iGEM 2014 — Team Kyoto

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# Team:Kyoto/Notebook

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# Notebook

## Calendar

| July | | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
|  |  | 1 | 2 | 3 | 4 | 5 |
| 6 | 7 | 8 | 9 | 10 | 11 | 12 |
| 13 | 14 | [15](#0715) | [16](#0716) | [17](#0717) | [18](#0718) | 19 |
| 20 | 21 | 22 | 23 | 24 | 25 | 26 |
| 27 | 28 | 29 | [30](#0730) | [31](#0731) |  |  |

| August | | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  | [1](#0801) | [2](#0802) |
| 3 | 4 | 5 | 6 | 7 | 8 | 9 |
| 10 | 11 | 12 | 13 | 14 | 15 | 16 |
| 17 | 18 | 19 | [20](#0820) | [21](#0821) | [22](#0822) | 23 |
| 24 | [25](#0825) | [26](#0826) | [27](#0827) | [28](#0828) | [29](#0829) | 30 |
| 31 |  |  |  |  |  |  |

| September | | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
|  | [1](#0901) | [2](#0902) | [3](#0903) | [4](#0904) | [5](#0905) | 6 |
| 7 | [8](#0908) | [9](#0909) | [10](#0910) | [11](#0911) | [12](#0912) | [13](#0913) |
| [14](#0914) | [15](#0915) | [16](#0916) | [17](#0917) | [18](#0918) | [19](#0919) | [20](#0920) |
| 21 | [22](#0922) | 23 | 24 | 25 | 26 | [27](#0927) |
| [28](#0928) | [29](#0929) | [30](#0930) |  |  |  |  |

| Octorber | | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
|  |  |  | [1](#1001) | [2](#1002) | [3](#1003) | [4](#1004) |
| [5](#1005) | [6](#1006) | [7](#1007) | [8](#1008) | [9](#1009) | [10](#1010) | 11 |
| 12 | [13](#1013) | [14](#1014) | [15](#1015) | [16](#1016) | [17](#1017) | 18 |
| 19 | 20 | 21 | 22 | 23 | 24 | 25 |
| 26 | 27 | 28 | 29 | 30 | 31 |  |

[アメフラシはこちら](https://2014.igem.org/Team%3AKyoto/Notebook/DMS_Synthesis)

### 7/15

#### transformation

Tatsui, Matsumoto and Sukegawa

| Sample/(µL) | | CompetentCells/(µl) | Total/(µl) | Medium |
| --- | --- | --- | --- | --- |
| BBa\_J23101(Pcon) | 2 | 20(2/23) | 22 | LB(CP) |
| BBa\_B0032(RBS) | 2 | 20(2/23) | 22 | LB(CP) |

### 7/16

#### transformation

Tatsui

| Sample/(µL) | | CompetentCells/(µl) | Total/(µl) | Medium |
| --- | --- | --- | --- | --- |
| BBa\_B0030(1) | 2 | 20(2/23) | 22 | LB(CP) |
| BBa\_B0030(2) | 2 | 20(bought) | 22 | LB(CP) |
| BBa\_B0032(1) | 2 | 20(2/23) | 22 | LB(CP) |
| BBa\_B0032(2) | 2 | 20(bought) | 22 | LB(CP) |

### 7/17

#### Colony PCR

Tatsui, Matsumoto, Li, and Yamauchi

| NameⅠ | NameⅡ |
| --- | --- |
| 7/16 BBa\_B0030(1) Colony\_1 | 7/16 BBa\_B0032(1) Colony\_1 |
| 7/16 BBa\_B0030(1) Colony\_2 | 7/16 BBa\_B0032(1) Colony\_2 |
| 7/16 BBa\_B0030(1) Colony\_3 | 7/16 BBa\_B0032(1) Colony\_3 |
| 7/16 BBa\_B0030(2) Colony\_1 | 7/16 BBa\_B0032(2) Colony\_1 |
| 7/16 BBa\_B0030(2) Colony\_2 | 7/16 BBa\_B0032(2) Colony\_2 |
| 7/16 BBa\_B0030(2) Colony\_3 | 7/16 BBa\_B0032(2) Colony\_3 |
| Negative Control | - |
| - | - |

#### Liquid Culture

Tatsui, Matsumoto, Li, and Yamauchi

| Name | medium |
| --- | --- |
| 7/16 BBa\_B0030(1) Colony\_1 | Plusgrow medium(CP) |
| 7/16 BBa\_B0030(1) Colony\_2 | Plusgrow medium(CP) |
| 7/16 BBa\_B0030(1) Colony\_3 | Plusgrow medium(CP) |
| 7/16 BBa\_B0030(2) Colony\_1 | Plusgrow medium(CP) |
| 7/16 BBa\_B0030(2) Colony\_2 | Plusgrow medium(CP) |
| 7/16 BBa\_B0030(2) Colony\_3 | Plusgrow medium(CP) |
| 7/16 BBa\_B0032(1) Colony\_1 | Plusgrow medium(CP) |
| 7/16 BBa\_B0032(1) Colony\_2 | Plusgrow medium(CP) |
| 7/16 BBa\_B0032(1) Colony\_3 | Plusgrow medium(CP) |
| 7/16 BBa\_B0032(2) Colony\_1 | Plusgrow medium(CP) |
| 7/16 BBa\_B0032(2) Colony\_2 | Plusgrow medium(CP) |
| 7/16 BBa\_B0032(2) Colony\_3 | Plusgrow medium(CP) |

### 7/18

#### Miniprep

Tatsui, Matsumoto, Tsujii, Stephane, and Yamauchi

| DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 7/16 BBa\_B0030(1) Colony\_1 | 229 | 1.96 | 1.40 |
| 7/16 BBa\_B0030(1) Colony\_2 | 209 | 1.95 | 1.50 |
| 7/16 BBa\_B0030(1) Colony\_3 | 108 | 2.12 | 2.12 |
| 7/16 BBa\_B0030(2) Colony\_1 | - | - | - |
| 7/16 BBa\_B0030(2) Colony\_2 | 174 | 2.02 | 1.56 |
| 7/16 BBa\_B0030(2) Colony\_3 | 158 | 2.01 | 1.68 |
| 7/16 BBa\_B0032(1) Colony\_1 | 179 | 2.00 | 2.06 |
| 7/16 BBa\_B0032(1) Colony\_2 | - | - | - |
| 7/16 BBa\_B0032(1) Colony\_3 | 221 | 1.90 | 1.37 |
| 7/16 BBa\_B0032(2) Colony\_1 | 225 | 1.89 | 1.37 |
| 7/16 BBa\_B0032(2) Colony\_2 | 270 | 1.98 | 1.63 |
| 7/16 BBa\_B0032(2) Colony\_3 | 194 | 1.89 | 1.33 |

### 7/30

#### Restriction Enzyme Digestion

No name

| Sample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffur/(µl) | BSA/(µl) | MilliQ/(µl) | TOTAL/(µl) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 7/16 BBa\_B0030(2) Colony\_1 | 1 | 0.3 | 0 | 0 | 0 | 1(H) | 0 | 7.7 | 10 |
| 7/16 BBa\_B0030(2) Colony\_1 | 1 | 0 | 0.3 | 0 | 0 | 1(M) | 1 | 6.7 | 10 |
| 7/16 BBa\_B0030(2) Colony\_1 | 1 | 0 | 0 | 0.3 | 0 | 1(M) | 0 | 7.7 | 10 |
| 7/16 BBa\_B0030(2) Colony\_1 | 1 | 0 | 0 | 0 | 0.3 | 1(H) | 0 | 7.7 | 10 |
| 7/16 BBa\_B0030(2) Colony\_1(Nega) | 1 | 0 | 0 | 0 | 0 | 1(M) | 0 | 8 | 10 |

#### Electrophoresis

No name

| Lane | Restriction Enzyme Digestion Products |
| --- | --- |
| 1 | 7/30 BBa\_B0030(2) Colony\_1(E CUT) |
| 2 | 7/30 BBa\_B0030(2) Colony\_1(X CUT) |
| 3 | 7/30 BBa\_B0030(2) Colony\_1(S CUT) |
| 4 | 7/30 BBa\_B0030(2) Colony\_1(P CUT) |
| 5 | 7/30 BBa\_B0030(2) Colony\_1(Nega) |
| 6 | 1kbp ladder |

### 7/31

#### Transformation

Hashitani, Tatsui, Li and Yamauchi

| Sample/(µL) | | CompetentCells/(µl) | Total/(µl) | Medium |
| --- | --- | --- | --- | --- |
| BBa\_E0240(RBS-GFP-DT) | 2 | 20 | 22 | LB(CP) |
| J23100(Pcon) | 2 | 20 | 22 | LB(AMP) |

#### Liquid Culture

No name

| Name | medium |
| --- | --- |
| 7/31 J23100(Pcon) Colony\_1 | Plusgrow medium(AMP) |
| 7/31 J23100(Pcon) Colony\_2 | Plusgrow medium(AMP) |
| 7/31 J23100(Pcon) Colony\_3 | Plusgrow medium(AMP) |
| 7/31 J23100(Pcon) Colony\_4 | Plusgrow medium(AMP) |
| 7/31 J23100(Pcon) Colony\_5 | Plusgrow medium(AMP) |
| 7/31 J23100(Pcon) Colony\_6 | Plusgrow medium(AMP) |

#### Restriction Enzyme Digestion

Hashitani, Tatsui, Li and Yamauchi

| 7/18 RBS Sample | DNA/(µl) | EcoR1/(µl) | Spe1/(µl) | Pst1/(µl) | Xba1/(µl) | Buffur/(µl) | BSA/(µl) | MilliQ/(µl) | TOTAL/(µl) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| BBa\_B0032 | 5 | 0.3 | 0 | 0 | 0 | 1(H) | 0 | 3.7 | 10 |
| BBa\_B0032 | 5 | 0 | 0.3 | 0 | 0 | 1(M) | 0 | 3.7 | 10 |
| BBa\_B0032 | 5 | 0 | 0 | 0.3 | 0 | 1(H) | 0 | 3.7 | 10 |
| BBa\_B0032 | 5 | 0 | 0 | 0 | 0.3 | 1(M) | 1 | 2.7 | 10 |
| BBa\_B0032(Nega) | 5 | 0 | 0 | 0 | 0 | 1(M) | 0 | 4 | 10 |

### 8/1

#### Electrophoresis

Honda, Li and Yamauchi

| Lane | Restriction Enzyme Digestion Products |
| --- | --- |
| 1 | 1kb ladder |
| 2 | 7/31 BBa\_B0032(E CUT) |
| 3 | 7/31 BBa\_B0032(S CUT)Error |
| 4 | - |
| 5 | - |
| 6 | 7/31 BBa\_B0032(P CUT) |
| 7 | 7/31 BBa\_B0032(X CUT) |
| 8 | 7/31 BBa\_B0032(Nega) |

#### PCR

Tatsui

| BBa\_E0240(RBS-GFP-DT) | Volume |
| --- | --- |
| 10×buffur/(µl) | 5 |
| 2mMdNTPs/(µl) 5 | |
| 25mM MgSO4/(µl) | 2 |
| VF Primer/(µl) | 1.5 |
| VR Primer/(µl) | 1.5 |
| template/(µl) | 0.5 |
| KOD-plus/(µl) | 1 |
| MilliQ/(µl) | 33.5 |
| TOTAL/(µl) | 50 |

| Steps | Temparature | Time | cycle |
| --- | --- | --- | --- |
| Pre Denature | 94℃ | 2min | 1time |
| Denature | 94℃ | 15sec | 30cycle |
| Annealing | 55℃ | 30sec | 30cycle |
| Extension | 68℃ | 1min | 30cycle |

| DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
| --- | --- | --- | --- |
| Product | 80 | 1.91 | 1.78 |

### 8/2

#### Miniprep

Tatsui, Honda, Matsumoto, Yamauchi, Li, Tsujii, and Nakamura

| DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 7/31 J23100(Pcon) Colony\_1 | 145 | 2.11 | 3.09 |
| 7/31 J23100(Pcon) Colony\_2 | - | - | - |
| 7/31 J23100(Pcon) Colony\_3 | 160 | 2.01 | 3.13 |
| 7/31 J23100(Pcon) Colony\_4 | 140 | 1.87 | 2.70 |
| 7/31 J23100(Pcon) Colony\_5 | 105 | 2.39 | 5.90 |
| 7/31 J23100(Pcon) Colony\_6 | 180 | 1.90 | 1.37 |

