ligated to *T7* promoter ([BBa\_I719005](http://parts.igem.org/Part%3ABBa_I719005)) in the pET28a cloning vector. Similarly, *coaBC* sequence ([BBa\_K3771001](http://parts.igem.org/Part%3ABBa_K3771001)) synthesized by IDT was amplified by PCR and ligated in the same cloning vector. Digestion results are shown in Fig. 19 and 20.

[![](https://static.igem.org/mediawiki/2021/c/c2/T--NCKU_Tainan--jjudigestion.png)](https://static.igem.org/mediawiki/2021/c/c2/T--NCKU_Tainan--jjudigestion.png)

Fig. 19. Confirmation of pET28a-T7-JJU by digestion (left). M: Marker; Lane 1: Colony of pET28a-T7-JJU (~6466 bp)

[![](https://static.igem.org/mediawiki/2021/5/59/T--NCKU_Tainan--CoaBC-digestion.png)](https://static.igem.org/mediawiki/2021/5/59/T--NCKU_Tainan--CoaBC-digestion.png)

Fig. 20. Confirmation of pET28a-T7-CoaBC by digestion (right). M: Marker; Lane 1~3: Different colonies of pET28a-T7-CoaBC (5398 bp)

##### Plasmid construction for *In Vivo* Enzyme Testing

  P*trc-csad* ([BBa\_K3771077](http://parts.igem.org/Part%3ABBa_K3771077)) plasmid was constructed by ligating the amplified *csad* ([BBa\_K3771003](http://parts.igem.org/Part%3ABBa_K3771003)) fragment to the *trc* promoter ([BBa\_K864400](http://parts.igem.org/Part%3ABBa_K864400)) in the pSUI cloning vector. P*trc-cdo1* ([BBa\_K3771075](http://parts.igem.org/Part%3ABBa_K3771075)), cloned into the pSB4K1 vector, and P*lacI-csad* ([BBa\_K3771076](http://parts.igem.org/Part%3ABBa_K3771076)), cloned into the pSAA vector, were constructed using the same process. Digestion results are shown in Fig. 21, 22, and 23.

[![](https://static.igem.org/mediawiki/2021/0/0d/T--NCKU_Tainan--2.png)](https://static.igem.org/mediawiki/2021/0/0d/T--NCKU_Tainan--2.png)

Fig. 21. Confirmation of pSUI-*Ptrc-csad* by digestion (left). M: Marker; Lane 1~3: Different colonies of pSUI-*Ptrc-csad* (3674 bp)

[![](https://static.igem.org/mediawiki/2021/a/aa/T--NCKU_Tainan--3.png)](https://static.igem.org/mediawiki/2021/a/aa/T--NCKU_Tainan--3.png)

Fig. 22. Confirmation of *cs* fragment by colony PCR (middle). M: Marker; Lane 1~6: Different colonies of pSUI-*Ptrc-cs* (1272 bp)

[![](https://static.igem.org/mediawiki/2021/0/0d/T--NCKU_Tainan--2.png)](https://static.igem.org/mediawiki/2021/0/0d/T--NCKU_Tainan--2.png)

Fig. 23. Confirmation of pSB4KI-*Ptrc-cdo1* by digestion (right).M: Marker; Lane 1~3: Different colonies of pSB4KI-*Ptrc-cdo1* (5470 bp)

### *In Vitro* Tests for Taurine Production by JJU-CoaBC

#### Achievements

1. *In vitro* Test 1&2: Chose suitable *E. coli* strains for taurine production of JJU ([BBa\_K3771000](http://parts.igem.org/Part%3ABBa_K3771000)) and CoaBC ([BBa\_K3771001](http://parts.igem.org/Part%3ABBa_K3771001))enzymes

2. Determined the taurine production of JJU and CoaBC enzymes[[2](#ref2),[3](#ref3)]

#### Results

##### *In Vitro* Test 1: Taurine Production of JJU and CoaBC in BL21(DE3)

  P*T7-jju* ([BBa\_K3771000](http://parts.igem.org/Part%3ABBa_K3771000)) and P*T7-coaBC* ([BBa\_K3771080](http://parts.igem.org/Part%3ABBa_K3771080)) were each transformed into the BL21(DE3) strain. Supernatants (S) were collected to confirm extracellular enzyme expression and later, taurine production. The proteins in supernatant samples contain folded proteins and enzymes ready to function. On the other hand, bacterial pellets were washed and lysed to prepare whole cell (WC) samples to determine intracellular proteins, which contain a higher concentration of unfolded proteins. JJU and CoaBC expression in both supernatants and whole cell samples were confirmed by SDS-PAGE (Fig. 24).

