iGEM 2010 — Team Chiba

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# Team:Chiba/Notebook 1

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* [Notebook 1](https://2010.igem.org/Team%3AChiba/Notebook)
* [Notebook 2](https://2010.igem.org/Team%3AChiba/Notebook_2)
* [Notebook 3](https://2010.igem.org/Team%3AChiba/Notebook_3)

### 2010-08-31

Making plate with Broth

```
Amp x 13
Kan x 1
Amp + IPTG x 1
```

Transformation

```
R0010 B0030 B0032 B0033 K145270 I746903 R0051 C0051 K145001 C0012 K145001 C0012 P0312 I719015
```

### 2010-09-02

Mini-prep(DNA transformated in August 31th)

```
I13522 C0040 C0052 C0053 C0080 C0072 I0500 Q04510 Q04400 Q04121 R0040 B0014
B1101 I715038 K081012 K082034 K113007 I763011
```

### 2010-09-03

Preparing for experiment

```
We inserted plasmid with cI promoter + GFP to E.coli. And
```

### 2010-09-04

Preparing for experiment

```
I746903
```

### 2010-09-05

```
K091107 R0065 K145150 Q01121 Q01511 Q03121 Q03400 Q03530 Q04511 J06800 J06801
```

### 2010-09-06

Co-transformation

```
Plux – gfp + Pc each 1uL
SOC 200uL
```

### 2010-09-07

```
E0240 R0061 I0500 C0080 C0072 Q04510 Q04400 Q04121
```

### 2010-09-08

Transformation

```
XL10GkanR 30uL DNA 1uL SOC 100uL
K145269 K145205 R1062 I0462 P0440 K091204 J01101 P0140 K091204 J01101 P0140 P0340 B0034
```

### 2010-09-14

```
1) K091204(2-8J) Pc – luxR function check
  [Pc – luxR(pSB1A2) + Plux – GFP(pAC)] each 1uL + XL10GkanR 50uL
  AHL : 30C8HSL(1/10000 of 1μM)
2) T9002
```

### 2010-09-16

```
 PCR
  T7/cI(OR1/2) – GFP
              (1)      (2)
  --------------------------------
   template   1uL
   primer     5uL
   F          5uL
   R          5uL
   10x buffer 5uL
   dNTP       5uL
   NFW        28uL
   ventP      1uL
  -------------------------------
             50uL     50uL
 Transformation of BBa_J04450(3A) -> incubation
```

### 2010-09-19

```
From 2010 Biobrick
 plasmid with Cm antibiotic   -------------
  1-3C 1-3A pSB1C3
 plasmid with Amp antibiotic  -------------
  1-1K pSB1A3
 ↓
To transformate total five types, we seeded to plates.
 ↓
PCR products
 ↓
Gel extraction
 ↓  ← ADB buffer
ZYMO purification
```

1. To prepare 2ml tube(for ZYMO purification) + column, add the solution and operate centrifuge in 1 minute. Throw away left solution.

2. Same to mini-prep

```
 * wash buffer 200uL -> centrifuge 30sec -> throwing away left solution.  X2
 * wash buffer 200uL -> centrifuge 1min -> throwing away left solution.  X1
 * operating centrifuge in 1min with empty tube -> throwing away left solution.  X1
```

3. NFW elution(10uL)
Add NFW and wait 1min. Operate centrifuge in 1min
4. Gel electrophoresis(each 1uL)

```
SOEing PCR
 ①T7/cI(OR2)-F-R
 ②T7/cI(OR1)-F-R
 ③Ptet-luxR-(OR2)
 ④Ptet-luxR-(OR1)
 ⑤Prom-luxR-(OR2)
 ⑥Prom-luxR-(OR1)
1. ①1uL, ③3uL
2. ②1uL, ④4uL
3. ①1uL, ⑤1uL
4. ②1uL, ⑥1uL
```

```
1. ①-③
2. ②-④
 --------------------------
 template    1uL-4uL
 primer Fwd  -
 primer Rev  -
 dNTP        5uL
 10xbuffer   5uL
 NFW         34uL
 VentP       1uL
 -------------------------
             50uL
```

