iGEM 2022 — Team kait-japan

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{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
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## What should this page contain?

---

## 7/13

Practicing experimental techniques

## 8/4

Team meeting

## 8/5

Collected data on the enzymes used for the experiment

## 8/8

Collected data on the enzymes used for the experiment

## 8/9

Collected data on the enzymes used for the experiment

Formed parts used in the experiment via snapgene.

## 8/10

Formed parts used in the experiment via snapgene.

## 8/11

Study session on the theme with the members

## 8/12

Sorted out practical difficulties with the help of the advisors.

## 8/19

Preparation of all solution that will be used during the experiment.

## 8/22

Brainstormed ideas for promotion video.

## 8/23

Protocol printouts and preparation of instruments.

## 8/24

Wrote down script for promotion video.

## 8/25

Re-writing the script for the promotion video.

## 8/29

Making slides for video

## 8/30

Making slides for video

## 8/31

Plasmid extraction

## 9/1

Electrophoresis.

Nanodrop.

Preparation to upload promotion video

## 9/2

Fixing technical errors of promotion video

## 9/5

A lesson on Fusion

A lesson on Snapgene

## 9/6

Plasmid extraction

Electrophoresis

Fusion

nanodrop

## 9/8

Restriction enzyme treatment

## 9/9

Electrophoresis

## 9/14

Calculation of bp via ImageJ

## 9/19

Gel extraction

Electrophoresis

## 9/30

SLiCE

Transformation of HMG-CoA reductase front

## 10/3

Restriction enzyme treatment of synthase and pTAC via Ecor1 and pst1

Colony PCR

Making of master plate

## 10/4

Restriction enzyme treatment of synthase and pTAC via Ecor1 and pst1

Plasmid extraction of Reductase front in pMD20 in E. coli

## 10/6

Electrophoresis

PCR and iPCR

Gel extraction

PCR product purification

## 10/7

Disposal of waste

Electrophoresis

ligation

## 10/10

PCR and iPCR

## 10/11

Electrophoresis of PCR and iPCR

Transformation (synthase in pTAC)

Wiki creation.

## Inspirations

---

* [2018 Munich](http://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

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{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
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#### Silver Medal Criterion #1

Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle. This achievement should be distinct from your Contribution for Bronze.

If you plan to show engineering success by creating a new Part that has been shown to work as expected, you must document your contribution on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page) for your team to be eligible for this criteria.

---

Please see the [2022 Medals Page](https://competition.igem.org/judging/medals) for more information.

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{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
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## SLICE Reaction

SLICE reactions were performed at a molar ratio of vector:insert=1:2.
1) Add 10x SLICE buffer 2.5 μL, SLiCE 2.5 μL, insert 150 fmol and vector 25fmol. Make the total volume 365μL with D2W, and gently pipette.
2) Incubate in a 37°C water bath for 15 minutes.
3) E. coli JM109 was transformed with the whole amount.

### Transformation

1. Plasmid and ligation sample are inserted into a microtube containing competence cell of 100μL.
2. Leave the mixture on ice for 30 mins.
3. Heat shock(temperature-42℃ for 45 sec).
4. Add 250μL of SOC broth and leave it on a shaking incubator for 1hour(37℃).
5. Add 100μL of incubated mixture is applied to a agar plate containing ampicillin.

InversePCR and PCR were performed using the primers and methods shown below.

##

### Inverse PCR (KOD-Plus-Neo, TOYOBO)

The conditions of iPCR were as follows: 94°C for 2 min, [98°C for 10 s, 55°C for 30 s, and 68°C for 150 s] repeats 35 cycles. Then, the sample was held at 4°C.

inversePCR Fw CTTATGGCGACGCTAACATT
Tm：53℃
inversePCR Rv GGGTACCGAGCTCGAATTCA
Tm：57℃

### PCR (KOD Fx Neo, TOYOBO)

The conditions of PCR were as follows: 94°C for 2 min, [98°C for 10 s, 55°C for 30 s, and 68°C for 90 s] repeats 35 cycles. Then, the sample was held at 4°C.