[![](https://static.igem.org/mediawiki/2021/8/80/T--NCKU_Tainan--invitro1-PAGE.png)](https://static.igem.org/mediawiki/2021/8/80/T--NCKU_Tainan--invitro1-PAGE.png)

Fig. 24. Confirmation of protein expression of JJU in BL21(DE3) and CoaBC in BL21(DE3) in soluble protein and whole cell samples. M: Marker; Lane 1: whole cell of JJU in BL21(DE3); Lane 2: soluble protein of JJU in BL21(DE3) (~43 kDa);Lane 3: whole cell of CoaBC in BL21(DE3); Lane 4: soluble protein of CoaBC in BL21(DE3) (~45 kDa)

[![](https://static.igem.org/mediawiki/2021/7/70/T--NCKU_Tainan--1223.png)](https://static.igem.org/mediawiki/2021/7/70/T--NCKU_Tainan--1223.png)

Fig. 25. Taurine production of JJU in BL21(DE3) and CoaBC in BL21(DE3) in supernatant and whole cell samples.

  2:1 ratio of JJU to CoaBC supernatant volume had the highest taurine production (Fig. 25). Because JJU concentrations were lower, two times as much JJU supernatant volume compared to CoaBC supernatant volume was required to produce a significant amount of taurine.

### *In Vitro* Test 2: Taurine Production of JJU in BD7G and CoaBC in BL21(DE3)

  Since JJU expression in BL21(DE3) strain was not prominent in the supernatant, we performed another *in vitro* test in which *PT7-coaBC* ([BBa\_K3771080](http://parts.igem.org/Part%3ABBa_K3771080)) was transformed into the BL21(DE3) strain, while *PT7-jju* ([BBa\_K3771000](http://parts.igem.org/Part%3ABBa_K3771000)) was transformed into the BD7G strain instead of the BL21(DE3) strain. The BD7G strain contains chaperone protein GroELS that aids in protein folding[[4](#ref4)]. SDS-PAGE results confirm JJU and CoaBC expression in all samples (Fig. 26).

[![](https://static.igem.org/mediawiki/2021/6/63/T--NCKU_Tainan--invitro2-PAGE.png)](https://static.igem.org/mediawiki/2021/6/63/T--NCKU_Tainan--invitro2-PAGE.png)

Fig. 26. Confirmation of protein expression of JJU in BD7G and CoaBC in BL21(DE3) in soluble protein and whole cell samples. M: Marker; Lane 1: whole cell of JJU in BD7G; Lane 2: soluble protein of JJU in BD7G (~43 kDa); Lane 3: whole cell of CoaBC in BL21(DE3); Lane 4: soluble protein of CoaBC in BL21(DE3) (~45 kDa)

[![](https://static.igem.org/mediawiki/2021/9/9f/T--NCKU_Tainan--taurinejjucoabc2.png)](https://static.igem.org/mediawiki/2021/9/9f/T--NCKU_Tainan--taurinejjucoabc2.png)

Fig. 27. Taurine production of JJU in BD7G and CoaBC in BL21(DE3) in soluble protein samples.

  P*T7-coaBC* ([BBa\_K3771080](http://parts.igem.org/Part%3ABBa_K3771080)) was transformed into the BL21(DE3) strain, while P*T7-jju* ([BBa\_K3771000](http://parts.igem.org/Part%3ABBa_K3771000)) was transformed into the BD7G strain instead of the BL21(DE3) strain. The BD7G strain contains chaperone protein GroELS that aids in protein folding[[4](#ref4)]. SDS-PAGE results confirm JJU and CoaBC expression in all samples (Fig. 26).