### 8/20

#### Transformation

Honda

| Sample/(µL) | | CompetentCells/(µl) | Total/(µl) | Medium |
| --- | --- | --- | --- | --- |
| 7/31 BBa\_E0240(RBS-GFD-DT) | 2 | 20 | 22 | LB(CP) |
| BBa\_I732095 | 2 | 20 | 22 | LB(CP) |

### 8/21

#### Colony PCR

Shimazaki

| NameⅠ |
| --- |
| 8/20 BBa\_I732095 Colony\_1 |
| 8/20 BBa\_I732095 Colony\_2 |
| 8/20 BBa\_I732095 Colony\_3 |
| Negative control |

#### Electrophoresis

Honda

| Lane | PCR Product |
| --- | --- |
| 1 | 1kb ladder |
| 2 | 8/21 BBa\_I732095 Colony\_1 |
| 3 | 8/21 BBa\_I732095 Colony\_2 |
| 4 | 8/21 BBa\_I732095 Colony\_3 |
| 5 | Negative Control |

#### Liquid Culture

Honda and Tatsui

| Name | medium |
| --- | --- |
| 8/20 BBa\_I732095 Colony\_1 | Plusgrow medium(CP) |
| 8/20 BBa\_I732095 Colony\_2 | Plusgrow medium(CP) |
| 8/20 BBa\_I732095 Colony\_3 | Plusgrow medium(CP) |

### 8/22

#### Miniprep

Honda

| DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 8/20 BBa\_I732095 Colony\_1 | 430 | 1.68 | 2.13 |
| 8/20 BBa\_I732095 Colony\_2 | 255 | 1.87 | 1.81 |
| 8/20 BBa\_I732095 Colony\_3 | 215 | 1.78 | 1.35 |

#### Restriction Enzyme Digestion

Nakamura

| Sample | DNA/(µl) | EcoR1/(µl) | Spe1/(µl) | Buffur/(µl) | MilliQ/(µl) | TOTAL/(µl) |
| --- | --- | --- | --- | --- | --- | --- |
| 8/22 BBa\_I732095 Colony\_1 | 4.7 | 1 | 1 | 3(H) | 20.3 | 30 |
| 8/22 BBa\_I732095 Colony\_2 | 7.8 | 1 | 1 | 3(H) | 17.2 | 30 |
| 8/22 BBa\_I732095 Colony\_3 | 9.3 | 1 | 1 | 3(H) | 15.7 | 30 |

#### Transformation

Tatsui and Yamauchi

| Sample/(µL) | | CompetentCells/(µl) | Total/(µl) | Medium |
| --- | --- | --- | --- | --- |
| BBa\_J04450(pSB3C5) | 2 | 20 | 22 | LB(CP) |

### 8/25

#### Transformation

Tatsui, Kita and Li

| Sample/(µL) | | CompetentCells/(µl) | Total/(µl) | Medium |
| --- | --- | --- | --- | --- |
| BBa\_J04450(pSB2K3) | 2 | 20 | 22 | LB(KAN) |
| BBa\_C0051(pSB1A2) | 2 | 20 | 22 | LB(AMP) |

#### Liquid Culture

Tatsui

| Name | medium |
| --- | --- |
| 8/22 BBa\_J04450(pSB3C5) | Plusgrow medium(CP)\*5 |

### 8/26

#### Miniprep

Tsujii, Honda and Matsumoto

| DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 8/25 BBa\_J04450(pSB3C5) | 575 | 1.37 | 1.37 |

#### Liquid Culture

Tatsui, Matsumoto and Nakamura

| Name | medium |
| --- | --- |
| 8/25 BBa\_J04450(pSB2K3) | Plusgrow medium(KAN)\*5 |
| 8/25 BBa\_J0051(pSB1A2) | Plusgrow medium(AMP)\*2 |

### 8/27

#### Miniprep

No Name

| DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 8/26 BBa\_J04450(pSB2K3) | 1045 | 1.43 | 1.11 |
| 8/26 BBa\_J0051(pSB1A2) | 790 | 1.39 | 0.90 |

#### Transformation

Tatsui

| Sample/(µL) | | CompetentCells/(µl) | Total/(µl) | Medium |
| --- | --- | --- | --- | --- |
| BBa\_J04450(pSB3T5) | 2 | 20 | 22 | LB(TET) |

### 8/28

#### Liquid Culture

Yamauchi

| Name | medium |
| --- | --- |
| 8/27 BBa\_J04450(pSB3T5) | Plusgrow medium(TET) |

### 8/29

#### Miniprep

Matsumoto

| DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 8/28 BBa\_J04450(pSB3T5) | 180 | 1.60 | 1.67 |

#### Transformation

Tatsui

| Sample/(µL) | | CompetentCells/(µl) | Total/(µl) | Medium |
| --- | --- | --- | --- | --- |
| BBa\_J04450(pSB4A5) | 2 | 20 | 22 | LB(AMP) |

### 9/1

#### Colony PCR

| NameⅠ |
| --- |
| - |
| - |
| - |
| 9/1 pSB4A5 Colony\_1 |
| 9/1 pSB4A5 Colony\_2 |
| Negative Control |

#### Restriction Enzyme Digestion

No name

| Sample | DNA/(µl) | EcoR1/(µl) | Pst1/(µl) | Buffur/(µl) | MilliQ/(µl) | TOTAL/(µl) |
| --- | --- | --- | --- | --- | --- | --- |
| 8/28 Miniprep pSB3T5 | 25 | 2 | 2 | 6(H) | 25 | 60 |
| Negative control\_1 | 3.7 | 0 | 0 | 1(H) | 5.3 | 10 |
| 8/22 Miniprep I732095 | 14 | 3 | 3 | 9(H) | 61 | 90 |
| Negative control\_2 | 1.5 | 0 | 0 | 1(H) | 7.5 | 10 |
| 8/27 Miniprep pSB2K3 | 6 | 3 | 3 | 9(H) | 69 | 90 |
| Negative control\_3 | 1 | 0 | 0 | 1(H) | 8 | 10 |
| 8/2 Miniprep Pcon Colony\_3 | 20 | 1.5 | 1.5 | 5(H) | 17 | 45 |
| Negative control\_4 | 4 | 0 | 0 | 1(H) | 5 | 10 |
| 8/2 Miniprep Pcon Colony\_1 | 20 | 1.5 | 1.5 | 5(H) | 17 | 45 |
| Negative control\_5 | 4 | 0 | 0 | 1(H) | 5 | 10 |

#### Liquid Culture

No name

| Name | medium |
| --- | --- |
| 8/29 BBa\_J04450(pSB4A5) Colony\_1 | Plusgrow medium(AMP)\*2 |
| 8/1 Pcon Colony\_1 | Plusgrow medium(AMP)\*2 |
| 8/27 pSB3T5 Colony\_1 | Plusgrow medium(TET)\*2 |

### 9/2

#### Miniprep

Honda, Yamauchi and Li

| DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 8/29 BBa\_J04450(pSB4A5) Colony\_1 | 210 | 1.61 | 1.18 |
| 8/1 Pcon Colony\_1 | 215 | 1.79 | 1.87 |
| 8/27 pSB3T5 Colony\_1 | 215 | 1.58 | 0.92 |

#### Gel Extraction

Matsumoto

| Lane | Restriction Enzyme Digestion ProductⅠ | Volume/(µl) |
| --- | --- | --- |
| 1 | 9/1 pSB3T5 Colony\_1 (Nega) | 10 |
| 2 | 9/1 pSB3T5 Colony\_1 (E+P)(Confirmation) | 5 |
| 3 | 9/1 pSB3T5 Colony\_1 (E+P)(Extraction) | 55 |
| 4 | 1kb ladder | 3 |
| 5 | 9/1 I732095 (Nega) | 10 |
| 6 | 9/1 I732095 (E+P)(Confirmation) | 5 |
| 7 | 9/1 I732095 (E+P)(Extraction) | 85 |
| 8 | 1kb ladder | 3 |
| 9 | 9/1 pSB2K3 (Nega) | 10 |
| 10 | 9/1 pSB2K3 (E+P)(Confirmation) | 5 |
| 11 | 9/1 pSB2K3 (E+P)(Extraction) | 85 |

| Lane | Sample | Volume/(µl) |
| --- | --- | --- |
| 1 | 9/1 Pcon Colony\_3 (E+P)(Confirmation) | 5 |
| 2 | 9/1 Pcon Colony\_3 (E+P)(Extraction) | 20 |
| 3 | 9/1 Pcon Colony\_3 (E+P)(Extraction) | 20 |
| 4 | 9/1 Pcon Colony\_3 (Nega) | 10 |
| 5 | 1kb ladder | 3 |
| 6 | 9/1 Pcon Colony\_1 (E+P)(Confirmation) | 5 |
| 7 | 9/1 Pcon Colony\_1 (E+P)(Extraction) | 20 |
| 8 | 9/1 Pcon Colony\_1 (E+P)(Extraction) | 20 |
| 9 | 9/1 Pcon Colony\_1 (Nega) | 10 |

### 9/3

#### Concentration Measurement

Matsumoto, Yamauchi and Li

| DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
| --- | --- | --- | --- |
| pSB2K3 | 40 | 1.48 | 0.10 |
| Pcon Colony\_1 | 30 | 1.76 | 0.62 |
| pSB3T5 | 50 | 2.35 | 0.50 |
| pSB1C3 | 65 | 2.14 | 0.10 |
| Pcon Colony\_3 | 45 | 1.56 | 0.58 |

#### Ligation

No name

| Insert/(µL) | | Vector/(µL) | | T4 Ligase/(µl) | 2\*Buffer/(µl) | MilliQ/(µl) | Total/(µl) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Pcon | 2.1 | pSB1C3 | 1.5 | 1 | 5 | 0.4 | 10 |
| -(Negative Control) | 0 | pSB1C3 | 1.5 | 1 | 5 | 2.5 | 10 |
| Pcon | 1.3 | pSB2K3 | 2.5 | 1 | 5 | 0.2 | 10 |
| -(Negative Control) | 0 | pSB2K3 | 2.5 | 1 | 5 | 1.5 | 10 |
| Pcon | 10 | pSB3T5 | 2.3 | 1 | 5 | 0.7 | 10 |
| -(Negative Control) | 0 | pSB3T5 | 2.3 | 1 | 5 | 1.7 | 10 |

#### Transformation

Matsumoto

| Sample/(µL) | | CompetentCells/(µl) | Total/(µl) | Medium |
| --- | --- | --- | --- | --- |
| 9/3 Ligation Pcon+pSB1C3 | 3 | 15 | 18 | LB(CP) |
| 9/3 Ligation pSB1C3(Negative Control) | 8 | 10 | 18 | LB(CP) |
| 9/3 Ligation Pcon+pSB2K3 | 3 | 15 | 18 | LB(KAN) |
| 9/3 Ligation pSB2K3(Negative Control) | 8 | 10 | 18 | LB(KAN) |
| 9/3 Ligation Pcon+pSB3T5 | 3 | 15 | 18 | LB(TET) |
| 9/3 Ligation pSB3T5(Negative Control) | 2 | 10 | 12 | LB(TET) |

