iGEM 2019 — Team NCTU_Formosa

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# Team:NCTU Formosa/Notebook

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  [Overview](https://2019.igem.org/Team%3ANCTU_Formosa/Experiments)

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* Model

  [Overview](https://2019.igem.org/Team%3ANCTU_Formosa/Model)

  [Computational
  Growth Model](https://2019.igem.org/Team%3ANCTU_Formosa/Growth_Model)

  [Quorum Sensing
  Model](https://2019.igem.org/Team%3ANCTU_Formosa/QS_Model)

  [Mutagenicity
  Prediction](https://2019.igem.org/Team%3ANCTU_Formosa/Mutagenicity_Prediction)
* Parts

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  [Overview](https://2019.igem.org/Team%3ANCTU_Formosa/Human_Practices)

  [Education &
  Public
  Engagement](https://2019.igem.org/Team%3ANCTU_Formosa/Public_Engagement)

  [Integrated HP](https://2019.igem.org/Team%3ANCTU_Formosa/Integrated)

  [Collaboration](https://2019.igem.org/Team%3ANCTU_Formosa/Collaborations)
* Team

  [Team Members](https://2019.igem.org/Team%3ANCTU_Formosa/Team)

  [Attributions](https://2019.igem.org/Team%3ANCTU_Formosa/Attributions)
* Notebook

  [Lab Note](https://2019.igem.org/Team%3ANCTU_Formosa/Notebook)

  [Protocol](https://2019.igem.org/Team%3ANCTU_Formosa/Protocol)
* [Achievements](https://2019.igem.org/Team%3ANCTU_Formosa/Achievement)

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[HOME](https://2019.igem.org/Team%3ANCTU_Formosa)

Project

[Description](https://2019.igem.org/Team%3ANCTU_Formosa/Description)
[Design](https://2019.igem.org/Team%3ANCTU_Formosa/Design)
[Demostration](https://2019.igem.org/Team%3ANCTU_Formosa/Demonstrate)

Wet Lab

[Overview](https://2019.igem.org/Team%3ANCTU_Formosa/Experiments)
[Experiments & Results](https://2019.igem.org/Team%3ANCTU_Formosa/Results)
[Contribution](https://2019.igem.org/Team%3ANCTU_Formosa/Contribution)
[Measurement](https://2019.igem.org/Team%3ANCTU_Formosa/Measurement)
[Safety](https://2019.igem.org/Team%3ANCTU_Formosa/Safety)
[Future Prospect](https://2019.igem.org/Team%3ANCTU_Formosa/Future_Prospect)

Model

[Overview](https://2019.igem.org/Team%3ANCTU_Formosa/Model)
[Computational Growth Model](https://2019.igem.org/Team%3ANCTU_Formosa/Growth_Model)
[Quorum Sensing Model](https://2019.igem.org/Team%3ANCTU_Formosa/QS_Model)
[Mutagenicity Prediction](https://2019.igem.org/Team%3ANCTU_Formosa/Mutagenicity_Prediction)

Parts

[Parts](https://2019.igem.org/Team%3ANCTU_Formosa/Parts)
[Basic Parts](https://2019.igem.org/Team%3ANCTU_Formosa/Basic_Part)
[Composite Parts](https://2019.igem.org/Team%3ANCTU_Formosa/Composite_Part)

[Hardware](https://2019.igem.org/Team%3ANCTU_Formosa/Hardware)

Human Practice

[Overview](https://2019.igem.org/Team%3ANCTU_Formosa/Human_Practices)
[Education and Public Engagement](https://2019.igem.org/Team%3ANCTU_Formosa/Public_Engagement)
[Integrated HP](https://2019.igem.org/Team%3ANCTU_Formosa/Integrated)
[Collaboration](https://2019.igem.org/Team%3ANCTU_Formosa/Collaborations)

Team

[Team Members](https://2019.igem.org/Team%3ANCTU_Formosa/Team)
[Attributions](https://2019.igem.org/Team%3ANCTU_Formosa/Attributions)

Notebook

[Lab Note](https://2019.igem.org/Team%3ANCTU_Formosa/Notebook)
[Protocol](https://2019.igem.org/Team%3ANCTU_Formosa/Protocol)

[Achievements](https://2019.igem.org/Team%3ANCTU_Formosa/Achievement)

Notebook

[

Your browser does not support the video tag.
](https://static.igem.org/mediawiki/2019/d/df/T--NCTU_Formosa--Notebook.mp4)

[Jul](#mJul)

[Aug](#mAug)

[Sep](#mSep)

[Oct](#mOct)

| SUN | MON | TUE | WED | THU | FRI | SAT |
| --- | --- | --- | --- | --- | --- | --- |
|  | [1](#July1) | [2](#July2) | [3](#July3) | [4](#July4) | [5](#July5) | [6](#July6) |
| [7](#July7) | [8](#July8) | [9](#July9) | [10](#July10) | [11](#July11) | [12](#July12) | [13](#July13) |
| [14](#July14) | [15](#July15) | [16](#July16) | [17](#July17) | [18](#July18) | [19](#July19) | [20](#July20) |
| [21](#July21) | [22](#July22) | [23](#July23) | [24](#July24) | [25](#July25) | [26](#July26) | [27](#July27) |
| [28](#July28) | [29](#July29) | [30](#July30) | [31](#July31) |

| SUN | MON | TUE | WED | THU | FRI | SAT |
| --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  | [1](#August1) | [2](#August2) | [3](#August3) |
| [4](#August4) | [5](#August5) | [6](#August6) | [7](#August7) | [8](#August8) | [9](#August9) | [10](#August10) |
| [11](#August11) | [12](#August12) | [13](#August13) | [14](#August14) | [15](#August15) | [16](#August16) | [17](#August17) |
| [18](#August18) | [19](#August19) | [20](#August20) | [21](#August21) | [22](#August22) | [23](#August23) | [24](#August24) |
| [25](#August25) | [26](#August26) | [27](#August27) | [28](#August28) | [29](#August29) | [30](#August30) | [31](#August31) |