pMD20 Fw　ATGACCATGATTACGCCAAGC
Tm:56℃
pMD20 Rv　TGTGCTGCAAGGCGATTAAG
Tm:55℃

## Gel purification

1. Cut out the desired band from the agarose gel using a clean cutter.
2.Place the gel strip in a micro tube and weigh it on an electronic balance.
3.Add Buffer QG at a 1:3 ratio of gel:Buffer QG (100 mg of gel = 100 μl).
4.Incubate at 50°C for 10 minutes. \*Vortex the tube every 2-3 minutes to dissolve the gel.
5.When the gel is completely dissolved, check that the mixture is yellow in color.
6. Add the same volume of isopropanol as the gel volume to the sample and mix.
7. Set the QIAquick Spin Column into the 2ml Collection Tube provided.
8. Apply the sample to the QIAquick Column and centrifuge for 1 minute to bind DNA.
9. Discard the flow-through and return the QIAquick Column to the same tube.
10. Add 500µl of Buffer QG to the QIAquick column and centrifuge for 1 minute.
11.Discard the flow-through and return the QIAquick column to the same tube.
12. Add 750µl of Buffer PE to the QIAquick column for washing and centrifuge for 1 minute.
13. Discard the flow-through and return the QIAquick column to the same tube.
14. Allow the column to stand for 2 minutes.
15. Centrifuge the QIAquick column for 1 minute in the provided 2 ml collection tube to remove any residual wash buffer.
16. Place the QIAquick column into a new 1.5 ml micro tube.
17. To increase the DNA concentration, add 30 μl of Buffer EB to the center of the QIAquick membrane, allow the column to stand for 1 minute, and centrifuge for 1 minute.

## Plasmid extraction

1. centrifuge (TIBITAN 1min) a 1.5ml tube containing 1ml of the pre-cultured bacteria solution
2.Discard the supernatant so as not to destroy the pellet.
3.Add 1ml of the pre-cultured bacteria solution and centrifuge (TIBITAN 1min).
4.Discard the supernatant twice, 900 μl at a time, so as not to destroy the pellet. 5.Add 200 μl of Solution I, and confirm that the bacteria are completely suspended with a vortex mixer.
6.Add 400 μl of Solution Ⅱ and mix slowly and gently by inversion, and confirm that the solution becomes clear.
7.Add 300 μl of Solution III and mix by vortexing and inverting, and confirm that the solution becomes cloudy and solids appear.
8. centrifuge (10,000G, 40min, 4℃)
9. take out the supernatant only and put it into a new 1.5ml tube to avoid precipitation.
If white precipitate enters the microtubes, centrifuge again.
If any liquid remains, repeat steps 7 and 8 several times.
10. add 2µl of RNase (10mg/ml) and allow to react for 30min at 37°C in a heat block.
11.Add 200 µl of phenol:chloroform (1:1) and stir vigorously.
12. centrifuge (1000G, 5min, 4℃)
13. remove all of the supernatant only, not the interlayer, and put it into a new 1.5ml tube
14. add 200µl of chloroform to the micro tube containing the supernatant, tap the tube, and agitate vigorously
15. centrifuge (10,000 G, 1 min, 4°C)
16. transfer the supernatant (up to 750ml) to a new 1.5ml tube after centrifugation
17.Add the same volume of PEG as the aspirated supernatant.
18.
centrifuge (20,400 G, 40 min, 4°C)
Discard the supernatant and add 700µl of 70% ethanol to it .

19.The white material is plasmid, so pour off the liquid from the opposite side of the white pellet to avoid peeling off the plasmid.
20.
centrifuge (10,000 rpm, 30 min, 4°C)

21. discard the supernatant, turn the microtubes upside down on a Kimwipe, and dry (5~10min)

22. add 30µl of 1×TE to the dried microtubes and dissolve the pellet by pipetting.

## Restriction Enzyme Treatment

1. Set heat block to 37°C.
2.Mix BST and 10×H buffer by tapping, and spin down with TIBITAN.
3.Transfer 18.6 μL of sample from a 1.5-ml tube containing the entire amount of Sample to a new tube.
4.Add 2.0 µL of 10×H buffer.
5.Add 0.2 µL of BSA.
6.Add 0.5 µL each of PSPⅠand EcoRⅠ.
7.After tapping, spin down using TIBITAN.
8.Incubate at 37°C for 3 hours (overnight is also acceptable).v

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{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
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# RESULTS

(Practice)Extraction of plasmid using the Alkaline extraction method (shown in fig.1).

![](https://static.igem.wiki/teams/4487/wiki/fusion-results/2022-09-05-ptac2-m-apeki-plasmid.png)

fig.1 pTAC2 including insert

Extraction of Plasmid, pTAC2 including insert, using the Alkaline extraction method,
RNase(100μg/mL) was used. RNA fragments were seen at the bottom of the gel, furthermore, desired band was observed (shown in fig.2).