### 9/4

#### Colony PCR

Honda and Yamauchi

| NameⅠ | NameⅡ | NameⅢ | NameⅣ |
| --- | --- | --- | --- |
| - | 9/3 pSB1C3(Nega) Colony\_7 | 9/3 Pcon+pSB3T5 Colony\_5 | 9/3 Pcon+pSB2K3 Colony\_1 |
| - | 9/3 pSB1C3(Nega) Colony\_8 | 9/3 Pcon+pSB3T5 Colony\_6 | 9/3 Pcon+pSB2K3 Colony\_2 |
| 9/3 pSB1C3(Nega) Colony\_1 | 9/3 Pcon+pSB3T5 Colony\_1 | 9/3 Pcon+pSB3T5 Colony\_7 | 9/3 Pcon+pSB2K3 Colony\_3 |
| 9/3 pSB1C3(Nega) Colony\_2 | 9/3 Pcon+pSB3T5 Colony\_2 | 9/3 Pcon+pSB3T5 Colony\_8 | 9/3 Pcon+pSB2K3 Colony\_4 |
| 9/3 pSB1C3(Nega) Colony\_3 | 9/3 Pcon+pSB3T5 Colony\_3 | 9/3 Pcon+pSB3T5 Colony\_9 | 9/3 Pcon+pSB2K3 Colony\_5 |
| 9/3 pSB1C3(Nega) Colony\_4 | 9/3 Pcon+pSB3T5 Colony\_4 | 9/3 Pcon+pSB3T5 Colony\_10 | 9/3 Pcon+pSB2K3 Colony\_6 |
| 9/3 pSB1C3(Nega) Colony\_5 | 9/3 pSB1C3(Nega) Colony\_9 | 9/3 pSB3T5(Nega) Colony\_1 | 9/3 Pcon+pSB2K3 Colony\_7 |
| 9/3 pSB1C3(Nega) Colony\_6 | 9/3 pSB1C3(Nega) Colony\_10 | 9/3 pSB3T5(Nega) Colony\_2 | 9/3 Pcon+pSB2K3 Colony\_8 |

| NameⅤ | NameⅥ | NameⅦ | NameⅧ |
| --- | --- | --- | --- |
| 9/3 Pcon+pSB2K3 Colony\_9 | 9/3 Pcon+pSB1C3 Colony\_2 | 9/3 Pcon+pSB1C3 Colony\_10 | 9/3 pSB1C3(Nega) Colony\_8 |
| 9/3 Pcon+pSB2K3 Colony\_10 | 9/3 Pcon+pSB1C3 Colony\_3 | 9/3 pSB1C3(Nega) Colony\_1 | 9/3 pSB1C3(Nega) Colony\_9 |
| 9/3 pSB2K3(Nega) Colony\_1 | 9/3 Pcon+pSB1C3 Colony\_4 | 9/3 pSB1C3(Nega) Colony\_2 | 9/3 pSB1C3(Nega) Colony\_10 |
| 9/3 pSB2K3(Nega) Colony\_2 | 9/3 Pcon+pSB1C3 Colony\_5 | 9/3 pSB1C3(Nega) Colony\_3 | 9/3 pSB1C3(Nega) Colony\_11 |
| 9/3 pSB2K3(Nega) Colony\_3 | 9/3 Pcon+pSB1C3 Colony\_6 | 9/3 pSB1C3(Nega) Colony\_4 | - |
| 9/3 pSB2K3(Nega) Colony\_4 | 9/3 Pcon+pSB1C3 Colony\_7 | 9/3 pSB1C3(Nega) Colony\_5 | - |
| 9/3 pSB2K3(Nega) Colony\_5 | 9/3 Pcon+pSB1C3 Colony\_8 | 9/3 pSB1C3(Nega) Colony\_6 | - |
| 9/3 Pcon+pSB1C3 Colony\_1 | 9/3 Pcon+pSB1C3 Colony\_9 | 9/3 pSB1C3(Nega) Colony\_7 | - |

| NameⅨ | NameⅩ |
| --- | --- |
| 9/3 Pcon+pSB1C3 Colony\_11 | 8/22 Miniprep I732095 Colony\_3 |
| Negative Control | - |

#### Restriction Enzyme Digestion

No name

| Sample | DNA/(µl) | EcoR1/(µl) | Pst1/(µl) | Buffur/(µl) | MilliQ/(µl) | TOTAL/(µl) |
| --- | --- | --- | --- | --- | --- | --- |
| 8/27 Miniprep pSB2K3 | 2 | 1 | 1 | 3(H) | 23 | 30 |
| 8/27 Miniprep pSB2K3(Nega) | 1 | 0 | 0 | 1(H) | 8 | 10 |
| 9/2 Miniprep pSB3T5 | 10 | 1 | 1 | 3(H) | 15 | 30 |
| 9/2 Miniprep pSB3T5(Nega) | 3 | 0 | 0 | 1(H) | 6 | 10 |
| 9/2 Miniprep pSB4A5 | 10 | 1 | 1 | 3(H) | 15 | 30 |
| 9/2 Miniprep pSB4A5(Nega) | 3 | 0 | 0 | 1(H) | 6 | 10 |
| 8/22 Miniprep pSB1C3 Colony\_2 | 2 | 1 | 1 | 1(H) | 5 | 10 |

### 9/5

#### Electrophoresis

Honda and Matsumoto

| Lane | Colony PCR ProductsⅠ Lane | Colony PCR ProductsⅠ | |
| --- | --- | --- | --- |
| 1 | 1kb ladder | 10 | 9/4 pSB1C3(Nega) Colony\_7 |
| 2 | - | 11 | 9/4 pSB1C3(Nega) Colony\_8 |
| 3 | - | 12 | 9/4 pSB1C3(Nega) Colony\_9 |
| 4 | 9/4 pSB1C3(Nega) Colony\_1 | 13 | 9/4 pSB1C3(Nega) Colony\_10 |
| 5 | 9/4 pSB1C3(Nega) Colony\_2 | 14 | 9/4 Pcon+pSB2K3 Colony\_1 |
| 6 | 9/4 pSB1C3(Nega) Colony\_3 | 15 | 9/4 Pcon+pSB2K3 Colony\_2 |
| 7 | 9/4 pSB1C3(Nega) Colony\_4 | 16 | 9/4 Pcon+pSB2K3 Colony\_3 |
| 8 | 9/4 pSB1C3(Nega) Colony\_5 | 17 | 1kb ladder |
| 9 | 9/4 pSB1C3(Nega) Colony\_6 |

| Lane | Colony PCR ProductsⅡ Lane | Colony PCR ProductsⅡ | |
| --- | --- | --- | --- |
| 1 | 1kb ladder | 10 | 9/4 pSB3T5(Nega) Colony\_2 |
| 2 | 9/4 Pcon+pSB2K3 Colony\_4 | 11 | 9/4 Pcon+pSB2K3 Colony\_1 |
| 3 | 9/4 Pcon+pSB2K3 Colony\_5 | 12 | 9/4 Pcon+pSB2K3 Colony\_2 |
| 4 | 9/4 Pcon+pSB2K3 Colony\_6 | 13 | 9/4 Pcon+pSB2K3 Colony\_3 |
| 5 | 9/4 Pcon+pSB2K3 Colony\_7 | 14 | 9/4 Pcon+pSB2K3 Colony\_4 |
| 6 | 9/4 Pcon+pSB2K3 Colony\_8 | 15 | 9/4 Pcon+pSB2K3 Colony\_5 |
| 7 | 9/4 Pcon+pSB2K3 Colony\_9 | 16 | 9/4 Pcon+pSB2K3 Colony\_6 |
| 8 | 9/4 Pcon+pSB3T5 Colony\_10 | 17 | 1kb ladder |
| 9 | 9/4 pSB3T5(Nega) Colony\_1 |

| Lane | Colony PCR ProductsⅢ Lane | Colony PCR ProductsⅢ | |
| --- | --- | --- | --- |
| 1 | 1kb ladder | 10 | 9/4 pSB2K3(Nega) Colony\_5 |
| 2 | 9/4 Pcon+pSB2K3 Colony\_7 | 11 | 9/4 pSB1C3(Nega) Colony\_1 |
| 3 | 9/4 Pcon+pSB2K3 Colony\_8 | 12 | 9/4 pSB1C3(Nega) Colony\_2 |
| 4 | 9/4 Pcon+pSB2K3 Colony\_9 | 13 | 9/4 pSB1C3(Nega) Colony\_3 |
| 5 | 9/4 Pcon+pSB2K3 Colony\_10 | 14 | 9/4 pSB1C3(Nega) Colony\_4 |
| 6 | 9/4 pSB2K3(Nega) Colony\_1 | 15 | 9/4 pSB1C3(Nega) Colony\_5 |
| 7 | 9/4 pSB2K3(Nega) Colony\_2 | 16 | 9/4 pSB1C3(Nega) Colony\_6 |
| 8 | 9/4 pSB2K3(Nega) Colony\_3 | 17 | 1kb ladder |
| 9 | 9/4 pSB2K3(Nega) Colony\_4 |

| Lane | Colony PCR ProductsⅣ Lane | Colony PCR ProductsⅣ | |
| --- | --- | --- | --- |
| 1 | 1kb ladder | 10 | 9/4 Pcon+pSB1C3 Colony\_5 |
| 2 | 9/4 pSB1C3(Nega) Colony\_7 | 11 | 9/4 Pcon+pSB1C3 Colony\_6 |
| 3 | 9/4 pSB1C3(Nega) Colony\_8 | 12 | 9/4 Pcon+pSB1C3 Colony\_7 |
| 4 | 9/4 pSB1C3(Nega) Colony\_9 | 13 | 9/4 Pcon+pSB1C3 Colony\_8 |
| 5 | 9/4 pSB1C3(Nega) Colony\_10 | 14 | 9/4 Pcon+pSB1C3 Colony\_9 |
| 6 | 9/4 Pcon+pSB1C3 Colony\_1 | 15 | 9/4 Pcon+pSB1C3 Colony\_10 |
| 7 | 9/4 Pcon+pSB1C3 Colony\_2 | 16 | 8/22 Miniprep BBa\_I732095 Colony\_3 |
| 8 | 9/4 Pcon+pSB1C3 Colony\_3 | 17 | 1kb ladder |
| 9 | 9/4 Pcon+pSB1C3 Colony\_4 |

| Lane | Colony PCR ProductsⅤ |
| --- | --- |
| 1 | 1kb ladder |
| 2 | 9/4 Pcon+pSB1C3 Colony\_11 |
| 3 | 9/4 Negative Control |
| 4 | 8/22 Miniprep BBa\_I732095 Colony\_3 |
| 5 | - |
| 6 | - |
| 7 | - |
| 8 | - |

#### Gel Extraction

No Name

| Lane | Restriction Enzyme Digestion ProductⅠ | Volume/(µl) |
| --- | --- | --- |
| 1 | 1kb ladder | 5 |
| 2 | 9/4 pSB2K3(E+P) | 5 |
| 3 | - | - |
| 4 | 9/4 pSB2K3(E+P) | 25 |
| 5 | - | - |
| 6 | 9/4 pSB2K3(Nega) | 10 |

| Lane | Restriction Enzyme Digestion ProductⅡ | Volume/(µl) |
| --- | --- | --- |
| 1 | 1kb ladder | 5 |
| 2 | 9/4 pSB3T5(E+P) | 5 |
| 3 | - | - |
| 4 | 9/4 pSB3T5(E+P) | 25 |
| 5 | - | - |
| 6 | 9/4 pSB3T5(Nega) | 10 |

| Lane | Restriction Enzyme Digestion ProductⅢ | Volume/(µl) |
| --- | --- | --- |
| 1 | 1kb ladder | 5 |
| 2 | 9/4 pSB4A5(E+P) | 5 |
| 3 | - | - |
| 4 | 9/4 pSB4A5(E+P) | 25 |
| 5 | - | - |
| 6 | 9/4 pSB4A5(Nega) | 10 |

| Lane | Restriction Enzyme Digestion ProductⅣ | Volume/(µl) |
| --- | --- | --- |
| 1 | 1kb ladder | 5 |
| 2 | - | - |
| 3 | - | - |
| 4 | 9/4 pSB1C3(E+P) | 10 |
| 5 | - | - |
| 6 | - | - |

| DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 9/4 pSB2K3(E+P) | 36 | 1.25 | 0.24 |
| 9/4 pSB3T5(E+P) | 10 | 1.00 | -/td> |
| 9/4 pSB4A5(E+P) | 40 | - | 0.01 |
| 9/4 pSB1C3(E+P) | 75 | 1.35 | 0.14 |

### 9/8

#### Cloning PCR

Honda and Li

| Target | mamB | mamQ | mamL |
| --- | --- | --- | --- |
| 2×KAPA HiFi Hotstart Ready Mix/(µl) | 12.5 | 12.5 | 12.5 |
| Fw Primer/(µl) | 1.5(mamB) | 1.5(mamQ) | 1.5(mamL) |
| Rv Primer/(µl) | 1.5(mamB) | 1.5(mamQ) | 1.5(mamL) |
| AMB-1 Genome/(µl) | 1 | 1 | 1 |
| MilliQ/(µl) | 10 | 10 | 10 |
| TOTAL/(µl) | 25 | 25 | 25 |

| Steps | Temparature | Time | cycle |
| --- | --- | --- | --- |
| Pre Denature | 98℃ | 5min | 1time |
| Denature | 98℃ | 20sec | 25cycle |
| Annealing | 65℃ | 15sec | 25cycle |
| Extension | 72℃ | 5min | 25cycle |
| Final elongation | 72℃ | 5min | 1time |
| Final hold | 4℃ | - | - |