| SUN | MON | TUE | WED | THU | FRI | SAT |
| --- | --- | --- | --- | --- | --- | --- |
| [1](#September1) | [2](#September2) | [3](#September3) | [4](#September4) | [5](#September5) | [6](#September6) | [7](#September7) |
| [8](#September8) | [9](#September9) | [10](#September10) | [11](#September11) | [12](#September12) | [13](#September13) | [14](#September14) |
| [15](#September15) | [16](#September16) | [17](#September17) | [18](#September18) | [19](#September19) | [20](#September20) | [21](#September21) |
| [22](#September22) | [23](#September23) | [24](#September24) | [25](#September25) | [26](#September26) | [27](#September27) | [28](#September28) |
| [29](#September29) | [30](#September30) |

| SUN | MON | TUE | WED | THU | FRI | SAT |
| --- | --- | --- | --- | --- | --- | --- |
|  |  | [1](#October1) | [2](#October2) | [3](#October3) | [4](#October4) | [5](#October5) |
| [6](#October6) | [7](#October7) | [8](#October8) | [9](#October9) | [10](#October10) | [11](#October11) | [12](#October12) |
| [13](#October13) | [14](#October14) | [15](#October15) | [16](#October16) | [17](#October17) | [18](#October18) | [19](#October19) |
| [20](#October20) | [21](#October21) | 22 | 23 | 24 | 25 | 26 |
| 27 | 28 | 29 | 30 | 31 |

## July 1

---

Build UV model

## July 2

---

Build mutation analytic program

## July 3

---

None

## July 4

---

None

## July 5

---

None

## July 6

---

None

## July 7

---

None

## July 8

---

None

## July 9

---

None

## July 10

---

None

## July 11

---

Fix mutation analytic program

## July 12

---

None

## July 13

---

None

## July 14

---

Fix hypermutation model

## July 15

---

IDT gblock resuspend

IDT Taq PCR

* Sample:All IDT parts.

## July 16

---

Dilute IDT solutions

* Sample:
  + hipA, LuxI-LuxR(without Pcon), LuxI-LuxR, GFP-Ampicillin.(Time set:
    95°C/5min, [95°C/30sec, 55°C/30sec, 72°C/143sec]x25cycles, 72°C/10min)
  + ccdB, MazF, yafQ, ydfD, chpBK, hipA. (Time set: 95°C/5min, [95°C/30sec, 55°C/30sec,
    72°C/91sec]x25cycles, 72°C/10min)

IDT dilution

* Dilute the gblock solution 50-fold

Taq PCR & gel electrophoresis

* Sample:
  + hipA, LuxI-LuxR(without Pcon), LuxI-LuxR, GFP-Ampicillin (Time set:
    95°C5/min, [95°C/30sec, 55°C/30sec, 72°C/143sec]x35cycles, 72°C/10min)
  + ccdB, MazF, yafQ, ydfD, chpBK, hipA. (Time set: 95°C/5min, [95°C/30sec, 55°C/30sec,
    72°C/91sec]x35cycles, 72°C/10min)
  + ccdB, yafQ. (Time set: 95°C/5min, [95°C/30sec, 48°C/30sec, 72°C/75sec]x35cycles,
    72°C/10min)
  + LuxI-LuxR(without Pcon). (Time set: 95°C/5min, [95°C/30sec 48°C/30sec,
    72°C/143sec]x35cycles, 72°C/10min)

## July 17

---

Taq PCR & gel electrophoresis

* Sample:LuxI-LuxR(without Pcon)

Pfu PCR & gel electrophoresis

* Sample:ccdB, yafQ

Gel extraction

* Sample:ccdB, yafQ

## July 18

---

Pfu PCR & gel electrophoresis

* Sample:ccdB, yafQ

Gel extraction

* Sample:ccdB, yafQ.

## July 19

---

Pfu PCR & gel electrophoresis

* Sample:ccdB, yafQ

Start studying machine learning

## July 20

---

None

## July 21

---

None

## July 22

---

Pfu PCR & gel electrophoresis

* Sample:ydfD, hipA,LuxI-LuxR, GFP-Ampicillin, mCherry.

Find dataset of mutation

## July 23

---

Pfu PCR & gel electrophoresis

* Sample: LuxI-LuxR, GFP-Ampicillin, ccdB, yafQ, chpBK, hipA, LuxI-LuxR(without
  Pcon)

Tramsformation

* Sample:pSB1C3, pSB1K3, pSB3C5 Backbone.

## July 24

---

Pfu PCR & gel electrophoresis

* Sample: LuxI-LuxR, mCherry, MazF, LuxI-LuxR(without Pcon)

Gel extraction

* Sample:MazF, LuxI-LuxR, GFP-Ampicillin, mCherry

Study the mechanism of mutation

## July 25

---

Pfu PCR & gel electrophoresis

- Sample:ccdB, yafQ, ydfD, hipA, LuxI-LuxR(without Pcon)

Digestion

* Sample:LuxI-LuxR, mCherry, MazF, pSB1A3

Ligation

* Sample:
  + LuxI-LuxR+pSB1A3
  + mCherry+pSB1A3
  + MazF+pSB1A3

Transformation

* Sample:
  + plasmid with LuxI-LuxR into DH5α
  + plasmid with mCherry into DH5α
  + plasmid with MazF into DH5α

Gel extraction

* Sample:ccdB, yafQ, ydfD, hipA

## July 26

---

Pfu PCR & gel electrophoresis

- Sample:LuxI-LuxR(without Pcon)

Digestion

* Sample:LuxI-LuxR, mCherry, MazF, pSB1A3

Ligation

* Sample:
  + LuxI-LuxR+pSB1A3
  + mCherry+pSB1A3
  + MazF+pSB1A3

## July 27

---

Transformation

* Sample:
  + plasmid with ccdB into DH5α
  + plasmid with yafQ into DH5α
  + plasmid with ydfD into DH5α
  + plasmid with hipA into DH5α

## July 28

---

Pfu PCR & gel electrophoresis

* Sample:LuxI-LuxR(without Pcon)