### 2010-09-20

Transformation

```
BBa_P0453(B0034-C2P22-T-T) RBS_C2P22 for PCR template
2-1 P    pSB1AK3
11:30 ~   DNA : 1uL    XL10GkanR : 30uL    SOC : 100uL
```

ZYMO purification

```
From PCR product from SOEing PCR (09-19)
1. Add PCR products(50uL) and DNA binding buffer(200uL) into 1.5mL tube(Commonly adding DNA binding buffer for double
amount of PCR products, but it must be 200uL at least). After that, mix this tube for a second with vortex and move
to 2mL tube with column. After operating centrifuge, transfer remaining a little PCR product to 2mL tube with column.
2. centrifuge 1min → throwing away left solution
3. Same to mini-prep
 * wash buffer 200uL -> centrifuge 30sec -> throwing away left solution.  X2
 * wash buffer 200uL -> centrifuge 1min -> throwing away left solution.  X1
 * operating centrifuge in 1min with empty tube -> throwing away left solution.  X1
4. NFW elution(10uL)
```

↓

```
PCR Condition
 94°C – 5min
 94°C – 15sec     --
 52°C – 30sec       │-10 cycles
 72°C – 1min      --
 72°C – 10min
 ----------------
 4°C   stop

After 10 cycles, we added primer to SOEing PCR product(completed ZYMO purification)
 -------------------------
 template      10uL
 primer Fwd     5uL
 primer Rev     5uL
 dNTP           5uL
 10xbuffer      5uL
 NFW            19uL
 VentP          1uL
 -------------------------
                50uL

Using primer
 1. ①-③ Fwd : Ptet-luxR∙FA-R  Rev : TcIg∙FA-R
 2. ②-④ Fwd : Ptet-luxR∙FA-R  Rev : TcIg∙FA-R
 3. ①-⑤ Fwd : PluxR∙FA-R     Rev : TcIg∙FA-R
 4. ②-⑥ Fwd : PluxR∙FA-R     Rev : TcIg∙FA-R
 5. control

Using template
 1. Ptet-LuxR-PT7/cI(OR2)-GFP
 2. Ptet-LuxR-PT7/cI(OR1)-GFP
 3. Prom-LuxR-Pt7/cI(OR2)-GFP
 4. Prom-LuxR-Pt7.cI(OR1)-GFP
 5. control

PCR condition(usingTAKARA PCR thermal cycler)
 94°C – 5min
 94°C – 15sec     --
 51°C – 30sec       │-25 cycles
 72°C – 1min      --
 72°C – 10min
```

↓
Gel Electrophoresis

```
Cm             Amp
1-3C            1-1K
1-3A            pSB1A3
pSB1C3
```

↓
Liquid Incubation(37°C) 7:15~

```
-------------------------
template       3uL
primer Fwd     5uL
primer Rev     5uL
dNTP           5uL
10xbuffer      5uL
NFW            26uL
VentP          1uL
-------------------------
               50uL

Using primer
 Fwd : pSB1C3FA-F
 Rev : pSB1C3FA-R

Using template
 1. 1-3C(J04450,pSB3C5)
 2. pSB1C3
 3. 1-3A(J04450,pSB1C3)
 4. 1-1K(J04450,J63010)
 5. control(pCI-GFP)   primer Fwd : PcI-GFP∙fwd Rev : PcI-GFP∙rev

PCR condition
 94°C – 5min
 94°C – 15sec    --
 51°C – 30sec      │-25 cycles
 72°C – 1min     --
 72°C – 10min
```

↓
Gel Electrophoresis

## Notebook

```
8/31 We've started experiment. First, we researched the property of T7 promoter.

9/5 We are researching promoter and inverters.

10/7 We are on the bench!! Design primers, PCR and experimentsssssssssss :D
```

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