#### Ligation

Honda

| Insert/(µL) | | Vector/(µL) | | T4 Ligase/(µl) | 2\*Buffer/(µl) | MilliQ/(µl) | Total/(µl) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Pcon | 1 | pSB3T5 | 10 | 1 | 11 | 0 | 22 |
| -(Negative Control) | 0 | pSB1C3 | 3 | 0 | 3 | 0 | 6 |

#### Electrophoresis

Li

| Lane | Cloning PCR Products |
| --- | --- |
| 1 | 1kb ladder |
| 2 | 9/8 Cloning PCR (AMB-1 mamB) |
| 3 | 9/8 Cloning PCR (AMB-1 mamQ) |
| 4 | 9/8 Cloning PCR (AMB-1 mamL) |
| 5 | 1kb ladder |

#### Transformation

Honda

| Sample/(µL) | | CompetentCells/(µl) | Total/(µl) | Medium |
| --- | --- | --- | --- | --- |
| 9/8 Ligation Pcon+pSB3T5 | 3 | 30 | 33 | LB(TET) |
| 9/8 Ligation pSB3T5(Negative Control) | 3 | 30 | 33 | LB(TET) |

#### Liquid Culture

No name

| Name | medium |
| --- | --- |
| 9/3 Transformation Pcon+pSB1C3 Colony\_8 | Plusgrow medium(CP)\*2 |

### 9/9

#### DNA Purification

Li

| DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 9/8 Cloning PCR (AMB-1 mamB) | 165 | 1.54 | 1.57 |
| 9/8 Cloning PCR (AMB-1 mamQ) | 250 | 1.69 | 1.92 |
| 9/8 Cloning PCR (AMB-1 mamL) | 190 | 1.56 | 1.79 |

#### Miniprep

Li

| DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 9/8 Liquid Culture Pcon+pSB1C3 | 90 | 1.65 | 1.17 |

#### Colony PCR

Honda and Li

| NameⅠ |
| --- |
| - |
| 9/8 Transformation Pcon+pSB3T5 Colony\_1 |
| 9/8 Transformation Pcon+pSB3T5 Colony\_2 |
| 9/8 Transformation Pcon+pSB3T5 Colony\_3 |
| 9/8 Transformation Pcon+pSB3T5 Colony\_4 |
| 9/8 Transformation Pcon+pSB3T5 Colony\_5 |
| 9/8 Transformation pSB3T5(Nega) Colony\_1 |
| Negative control |

#### Electrophoresis

Honda and Li

| Lane | Cloning PCR Products |
| --- | --- |
| 1 | 1kb ladder |
| 2 | 9/8 Colony PCR Pcon+pSB3T5 Colony\_1 |
| 3 | 9/8 Colony PCR Pcon+pSB3T5 Colony\_2 |
| 4 | 9/8 Colony PCR Pcon+pSB3T5 Colony\_3 |
| 5 | 9/8 Colony PCR Pcon+pSB3T5 Colony\_4 |
| 6 | 9/8 Colony PCR Pcon+pSB3T5 Colony\_5 |
| 7 | 9/8 Colony PCR pSB3T5(Nega) Colony\_1 |
| 8 | 9/8 Colony PCR Nega |

#### Restriction Enzyme Digestion

No name

| Sample | DNA/(µl) | Pst1/(µl) | Spe1/(µl) | Buffur/(µl) | MilliQ/(µl) | TOTAL/(µl) |
| --- | --- | --- | --- | --- | --- | --- |
| 9/9 Miniprep Pcon+pSB1C3 | 22 | 1 | 1 | 3(H) | 3 | 30 |
| 9/9 Miniprep Pcon+pSB1C3(Nega) | 1 | 0 | 0 | 1(H) | 8 | 10 |
| 8/2 Miniprep Pcon Colony\_4 | 14 | 1 | 1 | 3(H) | 11 | 30 |
| 8/2 Miniprep Pcon Colony\_4(Nega) | 1 | 0 | 0 | 1(H) | 8 | 10 |

### 9/10

#### Gel Extraction

Tatsui and Yamaura

| Lane | Restriction Enzyme Digestion ProductⅠ | Volume/(µl) |
| --- | --- | --- |
| 1 | 1kb ladder | - |
| 2 | - | - |
| 3 | 9/10 Digestion Pcon+pSB1C3(S+P) | - |
| 4 | - | - |
| 5 | 9/10 Digestion Pcon+pSB1C3(Nega) | - |
| 6 | - | - |
| 7 | Pcon(X+P) | - |
| 8 | - | - |
| 9 | Pcon(Nega) | - |
| 10 | 1kb ladder | - |
| 11 | - | - |

| DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
| Pcon(X+P) | 70 | 1.40 | 0.13 |

#### PCR

No Name

| Target | E+X+RBS+mamB+S | X+RBS+mamL+His+S+P | X+RBS+mamQ+His+S+P |
| --- | --- | --- | --- |
| 2×KAPA HiFi Hotstart Ready Mix/(µl) | 12.5 | 12.5 | 12.5 |
| Fw Primer/(µl) | 0.75(E+X+RBS+mamB) | 0.75(X+RBS+mamL) | 0.75(X+RBS+mamQ) |
| Rv Primer/(µl) | 0.75(S+mamB) | 0.75(P+S+His+mamL) | 0.75(P+S+His+mamQ) |
| 9/9 DNA Purification Product/(µl) | Trace | Trace | Trace |
| MilliQ/(µl) | 11 | 11 | 11 |
| TOTAL/(µl) | 25 | 25 | 25 |

| Steps | Temparature | Time | cycle |
| --- | --- | --- | --- |
| Pre Denature | 98℃ | 5min | 1time |
| Denature | 98℃ | 20sec | 25cycle |
| Annealing | 60℃ | 15sec | 25cycle |
| Extension | 72℃ | 15sec | 25cycle |
| Final elongation | 72℃ | 5min | 1time |
| Final hold | 12℃ | - | - |

#### Electrophoresis

Li

| Lane | Cloning PCR Products |
| --- | --- |
| 1 | 1kb ladder |
| 2 | 9/10 PCR E+X+RBS+mamB+S(922bp) |
| 3 | 9/10 PCR X+RBS+mamL+His+S+P(340bp) |
| 4 | - |
| 5 | 1kb ladder |
| 6 | 9/10 PCR X+RBS+mamQ+His+S+P(953bp) |

#### DNA Purification

Honda and Yamaura

| DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 9/10 PCR E+X+RBS+mamB+S | 200 | 1.84 | 2.15 |
| 9/10 PCR X+RBS+mamL+His+S+P | 120 | 1.69 | 1.84 |
| 9/10 PCR X+RBS+mamQ+His+S+P | 200 | 1.70 | 2.16 |

#### Restriction Enzyme Digestion

Shimizu

| Sample | DNA/(µl) | Xba1/(µl) | Pst1/(µl) | Buffur/(µl) | MilliQ/(µl) | TOTAL/(µl) |
| --- | --- | --- | --- | --- | --- | --- |
| 9/10 PCR E+X+RBS+mamB+S | 10 | 1 | 1 | 3(H) | 15 | 30 |
| 9/10 PCR X+RBS+mamL+His+S+P | 16.7 | 1 | 1 | 3(H) | 8.3 | 30 |
| 9/10 PCR X+RBS+mamQ+His+S+P | 10 | 1 | 1 | 3(H) | 15 | 30 |

#### Ligation

Tatsui and Murada

| Insert/(µL) | | Vector/(µL) | | T4 Ligase/(µl) | 2\*Buffer/(µl) | MilliQ/(µl) | Total/(µl) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 9/10 DNA Purification mamQ | 1 | YCplac111 | 1 | 1 | 5 | 2 | 10 |
| 9/10 DNA Purification mamL | 1 | YCplac111 | 1 | 1 | 5 | 2 | 10 |
| 9/10 DNA Purification mamB | 1 | YCplac111 | 1 | 1 | 5 | 2 | 10 |
| Control | 0 | YCplac111 | 1 | 1 | 5 | 2 | 9 |

#### Transformation

Tatsui and Murada

| Sample/(µL) | | CompetentCells/(µl) | Total/(µl) | Medium |
| --- | --- | --- | --- | --- |
| 9/10 Ligation mamQ+YCplac111 | - | - | - | - |
| 9/10 Ligation mamL+YCplac111 | - | - | - | - |
| 9/10 Ligation mamB+YCplac111 | - | - | - | - |

### 9/11

#### DNA Purification

No Name

| DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 9/10 Digestion mamB(X+P) | 65 | 1.68 | 0.51 |
| 9/10 Digestion mamL(X+P) | 30 | 1.95 | 0.81 |
| 9/10 Digestion mamQ(X+P) | 60 | 1.65 | 1.22 |

#### Electrophoresis

Murada

| Lane | Colony PCR Products |
| --- | --- |
|  |  |

| Lane | Colony PCR Products |
| --- | --- |
|  |  |

| Lane | Colony PCR Products |
| --- | --- |
|  |  |

| Lane | Colony PCR Products |
| --- | --- |
|  |  |

| Lane | Colony PCR Products |
| --- | --- |
|  |  |

#### Ligation

No Name

| Insert/(µL) | | Vector/(µL) | | T4 Ligase/(µl) | 2\*Buffer/(µl) | MilliQ/(µl) | Total/(µl) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| - | - | - | - | - | - | - | - |

#### Transformation

Yamauchi

| Sample/(µL) | | CompetentCells/(µl) | Total/(µl) | Medium |
| --- | --- | --- | --- | --- |
| 9/11 Ligation mamL | 2 | 10 | 12 | - |
| 9/11 Ligation mamQ | 1 | 5 | 6 | - |

#### Liquid Culture

Honda

| Name | medium |
| --- | --- |
| 9/8 Master Pcon+pSB1C3 Colony\_3 | Plusgrow medium(CP) |
| 9/10 Transformation mamL+YCplac111 | Plusgrow medium(AMP) |
| 9/10 Transformation mamQ+YCplac111 | Plusgrow medium(AMP) |

### 9/12

#### Colony PCR

Yamaura

| NameⅠ |
| --- |
|  |

#### Electrophoresis

Yamaura

| Lane | Colony PCR Products |
| --- | --- |
|  |  |

#### Miniprep

Honda

| DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 9/11 Liquid Culture Pcon+pSB1C3 | 110 | 1.83 | 2.63 |
| 9/11 Liquid Culture mamL+YCplac111 | 300 | 1.75 | 2.38 |
| 9/11 Liquid Culture mamQ+YCplac111 | 160 | 1.81 | 2.37 |

#### Restriction Enzyme Digestion

Yasuda

| Sample | DNA/(µl) | Xba1/(µl) | Pst1/(µl) | Spe1/(µl) | Buffur/(µl) | BSA | MilliQ/(µl) | TOTAL/(µl) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 9/12 Miniprep mamL+YCplac111 | 6.7 | 1 | 1 | 0 | 3(H) | 3 | 15.3 | 30 |
| 9/12 Miniprep mamQ+YCplac111 | 12.5 | 1 | 1 | 0 | 3(H) | 3 | 9.5 | 30 |
| 9/12 Miniprep Pcon+pSB1C3 | 18.1 | 0 | 1 | 1 | 3(H) | 0 | 6.9 | 30 |

#### Ligation

Honda

| Insert/(µL) | | Vector/(µL) | | T4 Ligase/(µl) | 2\*Buffer/(µl) | MilliQ/(µl) | Total/(µl) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 9/12 Digestion mamB(E+S) | 3 | 9/8 Digestion pSB1C3(E+S) | 1 | 1 | 5 | 0 | 10 |
| 9/10 mamB | 0.5 | YCplac111 | 1 | 1 | 5 | 2.5 | 10 |

#### DNA Purification

Tatsui

| DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
| --- | --- | --- | --- |
| mamB(E+S) | 50 | 1.75 | 2.04 |