## July 29

---

Pfu PCR & gel electrophoresis

* Sample:ccdB, yafQ, ydfD, hipA

Gel extraction

* Sample:ccdB, yafQ, ydfD, hipA

Digestion

* Sample:ccdB, yafQ, ydfD, hipA, pSB1C3

Ligation

* Sample:
  + ccdB+pSB1C3
  + yafQ+pSB1C3
  + ydfD+pSB1C3
  + hipA+pSB1C3

## July 30

---

Transformation

* Sample:
  + plasmid with ccdB into DH5α
  + plasmid with yafQ into DH5α
  + plasmid with ydfD into DH5α
  + plasmid with hipA into DH5α

Pfu PCR & gel electrophoresis

* Sample:chpBK

Colony PCR & gel electrophoresis

* Sample:LuxI-LuxR, mCherry, MazF transformation product

## July 31

---

Pfu PCR & gel electrophoresis

* Sample:chpBK

Digestion

* Sample:ccdB, yafQ, ydfD, hipA, pSB1A3

Ligation

* Sample:
  + ccdB+pSB1A3
  + yafQ+pSB1A3
  + ydfD+pSB1A3
  + hipA+pSB1A3

Taq PCR & gel electrophoresis

* Sample:LuxI-LuxR, mCherry, ccdB, ydfD transformation product

## August 1

---

Transformation

* Sample:
  + plasmid with ccdB into DH5α
  + plasmid with yafQ into DH5α
  + plasmid with ydfD into DH5α
  + plasmid with hipA into DH5α

Taq PCR & gel electrophoresis

* Sample:LuxI-LuxR, mCherry, ydfD

Start training AI

## August 2

---

Pfu PCR & gel electrophoresis

* Sample:LuxI-LuxR(without Pcon)

Taq PCR & gel electrophoresis

* Sample:LuxI-LuxR(without Pcon) (With new primer.)

Culturing

## August 3

---

Plasmid extraction & Sequencing

## August 4

---

Taq PCR & gel electrophoresis

* Sample:LuxI-LuxR(without Pcon)

Pfu PCR & gel electrophoresis

* Sample:ccdB, yafQ

Gel extraction

* Sample:ccdB, yafQ gel electrophoesis product

## August 5

---

Plasmid extraction & Sequencing

* Sample:LuxI-LuxR, mCherry

## August 6

---

Digestion

* Sample:ccdB, yafQ

## August 7

---

Digestion

* Sample:pSB1A3

Ligation

* Sample:
  + ccdB+pSB1A3
  + yafQ+pSB1A3

Taq PCR & gel electrophoresis

* Sample:LuxI-LuxR(without Pcon)

Transformation

* Sample:
  + Plasmid with ccdB into Rosetta gami
  + Plasmid with yafQ into Rosetta gami

## August 8

---

Taq PCR & gel electrophoresis

* Sample:LuxI-LuxR(without Pcon)

Pfu PCR & gel electrophoresis

* Sample:ccdB, yafQ

Gel extraction

* Sample:ccdB, yafQ, LuxI-LuxR(without Pcon) gel electrophoresis product

Digestion

* Sample:ccdB, yafQ, LuxI-LuxR(without Pcon), pSB1A3

Improving accuracy

## August 9

---

Ligation

* Sample:LuxI-LuxR(without Pcon)
  + ccdB+pSB1A3
  + yafQ+pSB1A3
  + LuxI-LuxR(without Pcon)+pSB1A3

Transformation

* Sample:
  + Plasmid with ccdB into Rosetta gami
  + Plasmid with yafQ into Rosetta gami
  + Plasmid with LuxI-LuxR(without Pcon) into Rosetta gami

Start using Keras

## August 10

---

Digestion

* Sample:ccdB, yafQ, 46, pSB1A3

Ligation

* Sample:
  + ccdB+pSB1A3
  + yafQ+pSB1A3
  + LuxI-LuxR(without Pcon)+pSB1A3

Colony PCR & gel electrophoresis

* Sample:
  + plasmid with ccdB.
  + plasmid with yafQ.

## August 11

---

Transformation

* Sample:
  + Plasmid with ccdB into Rosetta gami.
  + Plasmid with yafQ into Rosetta gami.
  + Plasmid with LuxI-LuxR(without Pcon) into DH5α.

Pfu PCR & gel electrophoresis

* Sample:MazF, ydfD, chpBK, hipA.

Gel extraction

* Sample:MazF, ydfD, chpBK, hipA gel electrophoresis product.

## August 12

---

Digestion

* Sample:MazF, ydfD, chpBK, pSB1A3.

Ligation

* Sample:
  + MazF+pSB1A3
  + ydfD+pSB1A3
  + chpBK+pSB1A3

  Pfu PCR & gel electrophoresis

  + Sample:
    - hipA+pSB1A3
    - LuxI-LuxR(without Pcon)+pSB1A3

  Gel extraction

  + Sample:LuxI-LuxR(without Pcon) gel electrophoresis product.

  Culturing

  Start studying Tensorflow

## August 13

---

Digestion

* Sample:MazF, ydfD, chpBK, pSB1A3.

Culturing

* Sample:
  + Rosetta gami with ccdB
  + Rosetta gami with yafQ

Ligation

* Sample:
  + MazF+pSB1A3
  + ydfD+pSB1A3
  + chpBK+pSB1A3

## August 14

---

Colony PCR

* Sample:LuxI-LuxR+mCherry.

Pfu PCR & gel electrophoresis

* Sample:ccdB, hipA.

Gel extraction

* Sample:ccdB, hipA.

Digestion

* Sample:ccdB, hipA, LuxI-LuxR+mCherry, pSB1A3, pSB1K3.

Transformation

* Sample:
  + Plasmid with MazF into BL21 DE3.
  + Plasmid with ydfD into BL21 DE3.
  + Plasmid with chpBK into BL21 DE3.