  The whole cell and supernatant JJU and CoaBC samples are collected and added in a JJU:CoaBC volume ratio of 1:1, 2:1, and 1:2. In *In Vitro* Test 1, a greater volume of JJU sample was needed to produce the maximum amount of taurine. However, this time when P*T7-jju* was transformed into the BD7G strain, 1:1 ratio of JJU to CoaBC supernatant had the highest taurine production (Fig. 27). This suggests the activity level of JJU does not significantly differ from that of CoaBC, and both are equally crucial and effective in converting L-cysteine to taurine.

### *In Vivo* Tests for Taurine Production by CS-CSAD-CDO1

#### Achievements

1. *In Vivo* Test 1: Selected the medium in which taurine production is greatest for the CDO1 ([BBa\_K3771004](http://parts.igem.org/Part%3ABBa_K3771004)), CSAD ([BBa\_K3771003](http://parts.igem.org/Part%3ABBa_K3771003)), and CS ([BBa\_K3771002](http://parts.igem.org/Part%3ABBa_K3771002)) enzymes[[5](#ref5)]

2. *In Vivo* Test 2: Determined extracellular taurine concentrations in *∆tauD*, *∆tauD-∆ompA* double knockout (DkO), and TOP10 strains

3. *In Vivo* Test 3: Determined intracellular taurine concentrations in DkO and TOP10 strains

4. *In Vivo* Test 4: Determined taurine production yield under the regulation of the oxidative stress sensing system

#### Results

##### *In Vivo* Test 1: Choosing a Growth Medium

  Since the intestine is not as nutrient-rich as LB (lysogeny broth), we chose to test taurine production in both LB and M9 (minimal salts) mediums. M9 consists of fewer nutrients than LB and more closely mimics the nutrient composition of the human intestine.

  First, P*trc-cdo1* ([BBa\_K3771075](http://parts.igem.org/Part%3ABBa_K3771075)), P*lacI-csad* ([BBa\_K3771076](http://parts.igem.org/Part%3ABBa_K3771076)), and P*trc-cs* ([BBa\_K3771078](http://parts.igem.org/Part%3ABBa_K3771078)) were transformed into DH5α strain. In another sample, only P*trc-cdo1* and P*trc-csad* ([BBa\_K3771077](http://parts.igem.org/Part%3ABBa_K3771077)) were transformed. The two samples were each grown in LB with IPTG inducer (LB+), M9 with IPTG inducer (M9+) , and M9 without IPTG inducer (M9-) mediums.

  The samples’ respective growth curves are shown in Fig. 28 and 29. Interestingly, the sample with all three enzymes (P*trc-cdo1* +P*lacI-csad* +P*trc-cs*) had a lower growth rate in M9+ medium than in M9- medium. In the sample with two enzymes (P*trc-cdo1*+P*trc-csad*), M9 medium with or without an inducer added produced similar growth curves. Both samples grew best in LB (Fig. 28, 29).

[![](https://static.igem.org/mediawiki/2021/b/b6/T--NCKU_Tainan--4.png)](https://static.igem.org/mediawiki/2021/b/b6/T--NCKU_Tainan--4.png)

Fig. 28. Growth curve of P*trc-cdo1* +P*lacI-csad* +P*trc-cs* in different growth mediums.

[![](https://static.igem.org/mediawiki/2021/6/6e/T--NCKU_Tainan--5-5.png)](https://static.igem.org/mediawiki/2021/6/6e/T--NCKU_Tainan--5-5.png)

Fig. 29. Growth curve of P*trc-cdo1*+P*trc-csad* in different growth mediums.

  Taurine concentrations were highest at 12 hours when grown in LB (Fig. 30, 31). However, at 24 hours, taurine levels in all three mediums were very low. To more easily detect and quantify taurine production, we decided to use LB medium for the rest of the in vivo taurine production experiments. In addition, because there is no difference in taurine produced with or without CS, we decided to conduct the rest of the experiments with CSAD and CDO1 enzymes. In the future, we hope to perform more experiments with the CS enzyme.