#### Transformation

Honda

| Sample/(µL) | | CompetentCells/(µl) | Total/(µl) | Medium |
| --- | --- | --- | --- | --- |
| 9/12 Ligation mamB+pSB1C3 | 1 | 10 | 11 | LB(CP) |
| 9/12 Ligation mamB+YCplac111 | 1 | 10 | 11 | LB(AMP) |

#### Colony PCR

Li

| NameⅠ | NameⅡ |
| --- | --- |
| 9/11 Transformation mamL+pSB1A2 Colony\_1 | 9/11 Transformation mamL+pSB1A2 Colony\_9 |
| 9/11 Transformation mamL+pSB1A2 Colony\_2 | 9/11 Transformation mamL+pSB1A2 Colony\_10 |
| 9/11 Transformation mamL+pSB1A2 Colony\_3 | 9/11 Transformation mamQ+pSB1A2 Colony\_1 |
| 9/11 Transformation mamL+pSB1A2 Colony\_4 | 9/11 Transformation mamQ+pSB1A2 Colony\_2 |
| 9/11 Transformation mamL+pSB1A2 Colony\_5 | 9/11 Transformation mamQ+pSB1A2 Colony\_3 |
| 9/11 Transformation mamL+pSB1A2 Colony\_6 | 9/11 Transformation mamQ+pSB1A2 Colony\_4 |
| 9/11 Transformation mamL+pSB1A2 Colony\_7 | 9/11 Transformation mamQ+pSB1A2 Colony\_5 |
| 9/11 Transformation mamL+pSB1A2 Colony\_8 | 9/11 Transformation mamQ+pSB1A2 Colony\_6 |

#### Electrophoresis

Li

| Lane | Colony PCR ProductsⅠ Lane | Colony PCR ProductsⅠ | |
| --- | --- | --- | --- |
| 1 | 9/12 mamL+pSB1A2 Colony\_1 | 10 | 9/12 mamL+pSB1A2 Colony\_9 |
| 2 | 9/12 mamL+pSB1A2 Colony\_2 | 11 | 9/12 mamL+pSB1A2 Colony\_10 |
| 3 | 9/12 mamL+pSB1A2 Colony\_3 | 12 | 9/12 mamQ+pSB1A2 Colony\_1 |
| 4 | 9/12 mamL+pSB1A2 Colony\_4 | 13 | 9/12 mamQ+pSB1A2 Colony\_2 |
| 5 | 9/12 mamL+pSB1A2 Colony\_5 | 14 | 9/12 mamQ+pSB1A2 Colony\_3 |
| 6 | 9/12 mamL+pSB1A2 Colony\_6 | 15 | 9/12 mamQ+pSB1A2 Colony\_4 |
| 7 | 9/12 mamL+pSB1A2 Colony\_7 | 16 | 9/12 mamQ+pSB1A2 Colony\_5 |
| 8 | 9/12 mamL+pSB1A2 Colony\_8 | 17 | 9/12 mamQ+pSB1A2 Colony\_6 |
| 9 | 1kb ladder |

#### Colony PCR

Honda

| NameⅠ |
| --- |
| 9/11 Transformation mamL+pSB1A2 Green\_1 |
| 9/11 Transformation mamL+pSB1A2 Green\_2 |
| 9/11 Transformation mamL+pSB1A2 Green\_3 |
| 9/11 Transformation mamL+pSB1A2 Green\_4 |
| 9/11 Transformation mamL+pSB1A2 Green\_5 |
| 9/11 Transformation mamL+pSB1A2 Black\_7 |
| 9/11 Transformation mamL+pSB1A2 Black\_8 |
| 9/11 Transformation mamQ+pSB1A2 Black\_1 |

#### Electrophoresis

Tatsui

| Lane | Colony PCR ProductsⅠ |
| --- | --- |
| 1 | 9/12 mamL+pSB1A2 Green\_1 |
| 2 | 9/12 mamL+pSB1A2 Green\_2 |
| 3 | 9/12 mamL+pSB1A2 Green\_3 |
| 4 | 9/12 mamL+pSB1A2 Green\_4 |
| 5 | 9/12 mamL+pSB1A2 Green\_5 |
| 6 | 9/12 mamL+pSB1A2 Black\_7 |
| 7 | 9/12 mamL+pSB1A2 Black\_8 |
| 8 | 9/12 mamQ+pSB1A2 Black\_1 |
| 9 | 1kb ladder |

#### Liquid Culture

Yamauchi

| Name | medium |
| --- | --- |
| 9/11 Transformation mamQ+pSB1A2 Colony\_1 | Plusgrow medium(AMP) |
| 9/11 Transformation mamQ+pSB1A2 Colony\_2 | Plusgrow medium(AMP) |
| 9/11 Transformation mamQ+pSB1A2 Colony\_3 | Plusgrow medium(AMP) |
| 9/11 Transformation mamQ+pSB1A2 Colony\_4 | Plusgrow medium(AMP) |
| 9/11 Transformation mamQ+pSB1A2 Colony\_5 | Plusgrow medium(AMP) |
| 9/11 Transformation mamQ+pSB1A2 Colony\_6 | Plusgrow medium(AMP) |

### 9/13

#### Miniprep

Tatsui

| DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 9/12 Liquid Culture mamQ+pSB1A2 Colony\_1 Error | - | - | - |
| 9/12 Liquid Culture mamQ+pSB1A2 Colony\_2 Error | - | - | - |
| 9/12 Liquid Culture mamQ+pSB1A2 Colony\_3 Error | - | - | - |
| 9/12 Liquid Culture mamQ+pSB1A2 Colony\_4 | 80 | 2.02 | 2.76 |
| 9/12 Liquid Culture mamQ+pSB1A2 Colony\_5 | 70 | 2.13 | 3.41 |
| 9/12 Liquid Culture mamQ+pSB1A2 Colony\_6 | 110 | 1.96 | 1.70 |
| 9/12 Liquid Culture Pcon+pSB1C3 | 110 | 1.96 | 1.70 |

#### PCR Labeling and Sequence

No Name

| Target | 9/13 mamQ+pSB1A2 Colony\_4 | 9/13 mamQ+pSB1A2 Colony\_5 | 9/13 mamQ+pSB1A2 Colony\_6 | Pcon+pSB1A2 | 9/13 mamQ+pSB1A2 Colony\_4 | 9/13 mamQ+pSB1A2 Colony\_5 | 9/13 mamQ+pSB1A2 Colony\_6 |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Template/(µl) | 5 | 4 | 6 | 4 | 5 | 6 | 4 |
| Big dye/(µl) | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 |
| 5×Buffer/(µl) | 1.75 | 1.75 | 1.75 | 1.75 | 1.75 | 1.75 | 1.75 |
| Primer/(µl) | 0.5(VF) | 0.5(VF) | 0.5(VF) | 0.5(VF) | 0.5(VR) | 0.5(VR) | 0.5(VR) |
| MilliQ/(µl) | 2.75 | 1.75 | 3.75 | 3.75 | 2.75 | 1.75 | 3.75 |
| TOTAL/(µl) | 10.5 | 10.5 | 10.5 | 10.5 | 10.5 | 10.5 | 10.5 |

| Target | 9/12 mamQ+YCplac111 | 9/12 mamL+YCplac111 | 9/12 mamQ+YCplac111 | 9/12 mamL+YCplac111 | 9/12 mamQ+YCplac111 |
| --- | --- | --- | --- | --- | --- |
| Template/(µl) | 3 | 1.5 | 3 | 1.5 | 3 |
| Big dye/(µl) | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 |
| 5×Buffer/(µl) | 1.75 | 1.75 | 1.75 | 1.75 | 1.75 |
| Primer/(µl) | 0.5(M13F) | 0.5(M13F) | 0.5(M13R) | 0.5(M13R) | 0.5(seqpLQB-2) |
| MilliQ/(µl) | 4.75 | 6.25 | 4.75 | 6.25 | 4.75 |
| TOTAL/(µl) | 10.5 | 10.5 | 10.5 | 10.5 | 10.5 |

#### Gel Extraction

No Name

| Lane | Restriction Enzyme Digestion ProductⅠ | Volume/(µl) |
| --- | --- | --- |
| 1 | 1kb ladder | - |
| 2 | - | - |
| 3 | 9/12 Digestion mamQ+YCpla111(X+P)(992bp) | - |
| 4 | - | - |
| 5 | - | - |
| 6 | - | - |
| 7 | 9/12 Digestion mamL+YCpla111(X+P)(340bp) | - |
| 8 | - | - |
| 9 | 9/12 Digestion Pcon+pSB1C3(Nega)(5600bp) | - |
| 10 | - | - |
| 11 | 9/12 Digestion Pcon+pSB1C3(S+P)(2070bp) | - |

| DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 9/12 Digestion mamL+YCpla111(X+P) | 10 | 1.00 | 0.70 |
| 9/12 Digestion Pcon+pSB1C3(S+P) | 150 | 1.25 | 0.71 |

#### Restriction Enzyme Digestion

Li

| Sample | DNA/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | EcoR1/(µl) | Buffur/(µl) | BSA | MilliQ/(µl) | TOTAL/(µl) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 9/13 Miniprep mamQ+pSB1A2 Colony\_4 | 12.5 | 0.5 | 0 | 0.5 | 0 | 1.7(M) | 1.6 | 0 | 16.8 |
| 9/13 Miniprep mamQ+pSB1A2 Colony\_5 | 14.3 | 0.5 | 0 | 0.5 | 0 | 1.9(M) | 1.86 | 0 | 19.06 |
| 9/13 Miniprep mamQ+pSB1A2 Colony\_6 | 9.1 | 0.5 | 0 | 0.5 | 0 | 1.5(M) | 1.5 | 1.9 | 15 |
| 9/13 Miniprep mamQ+pSB1A2 Colony\_6(Nega) | 3 | 0 | 0 | 0 | 0 | 0.5(M) | 0.5 | 1 | 5 |

#### Ligation

Yasuda

| Insert/(µL) | | Vector/(µL) | | T4 Ligase/(µl) | 2\*Buffer/(µl) | MilliQ/(µl) | Total/(µl) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 9/13 Gel Extraction mamL(X+P) | 3 | Pcon+pSB1C3(S+P) | 1 | 1 | 5 | 0 | 10 |

#### Miniprep

Honda

| DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 9/12 Liquid Culture Pcon+pSB1C3 | 240 | 1.78 | 1.59 |

### 9/14

#### Gel Extraction

Honda

| Lane | Restriction Enzyme Digestion ProductⅠ | Volume/(µl) |
| --- | --- | --- |
| 1 | 1kb ladder | 3 |
| 2 | - | - |
| 3 | 9/13 mamQ Colony\_4(X+P) | 16.8 |
| 4 | - | - |
| 5 | 1kb ladder | 3 |
| 6 | - | - |
| 7 | 9/13 mamQ Colony\_5(X+P) | 19.06 |
| 8 | - | - |
| 9 | 9/13 mamQ Colony\_6(Nega) | 5 |
| 10 | - | - |
| 11 | 9/13 mamQ Colony\_6(X+P) | 15 |

#### Ligation

Tatsui

| Insert/(µL) | | Vector/(µL) | | T4 Ligase/(µl) | 2\*Buffer/(µl) | MilliQ/(µl) | Total/(µl) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| mamB(E+S) | 4 | pSB1C3(E+S) | 3 | 1 | 8 | 0 | 16 |
| mamL(X+P) | 3.3 | pSB1C3(E+S) | 0.7 | 1 | 5 | 0 | 10 |

#### PCR

Tatsui

| Target | E+X+RBS+mamB+S |
| --- | --- |
| 2×KAPA HiFi Hotstart Ready Mix/(µl) | 12.5 |
| Fw Primer/(µl) | 0.75(E+X+RBS+mamB) |/tr>| Rv Primer/(µl) | 0.75(S+mamB) |
| 9/9 DNA Purification Product/(µl) | Trace |
| MilliQ/(µl) | 11 |
| TOTAL/(µl) | 25 |

| Steps | Temparature | Time | cycle |
| --- | --- | --- | --- |
| Pre Denature | 98℃ | 5min | 1time |
| Denature | 98℃ | 20sec | 25cycle |
| Annealing | 60℃ | 15sec | 25cycle |
| Extension | 72℃ | 15sec | 25cycle |
| Final elongation | 72℃ | 5min | 1time |
| Final hold | 12℃ | - | - |