## August 15

---

Ligation

* Sample:
  + ccdB+pSB1A3
  + hipA+pSB1A3
  + LuxI-LuxR+mCherry+pSB1K3

Transformation

* Sample:
  + Plasmid with ccdB into BL21 DE3.
  + Plasmid with hipA into BL21 DE3.
  + Plasmid with LuxI-LuxR+mCherry into DH5α.

Culturing

* Sample:
  + Culure with pSB1C3.
  + Culure with pSB1K3.
  + LuxI-LuxR(without Pcon).

Digestion

* Sample:pSB1K3

Fix growth model

## August 16

---

Plasmid extraction

* Sample:Plasmid with LuxI-LuxR(without Pcon).

Digestion

* Sample:LuxI-LuxR, mCherry, pSB1K3

Ligation

* Sample:LuxI-LuxR+mCherry+pSB1K3

Culturing

* Sample:
  + Culure with MazF.
  + Culure with ydfD.
  + Culure with chpBK.

Colony PCR & gel electrophoresis

* Sample:LuxI-LuxR+mCherry, ccdB, hipA.

## August 17

---

Culturing

Plasmid extraction

* Sample:
  + Plasmid with MazF.
  + Plasmid with ydfD.
  + Plasmid with chpBK.

Digestion

* Sample:pSB1C3

Ligation

* Sample:pSB1C3 backcone

## August 18

---

Transformation

* Sample:
  + pSB1C3 backcone into BL21 DE3.
  + LuxI-LuxR+mCherry+pSB1K3 into BL21 DE3.

## August 19

---

Colony PCR & gel electrophoresis

* Sample:
  + Plasmid with ccdB transformation product.
  + Plasmid with hipA transformation product.
  + Plasmid with LuxI-LuxR+mCherry transformation product.
  + Plasmid with pSB1C3 transformation product.

## August 20

---

Taq PCR & gel electrophoresis

* Sample:LuxI-LuxR+mCherry, ccdB, hipA, pSB1C3

Digestion

* Sample:LuxI-LuxR, mCherry, pSB1K3

## August 21

---

Ligation

* Sample:LuxI-LuxR+mCherry with pSB1K3

Digestion & gel electrophoresis

* Sample:pSB1C3 gel electrophoresis product.

Gel extraction

* Sample:pSB1C3

Ligation

* Sample:pSB1C3 backbon

Transformation

* Sample:LuxI-LuxR+mCherry+pSB1K3 into DH5α.

## August 22

---

Taq PCR & gel electrophoresis

* Sample:
  + ccdB.
  + hipA.
  + LuxI-LuxR+mCherry.
  + pSB1C3.
  + Beta carotene+pSB1A3.

Pfu PCR & gel electrophoresis

* Sample:
  + ccdB.
  + hipA.

Ligation

* Sample:LuxI-LuxR+mCherry with pSB1K3

Gel extraction

* Sample:ccdB, hipA.

Culturing

* Sample:hipA, K274000, pSB1C3.

Feature extraction

## August 23

---

Plasmid extraction

* Sample:
  + Plasmid with part hipA.
  + Plasmid with part K274000.
  + Plasmid with part pSB1C3.

Digestion

* Sample:LuxI-LuxR, mCherry, ccdB, hipA, pSB1A3, pSB1K3.

Ligation

* Sample:
  + ccdB+pSB1A3.
  + hipA+pSB1A3.

Transformation

* Sample:
  + ccdB into BL21.
  + hipA into BL21.

Gel electrophoresis & extraction

* Sample:LuxI-LuxR, mCherry.

Ligation

* Sample:LuxI-LuxR+mCherry with pSB1K3

## August 24

---

Transformation

* Sample:LuxI-LuxR+mCherry with pSB1K3 into DH5α.

## August 25

---

Digestion

* Sample:LuxI-LuxR, mCherry, pSB1C3.

Transformation

* Sample:
  + K592024 into BL21.
  + K1033917 into BL21.
  + K1633915 into BL21.
  + K864102 into BL21.

Culturing

* Sample:ccdB hipA.

## August 26

---

Gel electrophoresis & extraction

* Sample:LuxI-LuxR+mCherry.

Ligation

* Sample:LuxI-LuxR+mCherry with pSB1C3.

Plasmid extraction

* Sample:
  + Plasmid with part ccdB.
  + Plasmid with part hipA.

Transformation

* Sample:pSB1C3 with LuxI-LuxR+mCherry into DH5α.

Culturing

* Sample:Culture with part K274200.

Fix QS model

## August 27

---

Transformation

* Sample:
  + ccdB into BL21.
  + MazF into BL21.
  + yafQ into BL21.
  + ydfD into BL21.
  + chpBK into BL21.
  + hipA into BL21.

## August 27

---

Digestion & gel electrophoresis

* Sample:LuxI-LuxR, mCherry, pSB1C3

Ligation

* Sample:LuxI-LuxR+mCherry+pSB1C3

## August 29

---

Tramsformation

* Sample:Plasmid with LuxI-LuxR+mCherry into BL21.

Start using SVM & ANN

## August 30

---

Colony PCR & gel electrophoresis

* Sample:Plasmid with LuxI-LuxR+mCherry transformtion product

## August 31

---

Digestion & gel electrophoresis

* Sample:LuxI-LuxR, mCherry, pSB1C3

Ligation

* Sample:LuxI-LuxR+mCherry+pSB1C3

## September 1

---

Toxin gene function test

* Sample:ccdB, MazF, yafQ, ydfD, chpBK, hipA.

## September 2

---

Toxin gene function test

* Sample:ccdB, MazF, YafQ, ydfD, chpBK.

Tramsformation

* Sample:Plasmid with LuxI-LuxR+mCherry into BL21.

## September 3

---

Colony PCR & gel electrophoresis

* Sample:Plasmid with LuxI-LuxR+mCherry transformtion product

## September 4

---

Digestion & gel electrophoresis

* Sample:LuxI-LuxR, mCherry, pSB1C3

Ligation

* Sample:LuxI-LuxR+mCherry+pSB1C3

## September 5

---

Tramsformation

* Sample:Plasmid with LuxI-LuxR+mCherry into BL21.