[![](https://static.igem.org/mediawiki/2021/6/6f/T--NCKU_Tainan--6.png)](https://static.igem.org/mediawiki/2021/6/6f/T--NCKU_Tainan--6.png)

Fig. 30. Taurine production of P*trc-cdo1* +P*lacI-csad* +P*trc-cs* in different growth mediums.

[![](https://static.igem.org/mediawiki/2021/0/07/T--NCKU_Tainan--7.png)](https://static.igem.org/mediawiki/2021/0/07/T--NCKU_Tainan--7.png)

Fig. 31. Taurine production of P*trc-cdo1*+P*trc-csad* in different growth mediums.

##### *In Vivo* Test 2: Determining Extracellular Taurine Concentrations in Different *E. coli* Strains

  Once the taurine production enzymes are expressed, they can be found intracellularly and extracellularly, resulting in different taurine concentrations inside and outside the bacteria. To determine extracellular taurine, the supernatants of the bacteria culture samples are collected, and taurine production was compared between different knockout strains.

  For sample preparation, P*trc-cdo1* ([BBa\_K3771075](http://parts.igem.org/Part%3ABBa_K3771075)) and P*trc-csad* ([BBa\_K3771077](http://parts.igem.org/Part%3ABBa_K3771077)) are each transformed into *∆tauD* (taurine dioxygenase gene deletion), *∆tauD-∆ompA* (*tauD* and *ompA* gene deletion) double knockout (DkO), and TOP10 strains. In the *∆tauD* strain, the deletion of the taurine dioxygenase gene prevents taurine from degradation by catabolic enzymes in the cell. In addition to the *tauD* gene deletion, the *ompA* gene is also knocked out in the *∆tauD-∆ompA* double knockout strain, which prevents OmpA from interfering with IFN-γ binding to chimeric OmpA/OprF for the IFN-γ sensing system. TOP10 strain contains no gene deletions and serves as the control. More information about how the *tauD* and *ompA* genes were knocked out can be found in the [Engineering](https://2021.igem.org/Team%3ANCKU_Tainan/Engineering) page.

[![](https://static.igem.org/mediawiki/2021/5/5a/T--NCKU_Tainan--8-8.png)](https://static.igem.org/mediawiki/2021/5/5a/T--NCKU_Tainan--8-8.png)

Fig. 32. Growth curve of *Ptrc-cdo1*+*Ptrc-csad* in different strains.

  For *∆tauD* and DkO strains, OD600 of samples with no transformed plasmid were also recorded, serving as the negative control group (Fig. 33, 34). Even with a transformed plasmid, the bacteria still show adequate growth.

[![](https://static.igem.org/mediawiki/2021/9/96/T--NCKU_Tainan--9-9.png)](https://static.igem.org/mediawiki/2021/9/96/T--NCKU_Tainan--9-9.png)

Fig. 33. Growth curve of P*trc-cdo1*+P*trc-csad* in *∆tauD*.

[![]( https://static.igem.org/mediawiki/2021/c/cc/T--NCKU_Tainan--10-10.png)](https://static.igem.org/mediawiki/2021/c/cc/T--NCKU_Tainan--10-10.png)

Fig. 34. Growth curve of P*trc-cdo1*+P*trc-csad* in DkO.

  Extracellular CSAD and CDO1 expression at 8 and 10 hours after incubation were confirmed by SDS-PAGE (Fig. 35, 36).

[![](https://static.igem.org/mediawiki/2021/4/4a/T--NCKU_Tainan--invivo2-page.png)](https://static.igem.org/mediawiki/2021/4/4a/T--NCKU_Tainan--invivo2-page.png)

Fig. 35. Confirmation of protein expression of CDO1 and CSAD in *∆tauD*, DkO, and top10 at 8 hours. M: Marker; Lane 1: pSUI-P*trc-cdo1* and pSUI-P*trc-csad* transformed into *∆tauD*; Lane 2: pSUI-P*trc-cdo1* and pSUI-P*trc-csad* transformed into DkO; Lane 3: pSUI-P*trc-cdo1* and pSUI-P*trc-csad* transformed into top10

[![](https://static.igem.org/mediawiki/2021/4/4c/T--NCKU_Tainan--ex-PAGE2.png)](https://static.igem.org/mediawiki/2021/4/4c/T--NCKU_Tainan--ex-PAGE2.png)

Fig. 36. Confirmation of protein expression of CDO1 and CSAD in *∆tauD* and DkO at 10 hours.