#### Transformation

Tatsui

| Sample/(µL) | | CompetentCells/(µl) | Total/(µl) | Medium |
| --- | --- | --- | --- | --- |
| 9/14 Ligation Pcon+mamL+pSB1C3 | 1 | 10 | 11 | LB(CP) |
| 9/14 Ligation Pcon+mamL+pSB1C3(Nega) | 1 | 10 | 11 | LB(CP) |
| 9/14 Ligation mamB+pSB1C3 | 1 | 10 | 11 | LB(CP) |

### 9/15

#### Colony PCR

Tatsui

| NameⅠ | NameⅡ |
| --- | --- |
| 9/14 Transformation mamB+pSB1C3 Colony\_1 | 9/14 Transformation mamB+pSB1C3 Colony\_9 |
| 9/14 Transformation mamB+pSB1C3 Colony\_2 | 9/14 Transformation mamB+pSB1C3 Colony\_10 |
| 9/14 Transformation mamB+pSB1C3 Colony\_3 | 9/14 Transformation mamB+pSB1C3 Colony\_11 |
| 9/14 Transformation mamB+pSB1C3 Colony\_4 | 9/14 Transformation mamB+pSB1C3 Colony\_12 |
| 9/14 Transformation mamB+pSB1C3 Colony\_5 | 9/14 Transformation mamB+pSB1C3 Colony\_13 |
| 9/14 Transformation mamB+pSB1C3 Colony\_6 | 9/14 Transformation mamB+pSB1C3 Colony\_14 |
| 9/14 Transformation mamB+pSB1C3 Colony\_7 | 9/14 Transformation mamB+pSB1C3 Colony\_15 |
| 9/14 Transformation mamB+pSB1C3 Colony\_8 | - |

#### Electrophoresis

Tatsui

| Lane | Colony PCR ProductsⅠ |
| --- | --- |
| 1 | 9/14 mamB+pSB1C3 Colony\_1 |
| 2 | 9/14 mamB+pSB1C3 Colony\_2 |
| 3 | 9/14 mamB+pSB1C3 Colony\_3 |
| 4 | 9/14 mamB+pSB1C3 Colony\_4 |
| 5 | 9/14 mamB+pSB1C3 Colony\_5 |
| 6 | 9/14 mamB+pSB1C3 Colony\_6 |
| 7 | 9/14 mamB+pSB1C3 Colony\_7 |
| 8 | 9/14 mamB+pSB1C3 Colony\_8 |
| 9 | 1kb ladder |
| 10 | 9/14 mamB+pSB1C3 Colony\_9 |
| 11 | 9/14 mamB+pSB1C3 Colony\_10 |
| 12 | 9/14 mamB+pSB1C3 Colony\_11 |
| 13 | 9/14 mamB+pSB1C3 Colony\_12 |
| 14 | 9/14 mamB+pSB1C3 Colony\_13 |
| 15 | 9/14 mamB+pSB1C3 Colony\_14 |
| 16 | 9/14 mamB+pSB1C3 Colony\_15 |

#### Colony PCR

No Name

| NameⅠ |
| --- |
| 9/14 Transformation Pcon+mamL+SB1C3 Colony\_1 |
| 9/14 Transformation mamB+pSB1C3 Colony\_9 |

#### Transformation

Tatsui

| Sample/(µL) | | CompetentCells/(µl) | Total/(µl) | Medium |
| --- | --- | --- | --- | --- |
| mamB+YCplac111 | 1 | 10 | 11 | LB(AMP) |
| Pcon+mamL+pSB1C3 | 1 | 10 | 11 | LB(CP) |

#### Miniprep

Honda

| DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
| --- | --- | --- | --- |
| mamB+pSB1C3 Colony\_9 | - | - | - |
| mamB+pSB1C3 Colony\_10 | - | - | - |
| mamB+pSB1C3 Colony\_11 | - | - | - |
| mamL+pSB1A2 Colony\_1 | 170 | 1.74 | 1.41 |
| mamL+pSB1A2 Colony\_2 | 140 | 1.95 | 1.43 |
| mamL+pSB1A2 Colony\_3 | 135 | 1.95 | 1.60 |
| mamL+pSB1A2 Colony\_4 | - | - | - |
| mamL+pSB1A2 Colony\_5 | - | - | - |
| mamL+pSB1A2 Colony\_6 | - | - | - |

#### Liquid Culture

Honda

| Name | medium |
| --- | --- |
| 9/14 Transformation Pcon+mamL+pSB1C3 Colony\_1 | Plusgrow medium(CP) |
| 9/14 Transformation mamB+pSB1C3 Colony\_9 | Plusgrow medium(CP) |
| 9/14 Transformation mamB+pSB1C3 Colony\_10 | Plusgrow medium(CP) |
| 9/14 Transformation mamB+pSB1C3 Colony\_11 | Plusgrow medium(CP) |

#### Restriction Enzyme Digestion

Li

| Sample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | BSA | MilliQ/(µl) | Buffur/(µl) | TOTAL/(µl) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 9/15 DNA Purification mamB | 25 | 1 | 0 | 1 | 0 | 0 | 0 | 3(H) | 30 |
| 9/15 Mniprep mamL+pSB1A2 Colony\_1 | 11.8 | 0 | 1 | 0 | 1 | 3 | 10.2 | 3(M) | 30 |
| 9/15 Mniprep mamL+pSB1A2 Colony\_1(Nega) | 0.6 | 0 | 0 | 0 | 0 | 1 | 8.4 | 1(M) | 10 |
| 9/15 Mniprep mamL+pSB1A2 Colony\_2 | 14.3 | 0 | 1 | 0 | 1 | 3 | 7.7 | 3(M) | 30 |
| 9/15 Mniprep mamL+pSB1A2 Colony\_2(Nega) | 0.7 | 0 | 0 | 0 | 0 | 1 | 8.3 | 1(M) | 10 |
| 9/15 Mniprep mamL+pSB1A2 Colony\_3 | 14.8 | 0 | 1 | 0 | 1 | 3 | 7.2 | 3(M) | 30 |
| 9/15 Mniprep mamL+pSB1A2 Colony\_3(Nega) | 0.7 | 0 | 0 | 0 | 0 | 1 | 8.3 | 1(M) | 10 |
| 9/15 Mniprep mamL+pSB1A2 Colony\_4 | 13.3 | 0 | 1 | 0 | 1 | 3 | 8.7 | 3(M) | 30 |
| 9/15 Mniprep mamL+pSB1A2 Colony\_4(Nega) | 0.7 | 0 | 0 | 0 | 0 | 1 | 8.3 | 1(M) | 10 |
| 9/13 Pcon+pSB1C3 | 20 | 0 | 0 | 1 | 1 | 0 | 5 | 3(H) | 30 |
| 9/13 Pcon+pSB1C3(Nega) | 1 | 0 | 0 | 0 | 0 | 0 | 6 | 3(H) | 10 |

#### PCR

Honda

| Target | mamB Colony\_10 | mamB Colony\_11 |
| --- | --- | --- |
| 2×PrimeSTAR Max Premix/(µl) | 25 | 25 |
| Fw mamB Mutation Primer/(µl) | 1 | 1 |
| Rv mamB Mutation Primer/(µl) | 1 | 1 |
| 9/15 Miniprep mamB+pSB1C3/(µl) | Colony\_10(Trace) | Colony\_11(Trace) |
| MilliQ/(µl) | 23 | 23 |
| TOTAL/(µl) | 50 | 50 |

| Steps | Temparature | Time | cycle |
| --- | --- | --- | --- |
| Pre Denature | 98℃ | 2min | 1time |
| Denature | 98℃ | 10sec | 30cycle |
| Annealing | 55℃ | 15sec | 30cycle |
| Extension | 72℃ | 15sec | 30cycle |
| Final elongation | 72℃ | 5min | 1time |
| Final hold | 12℃ | - | - |

### 9/16

#### PCR Labeling and Sequence

No Name

| Target | 9/15 Pcon+mamL+pSB1C3 | 9/15 mamL+pSB1A2 Colony\_1 | 9/15 mamL+pSB1A2 Colony\_2 | 9/15 mamL+pSB1A2 Colony\_3 | 9/15 mamL+pSB1A2 Colony\_4 | 9/15 mamB+pSB1C3 Colony\_9 | 9/15 mamB+pSB1C3 Colony\_10 | 9/15 mamB+pSB1C3 Colony\_11 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Template/(µl) | 1.8 | 2.35 | 2.9 | 3.0 | 3.0 | 1.14 | 1.2 | 1.2 |
| Big dye/(µl) | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 |
| 5×Buffer/(µl) | 1.75 | 1.75 | 1.75 | 1.75 | 1.75 | 1.75 | 1.75 | 1.75 |
| Primer(VF)/(µl) | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 |
| MilliQ/(µl) | 5.95 | 5.4 | 4.85 | 4.75 | 4.75 | 6.61 | 6.55 | 6.55 |
| TOTAL/(µl) | 10.5 | 10.5 | 10.5 | 10.5 | 10.5 | 10.5 | 10.5 | 10.5 |

| Target | 9/15 Pcon+mamL+pSB1C3 | 9/15 mamL+pSB1A2 Colony\_1 | 9/15 mamL+pSB1A2 Colony\_2 | 9/15 mamL+pSB1A2 Colony\_3 | 9/15 mamB+pSB1C3 Colony\_9 | 9/15 mamB+pSB1C3 Colony\_10 | 9/15 mamB+pSB1C3 Colony\_11 |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Template/(µl) | 1.8 | 2.35 | 2.9 | 3.0 | 1.14 | 1.2 | 1.2 |
| Big dye/(µl) | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 |
| 5×Buffer/(µl) | 1.75 | 1.75 | 1.75 | 1.75 | 1.75 | 1.75 | 1.75 |
| Primer(VR)/(µl) | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 |
| MilliQ/(µl) | 5.95 | 5.4 | 4.85 | 4.75 | 6.61 | 6.55 | 6.55 |
| TOTAL/(µl) | 10.5 | 10.5 | 10.5 | 10.5 | 10.5 | 10.5 | 10.5 |

| Steps | Temparature | Time | cycle |
| --- | --- | --- | --- |
| Pre Denature | 96℃ | 2min | 1time |
| Denature | 96℃ | 10sec | 30cycle |
| Annealing | 55℃ | 5sec | 30cycle |
| Extension | 72℃ | 4min | 30cycle |
| Final elongation | 72℃ | 1min | 1time |
| Final hold | 4℃ | - | - |

#### Gel Extraction

Honda

| Lane | Restriction Enzyme Digestion ProductⅠ | Volume/(µl) |
| --- | --- | --- |
| 1 | 1kb ladder | - |
| 2 | 1kb ladder | - |
| 3 | 9/15 mamL Colony\_1(X+P) | - |
| 4 | - | - |
| 5 | 9/15 mamL Colony\_2(Nega) | - |
| 6 | - | - |
| 7 | 9/15 mamL Colony\_2(X+P) | - |
| 8 | 1kb ladder | - |
| 9 | 1kb ladder | - |
| 10 | 9/15 mamL Colony\_3(Nega) | - |
| 11 | 9/15 mamL Colony\_3(X+P) | - |

| Lane | Restriction Enzyme Digestion ProductⅡ | Volume/(µl) |
| --- | --- | --- |
| 1 | 1kb ladder | - |
| 2 | - | - |
| 3 | - | - |
| 4 | 9/15 mamQ(X+P) | - |
| 5 | - | - |
| 6 | 9/15 mamQ(Nega) | - |

| DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
| --- | --- | --- | --- |
| mamL Colony\_1 | 45 | 1.69 | 0.60 |
| mamL Colony\_2 | 60 | 1.33 | 0.70 |
| mamL Colony\_3 | 40 | 1.76 | 0.08 |
| mamQ | 65 | 1.35 | 0.83 |

#### Dephosphorylation

Honda

| Sample/(µL) | |
| --- | --- |
| 9/15 Digestion Pcon+pSB1C3(S+P) | - |
| 9/15 Gel Extraction Pcon+pSB1C3(S+P) | - |

#### DNA Purification

Honda

| DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 9/15 Mutation PCR mamB+pSB1C3 Colony\_10 | 225 | 1.64 | 1.74 |
| 9/15 Mutation PCR mamB+pSB1C3 Colony\_11 | 205 | 1.59 | 1.76 |