Studying SVM scoring function

## September 6

---

Colony PCR & gel electrophoresis

* Sample:Plasmid with LuxI-LuxR+mCherry transformtion product

## September 7

---

Digestion & gel electrophoresis

* Sample:LuxI-LuxR, mCherry, pSB1C3

Gel extraction

* Sample:LuxI-LuxR, mCherry, pSB1C3

Ligation

* Sample:LuxI-LuxR+mCherry+pSB1C3

Colony PCR & gel electrophoresis

* Sample:
  + Plasmid with ccdB transformation product.
  + Plasmid with MazF transformation product.
  + Plasmid with yafQ transformation product.
  + Plasmid with ydfD transformation product.
  + Plasmid with chpBK transformation product.
  + Plasmid with hipA transformation product.
  + Plasmid with LuxI-LuxR+mCherry transformation product.

## September 8

---

Transformation

* Sample:Plasmid with LuxI-LuxR+mCherry into BL21.

Pfu PCR & gel electrophoresis

* Sample:ccdB, MazF, yafQ, ydfD, hipA.

## September 9

---

Digestion & gel electrophoresis

* Sample:ccdB, MazF, yafQ, ydfD, hipA.

Ligation

* Sample:ccdB, MazF, yafQ, ydfD, hipA.

## September 10

---

Transformation

* Sample:ccdB, MazF, yafQ, ydfD, hipA.

## September 11

---

Colony PCR and electrophoresis

* Sample:ccdB, MazF, yafQ, ydfD, hipA.

## September 12

---

Gel electrophoresis & gel extraction

* Sample:
  + ccdB ligation product.
  + MazF ligation product.
  + yafQ ligation product.
  + ydfD ligation product.
  + hipA ligation product.

Plasmid extraction

* Sample:
  + ccdB transformation product.
  + MazF transformation product.
  + yafQ transformation product.
  + ydfD transformation product.
  + hipA transformation product.

Toxin gene function test

* Sample:ccdB, MazF, YafQ, ydfD, chpBK.

Pfu PCR & gel electrophoresis

* Sample:ccdB, MazF, yafQ, ydfD, hipA.

Transformation

* Sample:ccdB, MazF, yafQ, ydfD, hipA.

Troubleshooting of FNR

## September 13

---

Pfu PCR & gel electrophoresis

* Sample:ccdB, MazF, yafQ, ydfD, hipA.

## September 14

---

Taq PCR & gel electrophoresis

* Sample:ccdB, MazF, yafQ, ydfD, hipA.

## September 15

---

Toxin gene function test

* Sample:ccdB, MazF, yafQ, ydfD, chpBK, hipA.

Taq PCR & gel electrophoresis

* Sample:ccdB, MazF, yafQ, ydfD, hipA.

Colony PCR & gel electrophoresis

* Sample:ccdB, MazF, ydfD, hipA.

## September 16

---

Quorum sensing function test

SDS

* Sample:pSB1C3, gfaspurple.

## September 17

---

Digestion & gel electrophoresis

* Sample:ccdB, MazF, yafQ, ydfD, hipA, pSB1C3

Ligation

* Sample:
  + ccdB+pSB1C3.
  + MazF+pSB1C3.
  + yafQ+pSB1C3.
  + ydfD+pSB1C3.
  + chpBK+pSB1C3.
  + hipA+pSB1C3.

Taq PCR & gel electrophoresis

* Sample:ccdB, MazF, yafQ, ydfD, hipA.

Pfu PCR & gel electrophoresis

* Sample:ccdB, MazF, yafQ, ydfD, hipA.

Transformation

* Sample:Plasmid with LuxI-LuxR+mCherry into DH5α.

Gfaspurple absorbance specturm

## September 18

---

Transformation

* Sample:
  + Gfaspurple into DH5a.
  + pSB1A3 backbone into DH5α.
  + pSB1C3 backbone into DH5α.
  + K274200+pSB1A3 into DH5α.

## September 19

---

Toxin gene function test

* Sample:ccdB, MazF, YafQ, ydfD, chpBK.

Colony PCR & gel electrophoresis

* Sample:ccdB, MazF, yafQ, ydfD, chpBK, hipA.

Taq PCR & gel electrophoresis

* Sample:chpBK.

## September 20

---

Digestion & gel electrophoresis

* Sample:ccdB, MazF, yafQ, ydfD, hipA, pSB1C3

Ligation

* Sample:
  + ccdB+pSB1C3.
  + MazF+pSB1C3.
  + yafQ+pSB1C3.
  + ydfD+pSB1C3.
  + chpBK+pSB1C3.
  + hipA+pSB1C3.

Data balancing

## September 21

---

Plasmid extraction

* Sample:ccdB, MazF, yafQ, ydfD, hipA

Toxin gene function test

* ccdB, MazF, YafQ, ydfD, chpBK.

Colony PCR & gel electrophoresis

* Sample:RFP, PFP

Transformation

* Sample:
  + Plasmid with ccdB into BL21.
  + Plasmid with MazF into BL21.
  + Plasmid with yafQ into BL21.
  + Plasmid with ydfD into BL21.
  + Plasmid with chpBK into BL21.
  + Plasmid with hipA into BL21.

Make competent cell & testing.

* Competent cell type:BL21.

Design UI of AI

## September 22

---

Quorum sensing function test.

Colony PCR & gel electrophoresis

* Sample:ccdB, MazF, yafQ, ydfD, chpBK.

Colony PCR & gel electrophoresis

* Sample:ccdB, MazF, yafQ, ydfD, chpBK.

Import pubchempy

## September 23

---

Gel electrophoresis.

* Sample:k864102，k592024, gfaspurple.

## September 24

---

Chemical test

* Sample:MazF, ydfD, chpBK
* Chemical:NaN3
* Concentration:1μM

## September 25

---

Cotransformation.

* Sample:Plasmid with LuxI-LuxR+mCherry & plasmid with ydfD into BL21.
* Sample:Plasmid with LuxI-LuxR+mCherry & Plasmid with hipA into BL21.

Colony PCR.