![](https://static.igem.wiki/teams/4487/wiki/fusion-results/2022-09-06-ptac2-m-apeki-plasmid.png)

fig.2 pTAC2 including insert

Extraction of plasmid, pTAC2 including insert, using the PEG precipitation.
RNA was not seen while desired band was clearly visible (shown in fig.3).

![](https://static.igem.wiki/teams/4487/wiki/fusion-results/2022-09-08-ptac2-m-apeki-plasmid.png)

fig.3 pTAC2 including insert

Extraction of plasmid, pTAC2-M. ApeKI, using the PEG precipitation.
RNA was not seen while desired band was clearly visible (shown in fig.4).

![](https://static.igem.wiki/teams/4487/wiki/fusion-results/2022-09-09-ptac2-m-apeki-plasmid.png)

fig.4 pTAC2 including insert

Extraction of plasmid, pMD20 from E. coli　JM109, using the PEG precipitation.
RNA was not seen while desired band was clearly visible (shown in fig.5).

![](https://static.igem.wiki/teams/4487/wiki/fusion-results/2022-09-14-pmd20-in-e-coli-plasmid.png)

fig.5 pMD20 from E. coli

Previously extracted plasmid, pMD20, digested by the restriction enzyme, SmaI.
Calculations of molecular weight done via ImageJ (shown in fig.6 and table 1)

![](https://static.igem.wiki/teams/4487/wiki/fusion-results/2022-09-15-pmd20-in-e-coli-plasmid-sma1.png)

fig.6 pMD20 after digestion by SmaI

Gel extraction of digested plasmid, pMD20.

Table 1 pMD20 (SmaI+gel purification)

![](https://static.igem.wiki/teams/4487/wiki/fusion-results/bp-of-sma1.png)

Extraction of plasmid, pTAC2 in JM109, using the PEG precipitation.
RNA was not seen while desired band was clearly visible (shown in fig.7).

![](https://static.igem.wiki/teams/4487/wiki/fusion-results/2022-09-19-pmd20-in-e-coli-plasmid-sma1-gel-purification.png)

fig.7 pTAC2 from JM109

Desired band of HMG-CoA synthase is visible (shown in fig.8)

![](https://static.igem.wiki/teams/4487/wiki/fusion-results/2022-09-30-ptac2-in-jm109-plasmid.png)

fig.8 HMG-CoA Syntase

left side - Shows the bands of pMD20 reductase front plasmid.
The ride side – Shows the bands of pTAC digested by EcoRI, and PstI.
The result is shown in fig.9.
Bands of Synthase after being digested by EcoRI and PstI

![](https://static.igem.wiki/teams/4487/wiki/fusion-results/2022-10-03-hmg-coa-syntase.png)

fig.9 pMD20 reductase front plasmid, Synthetase and pTAC after digestion by EcoRI and Pst1

Gel extraction of digested plasmid, pTAC, and synthase (shown in fig.10).

![](https://static.igem.wiki/teams/4487/wiki/fusion-results/2022-10-05.png)

fig.10 Gel extraction of digested pTAC and Synthase

Left side-pTAC and Synthase
Right side-PCR product of Reductase front in pMD20 (To check whether recombination was successful) and iPCR to amplify pMD20 including a part of reductase to introduce the remaining sequence of reductase (shown in fig.11).(fail)

![](https://static.igem.wiki/teams/4487/wiki/fusion-results/2022-10-06-pcr-ipcr.png)

fig.11 pTAC and Synthase. PCR and iPCR of reductase front in pMD20.

iPCR to cut pMD20 to introduce the remaining sequence of reductase (shown in fig.12). (fail)

![](https://static.igem.wiki/teams/4487/wiki/fusion-results/2022-10-07-ptac-and-synthase-neo1-2-and-fx1-2-in-pmd20.png)

fig.12 iPCR of reductase front in pMD20

PCR of Reductase front in pMD20(To check whether recombination was successful )(shown in fig.13).

![](https://static.igem.wiki/teams/4487/wiki/fusion-results/2022-10-11-ipcr.png)

fig.13 PCR of reductase front in pMD20

![](https://static.igem.wiki/teams/4487/wiki/fusion-results/2022-10-11-pcr.png)

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