#### Gel Extraction

Yamauchi

| Lane | SampleⅠ | Volume/(µl) |
| --- | --- | --- |
| 1 | 1kb ladder | - |
| 2 | - | - |
| 3 | 9/15 Digestion Pcon+pSB1C3(S+P) | - |
| 4 | - | - |
| 5 | 1kb ladder | - |
| 6 | - | - |
| 7 | 9/15 Gel Extraction Pcon+pSB1C3(S+P) | - |
| 8 | - | - |
| 9 | 1kb ladder | - |
| 10 | - | - |
| 11 | - | - |

#### Transformation

Honda and Tatsui

| Sample/(µL) | | CompetentCells/(µl) | Total/(µl) | Medium |
| --- | --- | --- | --- | --- |
| 9/16 DNA Purification mamB+pSB1C3 Colony\_10 | 1 | 10 | 11 | LB(CP) |
| 9/16 DNA Purification mamB+pSB1C3 Colony\_11 | 1 | 10 | 11 | LB(CP) |

#### Restriction Enzyme Digestion

No Name

| Sample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | BSA | MilliQ/(µl) | Buffur/(µl) | TOTAL/(µl) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 9/15 mamQ+pSB1A2 Colony\_4 | 26.7 | 0 | 2 | 0 | 2 | 6 | 17.3 | 6(M) | 60 |
| 9/15 mamQ+pSB1A2 Colony\_4(Nega) | 0.7 | 0 | 0 | 0 | 0 | 1 | 7.3 | 1(M) | 10 |
| 9/15 Pcon+mamL+pSB1C3 | 15 | 0 | 0 | 2 | 2 | 0 | 35 | 6(H) | 60 |
| 9/15 Pcon+mamL+pSB1C3(Nega) | 0.5 | 0 | 0 | 0 | 0 | 0 | 8.5 | 1(H) | 10 |

#### PCR

Tatsui

| Target | E+Pcon+X+RBS+mamB+His+S(\*2) |
| --- | --- |
| 2×KAPA HiFi Hotstart Ready Mix/(µl) | 12.5 |
| Fw E+Pcon+X+RBS+mamB/(µl) | 0.75 |
| Rv S+His+mamB/(µl) | 0.75 |
| 9/9 DNA Purification mamB/(µl) | Trace |
| MilliQ/(µl) | 11 |
| TOTAL/(µl) | 25 |

#### Miniprep

Honda

| DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 9/15 mamB+pSB1C3 Colony\_9 | 350 | 1.68 | 1.81 |
| 9/15 mamB+pSB1C3 Colony\_10 | 325 | 1.65 | 1.50 |
| 9/15 mamB+pSB1C3 Colony\_11 | 340 | 1.67 | 1.68 |
| Pcon+mamL+pSB1C3 | 220 | 1.75 | 2.09 |

### 9/17

#### Colony PCR

Tatsui

| NameⅠ |
| --- |
| 9/16 Transformation Mutation mamB Colony\_11\_1 |
| 9/16 Transformation Mutation mamB Colony\_11\_2 |
| 9/16 Transformation Mutation mamB Colony\_11\_3 |
| 9/16 Transformation Mutation mamB Colony\_11\_4 |
| 9/16 Transformation Mutation mamB Colony\_10\_1 |
| 9/16 Transformation Mutation mamB Colony\_10\_2 |
| 9/16 Transformation Mutation mamB Colony\_10\_3 |
| 9/16 Transformation Mutation mamB Colony\_10\_4 |

#### Miniprep

Tatsui and Yamauchi

| DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 9/15 Pcon+pSB1C3 Colony\_3 | 110 | 1.66 | 1.48 |

#### Gel Extraction

Yamauchi

| Lane | SampleⅠ | Volume/(µl) |
| --- | --- | --- |
| 1 | 9/16 Mutation PCR Pcon+mamB+His | - |
| 2 | Pcon+mamL(Nega) | - |
| 3 | Pcon+mamL(S+P) | - |
| 4 | 1kb ladder | - |
| 5 | 9/16 Mutation PCR Pcon+mamB+His | - |
| 6 | - | - |
| 7 | mamQ+pSB1A2 Colony\_4(X+P) | - |
| 8 | 1kb ladder | - |
| 9 | mamQ(Nega) | - |
| 10 | - | - |
| 11 | mamQ+pSB1A2 Colony\_4(X+P) | - |

| DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
| --- | --- | --- | --- |
| Pcon+mamB+His | 245 | 1.87 | 2.10 |
| mamQ+pSB1A2 Colony\_4(X+P) | 35 | 1.87 | 0.580 |
| Pcon+mamL(S+P) | 285 Error | 1.14 | 0.85 |

#### Ligation

Tatsui

| Insert/(µL) | | Vector/(µL) | | T4 Ligase/(µl) | 2\*Buffer/(µl) | MilliQ/(µl) | Total/(µl) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| mamQ(X+P) | 3 | Pcon+mamL(S+P) | 1 | 1 | 5 | 0 | 10 |

#### Transformation

Tatsui

| Sample/(µL) | | CompetentCells/(µl) | Total/(µl) | Medium |
| --- | --- | --- | --- | --- |
| 9/17 Ligation Pcon+mamL+mamQ+pSB1C3 | 1 | 10 | 11 | LB(CP) |

#### Restriction Enzyme Digestion

Tatsui

| Sample | DNA/(µl) | EcoR1/(µl) | Spe1/(µl) | Xba1/(µl) | Pst1/(µl) | BSA | Buffur/(µl) | MilliQ/(µl) | TOTAL/(µl) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 9/17 Gel Extraction Pcon+mamB+His | 16.3 | 2 | 2 | 0 | 0 | 0 | 6(H) | 33.7 | 60 |
| 9/17 Gel Extraction Pcon+mamB+His(Nega) | 1 | 0 | 0 | 0 | 0 | 0 | 1(H) | 8 | 10 |
| 9/17 Miniprep Pcon+pSB1C3 | 36.4 | 0 | 2 | 0 | 2 | 0 | 6(H) | 13.6 | 60 |
| 9/17 Miniprep Pcon+pSB1C3(Nega) | 1 | 0 | 0 | 0 | 0 | 0 | 1(H) | 8 | 10 |

#### Liquid Culture

Tatsui

| Name | medium |
| --- | --- |
| Mutation mamB Colony\_10\_3 | Plusgrow medium(CP) |
| Mutation mamB Colony\_10\_4 | Plusgrow medium(CP) |
| Mutation mamB Colony\_11\_3 | Plusgrow medium(CP) |
| Mutation mamB Colony\_11\_4 | Plusgrow medium(CP) |
| mamQ+pSB1A2 Colony\_4 | Plusgrow medium(AMP)\*2 |
| Pcon+mamL Colony\_1 | Plusgrow medium(CP)\*3.5 |

### 9/18

#### Miniprep

Tatsui and Honda

| DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 9/17 Liquid Culture Mutation mamB Colony\_10\_3 | 355 | 1.69 | 2.02 |
| 9/17 Liquid Culture Mutation mamB Colony\_10\_4 | 265 | 1.52 | 1.55 |
| 9/17 Liquid Culture Mutation mamB Colony\_11\_3 | 355 | 1.62 | 1.77 |
| 9/17 Liquid Culture Mutation mamB Colony\_11\_4 | 250 | 1.62 | 1.51 |
| 9/17 Liquid Culture mamQ+pSB1A2 Colony\_4 | 290 | 1.41 | 1.10 |
| 9/17 Liquid Culture Pcon+mamL+pSB1C3 Colony\_1 | 85 | 1.47 | 1.56 |

#### PCR Labeling and Sequence

No Name

| Target | 9/18 Miniprep Mutation mamB Colony\_10\_3 | 9/18 Miniprep Mutation mamB Colony\_10\_4 | 9/18 Miniprep Mutation mamB Colony\_11\_3 | 9/18 Miniprep Mutation mamB Colony\_11\_4 | 9/18 Miniprep Mutation mamB Colony\_10\_3 | 9/18 Miniprep Mutation mamB Colony\_10\_4 | 9/18 Miniprep Mutation mamB Colony\_11\_3 | 9/18 Miniprep Mutation mamB Colony\_11\_4 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Template/(µl) | 1.1 | 1.5 | 1.1 | 1.6 | 1.1 | 1.5 | 1.1 | 1.6 |
| Big dye/(µl) | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 |
| 5×Buffer/(µl) | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
| Primer/(µl) | 0.16(VF) | 0.16(VF) | 0.16(VF) | 0.16(VF) | 0.16(VR) | 0.16(VR) | 0.16(VR) | 0.16(VR) |
| MilliQ/(µl) | 5.74 | 5.34 | 5.74 | 5.24 | 5.74 | 5.34 | 5.74 | 5.24 |
| TOTAL/(µl) | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 |

| Target | 9/18 Miniprep Mutation mamB Colony\_10\_3 | 9/18 Miniprep Mutation mamB Colony\_10\_4 | 9/18 Miniprep Mutation mamB Colony\_11\_3 | 9/18 Miniprep Mutation mamB Colony\_11\_4 |
| --- | --- | --- | --- | --- |
| Template/(µl) | 1.1 | 1.5 | 1.1 | 1.6 |
| Big dye/(µl) | 2 | 2 | 2 | 2 |
| 5×Buffer/(µl) | 1 | 1 | 1 | 1 |
| Primer/(µl) | 0.16(pQLB seq\_2) | 0.16(pQLB seq\_2) | 0.16(pQLB seq\_2) | 0.16(pQLB seq\_2) |
| MilliQ/(µl) | 5.74 | 5.34 | 5.74 | 5.24 |
| TOTAL/(µl) | 10 | 10 | 10 | 10 |

| Steps | Temparature | Time | cycle |
| --- | --- | --- | --- |
| Pre Denature | 96℃ | 2min | 1time |
| Denature | 96℃ | 10sec | 30cycle |
| Annealing | 50℃ | 5sec | 30cycle |
| Extension | 60℃ | 4min | 30cycle |
| Final elongation | 4℃ | 1min | 1time |
| Final hold | 4℃ | - | - |

#### Colony PCR

Tatsui

| NameⅠ | NameⅡ |
| --- | --- |
| 9/17 Transformation Pcon+mamL+mamQ+pSB1C3 Colony\_1 | 9/17 Transformation Pcon+mamL+mamQ+pSB1C3 Colony\_9 |
| 9/17 Transformation Pcon+mamL+mamQ+pSB1C3 Colony\_2 | 9/17 Transformation Pcon+mamL+mamQ+pSB1C3 Colony\_10 |
| 9/17 Transformation Pcon+mamL+mamQ+pSB1C3 Colony\_3 | 9/17 Transformation Pcon+mamL+mamQ+pSB1C3 Colony\_11 |
| 9/17 Transformation Pcon+mamL+mamQ+pSB1C3 Colony\_4 | 9/17 Transformation Pcon+mamL+mamQ+pSB1C3 Colony\_12 |
| 9/17 Transformation Pcon+mamL+mamQ+pSB1C3 Colony\_5 | 9/17 Transformation Pcon+mamL+mamQ+pSB1C3 Colony\_13 |
| 9/17 Transformation Pcon+mamL+mamQ+pSB1C3 Colony\_6 | 9/17 Transformation Pcon+mamL+mamQ+pSB1C3 Colony\_14 |
| 9/17 Transformation Pcon+mamL+mamQ+pSB1C3 Colony\_7 | 9/17 Transformation Pcon+mamL+mamQ+pSB1C3 Colony\_15 |
| 9/17 Transformation Pcon+mamL+mamQ+pSB1C3 Colony\_8 | 9/17 Transformation Pcon+mamL+mamQ+pSB1C3 Colony\_16 |