* Sample:hipA

## September 26

---

Digestion & gel electrophoresis

* Sample:LuxI-LuxR, mCherry, pSB1C3

Ligation

* Sample:LuxI-LuxR+mCherry+pSB1C3

## September 27

---

Quorum sensing function test

Transformation

* Sample:plasmisd with LuxI-LuxR+mCherry into BL21.

## September 28

---

Transformation

* Sample:plasmisd with J0yafQ1 into DH5α.

Cotransformation

* Sample:
  + plasmisd with LuxI-LuxR+mCherry & plasmid with ydfD into BL21.
  + plasmisd with LuxI-LuxR+mCherry & plasmid with chpBK into BL21.

Colony PCR & gel electrophoresis

* Sample:plasmisd with LuxI-LuxR+mCherry transformation product.

## September 29

---

Toxin gene function test

* Sample:ccdB, MazF, yafQ, ydfD, chpBK, hipA.

## September 30

---

Digestion & gel electrophoresis

* Sample:LuxI-LuxR, mCherry, pSB1C3

Ligation

* Sample:LuxI-LuxR+mCherry+pSB1C3

## October 1

---

Transformation

* Sample:
  + plasmisd with J04500 into DH5α.
  + plasmisd with LuxI-LuxR+mCherry into BL21.

Chemical test

* Sample:ccdB, MazF, yafQ, ydfD, chpBK.
* Chemical:EtBr
* Concentration:0.1μM

## October 2

---

Plasmid extraction

* Sample:ccdB, MazF, yafQ, ydfD, chpBK.

Colony PCR

* Sample:
  + plasmisd with J04500 transformation product.
  + plasmisd with LuxI-LuxR+mCherry transformation product.

## October 3

---

Toxin gene function test

* Sample:ydfD, chpBK.

Chemical test

* Sample:ydfD.
* Chemical:EtBr
* Concentration:0.05μM, 0.5μM, 1μM.

## October 4

---

Chemical test

* Sample:ydfD, chpBK.
* Chemical:EtBr
* Concentration:0.05μM, 0.5μM, 1μM.

## October 5

---

Chemical test

* Sample:ydfD.
* Chemical:EtBr
* Concentration:0.05μM, 0.5μM, 1μM.

UV test

* Sample:ydfD.
* UV type:UVB
* Strength:1mW/cm2, 2mW/cm2, 3mW/cm2.

Transformation

* Sample:Plasmid with ydfD into BL21.

Toxin gene function test

* Sample:ccdB, MazF, YafQ, ydfD, chpBK.

Colony PCR & gel electrophoresis

* Sample:MazF, yafQ, ydfD, YFP.

Make competent cell

* Sample:QS system.

## October 6

---

Plasmid extraction

* Sample:ydfD.

Transformation

* Sample:
  + Plasmid with ccdB into BL21.
  + Plasmid with MazF into BL21.
  + Plasmid with yafQ into BL21.
  + Plasmid with ydfD into BL21.
  + Plasmid with chpBK into BL21.

Cotransformation

* Sample:Plasmid with LuxI-LuxR+mCherry & plasmid ith ydfD into BL21.

Colony PCR & gel electrophoresis

* Sample:
  + Plasmid with LuxI-LuxR+mCherry transformation product.
  + Plasmid with ydfD transformation product.

Chemical test

* Sample:ydfD.
* Chemical:EtBr
* Concentration:
  + 0.5uM, 1uM.
  + 0.05uM, 0.5uM, 1uM.
  + 0.05uM, 0.1uM, 0.5uM, 1uM.

UV test

* Sample:ydfD.
* UV type:UVB
* Strength:1mW/cm2, 2mW/cm2, 3mW/cm2.

## October 7

---

Cotransformation

* Sample:Plasmid with LuxI-LuxR+mCherry & plasmid ith ydfD into BL21.

Colony PCR & gel electrophoresis

* Sample:QS-ydfD cotransformation product.

QS system function test

## October 8

---

Colony PCR & gel electrophoresis

* Sample:QS, ccdB, MazF, yafQ, ydfD, chpBK.

## October 9

---

Cotransformation function test

* Sample:QS-ydfD.

Toxin gene function test

* Sample:ccdB, MazF, YafQ, ydfD, chpBK.

## October 10

---

Cotransformation function test

* Sample:QS-ydfD.

## October 11

---

Transformation

* Sample:BBa\_I6060 into BL21.

## October 12

---

UV & EtBr mutagenicity test

* Sample:BL21 with ydfD inserted.

## October 13

---

Toxin gene function test

* Sample:ccdB, YafQ, ydfD, chpBK.

## October 14

---

UV & EtBr mutagenicity test

* Sample:ccdB, YafQ, ydfD, chpBK.

## October 15

---

Colony PCR

* Sample:BL21 with BBa\_I6060 inserted.

SDS Page

* Sample:BL21 with BBa\_I6060 inserted.

Characterization Experiment

* Sample:BBa\_I6060.

## October 16

---

UV & EtBr mutagenicity test

* Sample:ccdB, YafQ, ydfD, chpBK.

## October 17

---

Transformation

* Sample:ccdB into BL21.

## October 18

---

Toxin gene function test

* Sample:ccdB.

## October 19

---

EtBr mutagenicity test

* Sample:ccdB.

![](https://static.igem.org/mediawiki/2019/f/f7/T--NCTU_Formosa--footer2.png)


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# Team:NCTU Formosa/Experiments

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Experiment Design

[

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](https://static.igem.org/mediawiki/2019/2/2f/T--NCTU_Formosa--Exp.mp4)

Overview

   Integrating the designed gene circuit and model, we performed qualitative and quantitative analysis. What we have done, specifically, was to transform the toxin gene into *E. coli* BL21(DE3) and observed its growth curve. Based on the design of the toxin gene circuit and its selection logic, when the engineered *E. coli* encounters a growing environment with mutagen, the growth will be affected accordingly. After we documented the results from different situations and model analysis, we could thus build up a qualitative and quantitative analysis of mutagenic factors of interest.

   The experiments consisted of two main parts. One was constructing the vectors with iGEM standard cloning, and the other was the functional test of biobricks.