  Taurine was successfully produced in *∆tauD*, DKO, and TOP10 strains (Fig. 37) Taurine concentration is highest at 10 hours after incubation for both the ∆tauD and DKO strains (Fig. 38, 39). After confirming extracellular taurine concentrations by the DkO strain, we continued the following *in vivo* tests with the DkO strain.

[![](https://static.igem.org/mediawiki/2021/1/18/T--NCKU_Tainan--TP.png)](https://static.igem.org/mediawiki/2021/1/18/T--NCKU_Tainan--TP.png)

Fig. 37. Taurine production (extracellular) of CDO1 and CSAD in different strains.

[![](https://static.igem.org/mediawiki/2021/2/2f/T--NCKU_Tainan--12-12.png)](https://static.igem.org/mediawiki/2021/2/2f/T--NCKU_Tainan--12-12.png)

Fig. 38. Taurine production (extracellular) of CDO1 and CSAD in ∆tauD.

[![](https://static.igem.org/mediawiki/2021/9/93/T--NCKU_Tainan--11-11.png)](https://static.igem.org/mediawiki/2021/9/93/T--NCKU_Tainan--11-11.png)

Fig. 39. Taurine production (extracellular) of CDO1 and CSAD in DkO.

##### *In Vivo* Test 3: Determining Intracellular Taurine Concentration in the DKO Strain

  To further understand the bacteria’s taurine production, we conducted another *in vivo* experiment to determine intracellular taurine concentration in the DkO and TOP10 strains. Instead of measuring taurine concentrations in the supernatants of the samples as we did for extracellular concentrations, intracellular taurine concentrations were determined by conducting HPLC on lysed bacterial pellets of each sample.

  For sample preparation, P*trc-cdo1* ([BBa\_K3771075](http://parts.igem.org/Part%3ABBa_K3771075)) and P*trc-csad* ([BBa\_K3771077](http://parts.igem.org/Part%3ABBa_K3771077)) are each transformed into the DKO strain and TOP10 strain. Growth curves in each of the strains are shown in Fig. 40.

[![](https://static.igem.org/mediawiki/2021/4/4d/T--NCKU_Tainan--13-13.png)](https://static.igem.org/mediawiki/2021/4/4d/T--NCKU_Tainan--13-13.png)

Fig. 40. Growth curves of P*trc-cdo1*+P*trc-csad* in DkO and top10.

  Expression of CSAD and CDO1 in both supernatant and whole cell samples was confirmed by SDS-PAGE (Fig. 41).

[![]( https://static.igem.org/mediawiki/2021/a/a3/T--NCKU_Tainan--in-PAGE.png)](https://static.igem.org/mediawiki/2021/a/a3/T--NCKU_Tainan--in-PAGE.png)

Fig. 41. Confirmation of protein expression of CDO1 and CSAD in DkO and TOP10.

[![](https://static.igem.org/mediawiki/2021/c/c1/T--NCKU_Tainan--14-14.png)](https://static.igem.org/mediawiki/2021/c/c1/T--NCKU_Tainan--14-14.png)

Fig. 42. Taurine production (intracellular and extracellular) of CDO1 and CSAD in DkO and TOP10.

  To determine intracellular taurine concentration, HPLC was conducted on the whole cell samples, which contain all soluble proteins within the bacteria. Although CSAD expression is higher in the supernatant sample of the TOP10 strain than in the DKO strain according to SDS-PAGE results (Fig. 41), DKO strain still produced more taurine than the TOP10 strain (Fig. 42). This proves that the deletion of the *tauD* gene is crucial in increasing taurine concentrations.