   In the experiment part, we first selected the toxin gene, which could steadily exist when it was not induced. Then we cloned five toxin genes into *E. coli* BL21(DE3), a strain that could produce LacI protein, respectively. So, the toxin gene would not express until we added IPTG for induction.

   We ran the electrophoresis of Taq PCR products to verify that our target gene was correctly cloned into the *E. coli*, we carried out the functional test of each toxin gene. Then we chose the ccdB gene and the ydfD gene to perform the mutagen bioassay with two well-known mutagens that cause DNA damages, EtBr and UV light.

   Altogether, we combined our experiment data points with model simulation to fit the growth curve and also calculated the mutation rate(M2) that quantified the mutagenic intensity and strength of the two mutagens.

![](https://static.igem.org/mediawiki/2019/3/3f/T--NCTU_Formosa--exp_over.png)

Figure 1: The workflow of our experiment

Go to >>>

[Experiments & Results](https://2019.igem.org/Team%3ANCTU_Formosa/Results)

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Wet Lab

[Overview](https://2019.igem.org/Team%3ANCTU_Formosa/Experiments)
[Experiments & Results](https://2019.igem.org/Team%3ANCTU_Formosa/Results)
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Model

[Overview](https://2019.igem.org/Team%3ANCTU_Formosa/Model)
[Computational Growth Model](https://2019.igem.org/Team%3ANCTU_Formosa/Growth_Model)
[Quorum Sensing Model](https://2019.igem.org/Team%3ANCTU_Formosa/QS_Model)
[Mutagenicity Prediction](https://2019.igem.org/Team%3ANCTU_Formosa/Mutagenicity_Prediction)

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Human Practice

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[Attributions](https://2019.igem.org/Team%3ANCTU_Formosa/Attributions)

Notebook

[Lab Note](https://2019.igem.org/Team%3ANCTU_Formosa/Notebook)
[Protocol](https://2019.igem.org/Team%3ANCTU_Formosa/Protocol)

[Achievements](https://2019.igem.org/Team%3ANCTU_Formosa/Achievement)

Experiments & Results

[

Your browser does not support the video tag.
](https://static.igem.org/mediawiki/2019/8/8e/T--NCTU_Formosa--Results.mp4)

![](https://static.igem.org/mediawiki/2019/0/03/T--NCTU_Formosa--circle2.png)

Toxin Gene

Mutagens Bioassay
 Function Test

Toxin Gene

Toxin Gene Sequence Modifications

   All of the toxin genes we chose were toxic to *E. coli*. Thus, during the cloning experiment, we could hardly get the intact sequence of toxin genes. Since they cause growth arrest in *E. coli*, most of the *E. coli* would go through natural selection with some mutation to lower the toxicity of the gene itself. Therefore, we expected that the natural selection (mutations) might happen during the cloning step (Table 1). However, it did not stop us from the progress of the functional test. It is expected that the naturally “modified” sequence would not lose its function but rather mild ones for survival. This logic supported the idea of the project design, for toxin genes were not to kill all the *E. coli*. Therefore, we went on to the subsequent functional test to check whether if there were functional colonies from the plates. Fortunately, all the toxin genes showed functional colonies. These colonies were sequenced, and the modified amino acid sequence of the four genes are listed below.

Table 1: Modified amino acid sequence of toxin genes. WT: Wild Type, MT: Mutation Type.

![](https://static.igem.org/mediawiki/2019/0/05/T--NCTU_Formosa--amino_acid_sequence_new.png)

   We submitted the parts with modified sequences for future iGEMers to easily passed on the work we have done for reference.

Cloning

   We conducted colony PCR to verify that our target gene was correctly cloned into the *E. coli* BL21 (DE3).

   Figure 1 was the electrophoresis results of the colony PCR with a marker on the left side and the target gene on the right side. The lengths are labeled beside each band. As a result, we successfully cloned five target genes, respectively, into *E. coli*.

![](https://static.igem.org/mediawiki/2019/1/19/T--NCTU_Formosa--table1.png)

Figure 1: Colony PCR result of toxin genes after cloning into *E. coli* BL21(DE3). (A) ccdB\* BBa\_K3256440 (B) MazF\* BBa\_K3256441 (C) YafQ\* BBa\_K3256442 (D) ydfD BBa\_K3256443 (E) ChpBK\* BBa\_K3256444.

Functional Test

   After confirming the cloning of target genes, we tested their function by measuring the O.D. value after IPTG induction when the O.D. value reached 0.3 and compared with the O.D. value of the control. The O.D. values were documented in every five minutes for seven hours in total. All experiments were completed in triplicates. (Figure 2~6)

![](https://static.igem.org/mediawiki/2019/4/48/T--NCTU_Formosa--ccdB_functional.png)

Figure 2: Growth curve of *E. coli* BL21(DE3) with 500uM IPTG induction ccdB toxin gene (blue), and control (orange)

![](https://static.igem.org/mediawiki/2019/3/38/T--NCTU_Formosa--MazF_functional.png)

Figure 3: Growth curve of *E. coli* BL21(DE3) with 500uM IPTG induction MazF toxin gene (blue), and control (orange).

![](https://static.igem.org/mediawiki/2019/f/f0/T--NCTU_Formosa--YafQ_functional.png)

Figure 4: Growth curve of *E. coli* BL21(DE3) with 500uM IPTG induction YafQ toxin gene (blue), and control (orange).

![](https://static.igem.org/mediawiki/2019/4/4a/T--NCTU_Formosa--ydfD_functional.png)

Figure 5: Growth curve of *E. coli* BL21(DE3) with 500uM IPTG induction YdfD toxin gene (blue), and control (orange).

![](https://static.igem.org/mediawiki/2019/5/5c/T--NCTU_Formosa--chpBK_Functional.png)

Figure 6: Growth curve of *E. coli* BL21(DE3) with 500uM IPTG induction ChpBK toxin gene (blue), and control (orange)

Calculating Toxicity of Toxin Genes

  We first fit the control’s experiment data to the following equation,
$$\frac{dB\_{T}}{dt}= g\cdot B\_{T} (1-\frac{B\_{T}}{B\_{Max}})$$   and we fit the induced data to another equation,
$$\frac{dB\_{T}}{dt}= g\cdot B\_{T} (1-\frac{B\_{T}}{B\_{Max}})-T\_{toxin}\cdot B\_{N}\cdot [toxin]$$  Next, we compared the two equations to calculate Ttoxin, the toxicity of the toxin gene (Figure 7). In the end, we chose the ydfD gene, which had the most significant toxicity, and the ccdB gene, which had relatively weak toxicity, moving on to the functional test with mutagens.

![](https://static.igem.org/mediawiki/2019/3/3b/T--NCTU_Formosa--Growth_Difference.png)

Figure 7: Toxicity of different toxin genes. Ttoxin: The toxicity of toxin gene.

Mutagens Bioassay Function Test

  According to the toxin gene functional test, with selected ccdB and ydfD for mutagens bioassay. To see whether our design can work as expected, we put the transformed *E. coli* into a different dose of mutagens (UV light and EtBr), respectively. Since the mutagen might cause growth inhibition, it was difficult to tell how strong the mutation rate caused by the mutagen was only through the raw data of O.D.600. Therefore, we took the maximum O.D value of the controlled group (which represented the none-induced *E. coli* treated with the same dose of mutagen) as 100%, and the minimum value is 0% to normalize the data, and we called the normalized data growth population percentage.

Functional Test of Mutagenic Bioassay on UV Light

  In the environment, UVB (275～320nm) is the most carcinogenic wave band that humans usually contact according to the reference, so we chose it to be our physical mutagen for the functional test of bioassay. In the experiment, we exposed them to the different intensity of UV light for 5 seconds and induced the toxin gene when its O.D reached 0.3. After that, we documented the O.D. of the *E. coli* every 5 minutes for 7 hours. The results of the two engineered *E. coli* with ydfD and ccdB, respectively, under different UV light intensity, were shown below (Figure 9, 10).

![](https://static.igem.org/mediawiki/2019/1/12/T--NCTU_Formosa--UV_ydfD.png)

Figure 8: The growth curve of ydfD with different intensity of UV Light.

![](https://static.igem.org/mediawiki/2019/2/26/T--NCTU_Formosa--UV_ccdB.png)

Figure 9: The growth curve of ccdB with different intensity of UV Light.

Functional Test of Mutagenic Bioassay on EtBr

  We chose EtBr, a chemical compound that causes mutation and usually seen in laboratories to be our chemical mutagen. First, we made them added different concentrations of EtBr for creating different mutagenic strengths and added 500uM IPTG to induce the toxin gene when its O.D reached 0.3. After that, we documented the O.D value of *E. coli* every 5 minutes for 7 hours. Below was the EtBr mutagenic effect on the two toxin genes.

![](https://static.igem.org/mediawiki/2019/e/e6/T--NCTU_Formosa--EtBr_ydfD_NEW.png)

Figure 10: The growth curve of ydfD with different dose of EtBr

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

Figure 11: The growth curve of ccdB with different dose of EtBr

  From the experiment result, we can see that the curve of ydfD has a more significant drop than ccdB after we induced the toxin gene. This phenomenon can be explained, for the sequence of ccdB has been modified (Figure 11, 12). Therefore, its toxicity has been weakened compare with the intact ydfD sequence.

Mutation rate analysis

  According to the growth curve of two mutagenic factors with two selected toxin genes respectively for building up mutagenic bioassay, we further converted the raw data above to mutation rate of the mutagen which we called M2, we used the following differential equation from modeling to fit the experiment data:
$$\frac{dB\_{N}}{dt}=[ g\cdot B\_{N}(1-\frac{B\_{T}}{B\_{Max}}) -T\_{chem}\cdot B\_{N}](1-M\_{1}-M\_{2})-T\_{toxin}\cdot B\_{N}\cdot [toxin]$$
$$\frac{dB\_{M}}{dt}=[g\cdot B\_{N}(1-\frac{B\_{T}}{B\_{Max}})-T\_{chem}\cdot B\_{N} ] (M\_{1}+M\_{2})+[g\cdot B\_{M}(1-\frac{B\_{T}}{B\_{Max}})-T\_{chem}\cdot B\_{M}]$$
$$\frac{dB\_{T}}{dt}=[g\cdot B\_{T}(1-\frac{B\_{T}}{B\_{Max}})-T\_{chem}\cdot B\_{T} ] -T\_{toxin}\cdot B\_{N}\cdot [toxin]$$

  We were first fixed the parameters from the isolated experiments and essays, and then used Matlab to estimate the mutation rate, M2, with the most appropriate curve. Below is the linear regression of M2 and dose of mutagen (Figure 12~15).

![](https://static.igem.org/mediawiki/2019/4/4e/T--NCTU_Formosa--UV_M2_ydfD_YA.png)

Figure 12: Linear correlation of mutation rate in UV intensity (ydfD)

![](https://static.igem.org/mediawiki/2019/0/01/T--NCTU_Formosa--UV-M2_ccdB_NEW.png)

Figure 13: Linear correlation of mutation rate in UV intensity (ccdB)

![](https://static.igem.org/mediawiki/2019/e/ee/T--NCTU_Formosa--EtBr_M2_ydfD_YA.png)

Figure 14: Linear correlation of mutation rate in EtBr concentration (ydfD)

![](https://static.igem.org/mediawiki/2019/2/22/T--NCTU_Formosa--EtBr_M2_ccdB_YA.png)

Figure 15: Linear Correlation of Mutation Rate in EtBr concentration (ccdB)

  We can see that the M2 we calculate was a highly positive correlation with the dose of mutagens. It verified that by using our designed gene circuit and model, the mutagenic bioassay system we constructed is highly credible and can be further used for mutagenic studies for unknown substances. For the detail of how we build up the simulation equation and the verification of the growth curve model, please see the [growth curve model](https://2019.igem.org/Team%3ANCTU_Formosa/Growth_Model).

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