##### *In Vivo* Test 4: Taurine Production Under the Regulation of the Oxidative Stress Sensing System

  From the previous *in vivo* tests, we proved that the DKO strain with our transformed plasmids can successfully express CDO1 and CSAD and produce taurine. In the final *in vivo* test, the *trc* promoter in P*trc-csad* was changed to the *soxS* promoter to determine extracellular taurine production under the regulation of the oxidative stress sensing system.

  For sample preparation, P*trc-cdo1* ([BBa\_K3771077](http://parts.igem.org/Part%3ABBa_K3771077)) and *soxR*-P*soxS-csad* of the oxidative stress sensing system were both transformed into the DKO strain. Since IPTG is not naturally found in the human body, lactose was also added as an inducer instead of IPTG to consider taurine production in the intestine. Paraquat was added to induce the oxidative stress system. The growth curve is shown in Fig. 43 below.

[![](https://static.igem.org/mediawiki/2021/d/de/T--NCKU_Tainan--15-15.png)](https://static.igem.org/mediawiki/2021/d/de/T--NCKU_Tainan--15-15.png)

Fig. 43. Growth curve of P*trc-cdo1* + *soxR*-P*soxS-csad* in DkO.

  CSAD and CDO1 expression after 8 hours of incubation was successful as shown in the SDS-PAGE results (Fig. 44).

[![](https://static.igem.org/mediawiki/2021/e/ea/T--NCKU_Tainan--invivo4-PAGE.png)](https://static.igem.org/mediawiki/2021/e/ea/T--NCKU_Tainan--invivo4-PAGE.png)

Fig. 44. Confirmation of protein expression of CDO1 and CSAD with expression of CSAD regulated by *soxS* promoter in DkO.

[![](https://static.igem.org/mediawiki/2021/b/bd/T--NCKU_Tainan--16-16.png)](https://static.igem.org/mediawiki/2021/b/bd/T--NCKU_Tainan--16-16.png)

Fig. 45. Taurine production (extracellular) of CDO1 and CSAD with expression of CSAD regulated by *soxS* promoter in DkO.

  The oxidative stress sensing system successfully expressed CSAD which aided in the production of taurine. Although having a lower taurine concentration than the IPTG-induced sample, the lactose-induced sample proves that the oxidative sensing system also has the potential to activate the bacteria’s taurine production in the human intestine (Fig. 45).

  In conclusion, from the experiment data collected through four *in vivo* tests, we proved that our bacteria with the *tauD* gene deletion can successfully produce taurine when under oxidative stress. To increase *E. coli*’s specificity in detecting and responding to depressive symptoms, we hope to exchange the lactose-induced *trc* promoter with the biobrick of the IFN-γ sensing system for the regulation of CDO1 expression.

## References

1. Aurand TC, March JC. Development of a synthetic receptor protein for sensing inflammatory mediators interferon‐γ and tumor necrosis factor‐α. *Biotechnology and Bioengineering*. 2016;113(3):492-500. doi:10.1002/bit.25832
2. BRENDA - Information on EC 4.4.1.10 - cysteine lyase. Brenda-enzymes.org. Published 2021. Accessed October 6, 2021. <https://www.brenda-enzymes.org/enzyme.php?ecno=4.4.1.10>
3. Mendes V, Green SR, Evans JC, et al. Inhibiting Mycobacterium tuberculosis CoaBC by targeting an allosteric site. *Nature Communications*. 2021;12(1). doi:10.1038/s41467-020-20224-x
4. Yu T-H, Yi Y-C, Shih I-Tai, Ng I-Son. Enhanced 5-Aminolevulinic Acid Production by Co-expression of Codon-Optimized *hemA* Gene with Chaperone in Genetic Engineered *Escherichia coli*. *Applied Biochemistry and Biotechnology*. 2019;191(1):299-312. doi:10.1007/s12010-019-03178-9
5. Sung Ok Han et. al. Creating a New Pathway in Corynebacterium glutamicum for the Production of Taurine as a Food Additive. *J. Agric. Food Chem*. 2018, 66, 13454−13463Sung Ok Han et. al. Creating a New Pathway in Corynebacterium glutamicum for the Production of Taurine as a Food Additive. J. Agric. Food Chem. 2018, 66, 13454−13463

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