#### Electrophoresis

Tatsui

| Lane | Colony PCR ProductsⅠ |
| --- | --- |
| 1 | 9/18 Pcon+mamL+mamQ+pSB1C3 Colony\_1 |
| 2 | 9/18 Pcon+mamL+mamQ+pSB1C3 Colony\_2 |
| 3 | 9/18 Pcon+mamL+mamQ+pSB1C3 Colony\_3 |
| 4 | 9/18 Pcon+mamL+mamQ+pSB1C3 Colony\_4 |
| 5 | 9/18 Pcon+mamL+mamQ+pSB1C3 Colony\_5 |
| 6 | 9/18 Pcon+mamL+mamQ+pSB1C3 Colony\_6 |
| 7 | 9/18 Pcon+mamL+mamQ+pSB1C3 Colony\_7 |
| 8 | 9/18 Pcon+mamL+mamQ+pSB1C3 Colony\_8 |
| 9 | 1kb ladder |
| 10 | 9/18 Pcon+mamL+mamQ+pSB1C3 Colony\_9 |
| 11 | 9/18 Pcon+mamL+mamQ+pSB1C3 Colony\_10 |
| 12 | 9/18 Pcon+mamL+mamQ+pSB1C3 Colony\_11 |
| 13 | 9/18 Pcon+mamL+mamQ+pSB1C3 Colony\_12 |
| 14 | 9/18 Pcon+mamL+mamQ+pSB1C3 Colony\_13 |
| 15 | 9/18 Pcon+mamL+mamQ+pSB1C3 Colony\_14 |
| 16 | 9/18 Pcon+mamL+mamQ+pSB1C3 Colony\_15 |
| 17 | 9/18 Pcon+mamL+mamQ+pSB1C3 Colony\_16 |

#### DNA Purification

Tatsui

| DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 9/17 Digestion Pcon+mamB+His(E+S) | 165 | - | - |

#### Gel Extraction

Tatsui

| Lane | SampleⅠ | Volume/(µl) |
| --- | --- | --- |
| 1 | - | - |
| 2 | 1kb ladder | - |
| 3 | 9/17 Digestion Pcon+pSB1C3(Nega) | - |
| 4 | 9/17 Digestion Pcon+pSB1C3(S+P) | - |
| 5 | - | - |
| 6 | - | - |

| Lane | SampleⅡ | Volume/(µl) |
| --- | --- | --- |
| 1 | - | - |
| 2 | - | - |
| 3 | 1kb ladder | - |
| 4 | 9/17 Digestion Pcon+pSB1C3(S+P) | - |
| 5 | - | - |
| 6 | - | - |

| DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 9/17 Digestion Pcon+pSB1C3 | - | - | - |

#### Ligation

Tatsui

| Insert/(µL) | | Vector/(µL) | | T4 Ligase/(µl) | 10\*Buffer/(µl) | MilliQ/(µl) | Total/(µl) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| mamQ(X+P) | 6 | 9/18 Gel Extraction Pcon+pSB1C3(S+P) | Trace | 0.5 | 1 | 2.5 | 10 |
| 9/18 DNA Purification Pcon+mamB+His(E+S) | 5 | 9/14 pSB3T5(E+S) | 2 | 0.5 | 1 | 1.5 | 10 |

#### Transformation

Tatsui

| Sample/(µL) | | CompetentCells/(µl) | Total/(µl) | Medium |
| --- | --- | --- | --- | --- |
| 9/18 Ligation Pcon+mamQ+pSB1C3 | 1 | 10 | 11 | LB(CP) |
| 9/18 Ligation Pcon+mamB+His+pSB3T5 | 1 | 10 | 11 | LB(TET) |

#### Liquid Culture

Tatsui

| Name | medium |
| --- | --- |
| pSB3T5 | Plusgrow medium(TET)\*2 |
| 9/17 Transformation Pcon+mamL+mamQ+pSB1C3 | Plusgrow medium(CP) |

#### Restriction Enzyme Digestion

No Name

| Sample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | BSA | Buffur/(µl) | MilliQ/(µl) | TOTAL/(µl) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 9/18 Miniprep Mutation mamB Colony\_10\_3 | 11.2 | 0 | 2 | 0 | 2 | 0.8 | 8(D) | 56 | 80 |
| 9/18 Miniprep Mutation mamB Colony\_10\_3(Nega) | 1 | 0 | 0 | 0 | 0 | 0 | 1(D) | 8 | 10 |
| 9/18 Miniprep Mutation mamB Colony\_10\_4 | 15.1 | 0 | 2 | 0 | 2 | 0.8 | 8(D) | 52.1 | 80 |
| 9/18 Miniprep Mutation mamB Colony\_10\_4(Nega) | 1 | 0 | 0 | 0 | 0 | 0 | 1(D) | 8 | 10 |
| 9/18 Miniprep Mutation mamB Colony\_11\_3 | 11.2 | 0 | 2 | 0 | 2 | 0.8 | 8(D) | 56 | 80 |
| 9/18 Miniprep Mutation mamB Colony\_11\_3(Nega) | 1 | 0 | 0 | 0 | 0 | 0 | 1(D) | 8 | 10 |
| 9/18 Miniprep Mutation mamB Colony\_11\_4 | 16 | 0 | 2 | 0 | 2 | 0.8 | 8(D) | 51.2 | 80 |
| 9/18 Miniprep Mutation mamB Colony\_11\_4(Nega) | 1 | 0 | 0 | 0 | 0 | 0 | 1(D) | 8 | 10 |
| 9/18 Miniprep mamQ+pSB1A2 | 34.5 | 0 | 5 | 0 | 5 | 2 | 20(D) | 133.5 | 200 |
| 9/18 Miniprep mamQ+pSB1A2(Nega) | 1 | 0 | 0 | 0 | 0 | 0 | 1(D) | 8 | 10 |
| 9/17 Miniprep Pcon+pSB1C3 | 40 | 0 | 0 | 2 | 2 | 0.8 | 8(B) | 27.2 | 80 |
| 9/17 Miniprep Pcon+pSB1C3(Nega) | 1 | 0 | 0 | 0 | 0 | 0 | 1(B) | 8 | 10 |
| 9/18 Miniprep Pcon+mamL+pSB1C3 | 47 | 0 | 0 | 2 | 2 | 0.8 | 8(B) | 20.2 | 80 |
| 9/18 Miniprep Pcon+mamL+pSB1C3(Nega) | 1 | 0 | 0 | 0 | 0 | 0 | 1(B) | 8 | 10 |

### 9/19

#### Miniprep

Tatsui

| DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 9/18 Liquid Culture pSB3T5 | 115 | 1.52 | 1.22 |
| 9/18 Liquid Culture Pcon+mamL+mamQ+pSB1C3 | 75 | 1.59 | 1.20 |

#### PCR Labeling and Sequence

No Name

| Target | 9/19 Miniprep Pcon+mamL+mamQ+pSB1C3 | 9/19 Miniprep Pcon+mamL+mamQ+pSB1C3 | 9/19 Miniprep Pcon+mamL+mamQ+pSB1C3 | 9/19 Miniprep Pcon+mamL+mamQ+pSB1C3 |
| --- | --- | --- | --- | --- |
| Template/(µl) | 3.5 | 3.5 | 3.5 | 3.5 |
| Big dye/(µl) | 0.5 | 0.5 | 0.5 | 0.5 |
| 5×Buffer/(µl) | 1.75 | 1.75 | 1.75 | 1.75 |
| Primer/(µl) | 0.5(VF) | 0.5(VR) | 0.5(seq\_1) | 0.5(seq\_2) |
| MilliQ/(µl) | 4.25 | 4.25 | 4.25 | 4.25 |
| TOTAL/(µl) | 10.5 | 10.5 | 10.5 | 10.5 |

| Steps | Temparature | Time | cycle |
| --- | --- | --- | --- |
| Pre Denature | 96℃ | 2min | 1time |
| Denature | 96℃ | 10sec | 30cycle |
| Annealing | 50℃ | 5sec | 30cycle |
| Extension | 60℃ | 4min | 30cycle |
| Final elongation | 4℃ | 1min | 1time |
| Final hold | 4℃ | - | - |

#### Gel Extraction

Tatsui

| Lane | SampleⅠ | Volume/(µl) |
| --- | --- | --- |
| 1 | - | - |
| 2 | - | - |
| 3 | 9/18 Digestion Mutation mamB Colony\_10\_3(X+P) | 40 |
| 4 | 9/18 Digestion Mutation mamB Colony\_10\_3(Nega) | 10 |
| 5 | 1kb ladder | - |
| 6 | - | - |
| 7 | 9/18 Digestion Mutation mamB Colony\_10\_3(X+P) | 40 |
| 8 | - | - |
| 9 | 1kb ladder | - |
| 10 | 9/18 Digestion Mutation mamB Colony\_10\_4(Nega) | 10 |
| 11 | 9/18 Digestion Mutation mamB Colony\_10\_4(X+P) | 40 |

| Lane | SampleⅡ | Volume/(µl) |
| --- | --- | --- |
| 1 | 9/18 Digestion mamQ+pSB1A2(X+P) | 200 |
| 2 | - | - |
| 3 | - | - |
| 4 | - | - |
| 5 | - | - |
| 6 | - | - |

| Lane | SampleⅢ | Volume/(µl) |
| --- | --- | --- |
| 1 | 100b ladder | - |
| 2 | 9/18 Digestion Pcon+pSB1C3(Nega) | 10 |
| 3 | 9/18 Digestion Pcon+pSB1C3(S+P) | 80 |

| DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
| --- | --- | --- | --- |
| mamB Colony\_10\_3(X+P) | - | - | - |
| mamB Colony\_10\_4(X+P) | - | - | - |
| mamQ+pSB1A2(X+P) | - | - | - |
| Pcon+pSB1C3(S+P) | - | - | - |

#### Colony PCR

Tatsui

| NameⅠ |
| --- |
| 9/18 Transformation Pcon+mamQ+pSB1C3 Colony\_1 |
| 9/18 Transformation Pcon+mamQ+pSB1C3 Colony\_2 |
| 9/18 Transformation Pcon+mamQ+pSB1C3 Colony\_3 |
| 9/18 Transformation Pcon+mamQ+pSB1C3 Colony\_4 |
| 9/18 Transformation Pcon+mamQ+pSB1C3 Colony\_5 |

#### Electrophoresis

Tatsui

| Lane | Colony PCR ProductsⅠ |
| --- | --- |
| 1 | - |
| 2 | - |
| 3 | Pcon+mamQ+pSB1C3 Colony\_1 |
| 4 | Pcon+mamQ+pSB1C3 Colony\_2 |
| 5 | Pcon+mamQ+pSB1C3 Colony\_3 |
| 6 | Pcon+mamQ+pSB1C3 Colony\_4 |
| 7 | Pcon+mamQ+pSB1C3 Colony\_5 |
| 8 | - |

#### Restriction Enzyme Digestion

Tatsui

| Sample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | BSA | Buffur/(µl) | MilliQ/(µl) | TOTAL/(µl) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 9/19 Miniprep Pcon+mamL+mamQ+pSB1C3 | 34.7 | 0 | 0 | 2 | 2 | 0.8 | 8(B) | 32.5 | 80 |
| 9/19 Miniprep pSB3T5 | 53.3 | 2 | 0 | 2 | 0 | 0.8 | 8(B) | 14 | 80 |
| 9/19 Miniprep pSB3T5(Nega) | 1 | 0 | 0 | 0 | 0 | 0 | 1(B) | 8 | 10 |

#### PCR

Tatsui

| Target | Pcon+mamB+His |
| --- | --- |
| KOD Plus/(µl) | 1 |
| 10×Buffer/(µl) | 5 |
| 2mM dNTPS/(µl) | 5 |
| 25mM MgSO4/(µl) | 3 |
| Fw Primer(E+Pcon+X+RBS+mamB)/(µl) | 2 |
| Rv Primer(S+His+mamB)/(µl) | 2 |
| Template/(µl) | 0.5 |
| MilliQ/(µl) | 31.5 |
| Total/(µl) | 50 |

| Steps | Temparature | Time | cycle |
| --- | --- | --- | --- |
| Pre Denature | 98℃ | 2min | 1time |
| Denature | 98℃ | 10sec | 25cycle |
| Annealing | 55℃ | 15sec | 25cycle |
| Extension | 72℃ | 1min | 25cycle |
| Final elongation | 72℃ | 5min | 1time |
| Final hold | 12℃ | - | - |

#### DNA Purification

Honda

| DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 9/18 Digestion mamL+pSB1C3(S+P) | 55 | 1.66 | 1.66 |
| 9/19 PCR Pcon+mamB+His | 150 | 1.63 | 1.52 |

#### Restriction Enzyme Digestion

Honda

| Sample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | BSA | Buffur/(µl) | MilliQ/(µl) | TOTAL/(µl) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 9/19 DNA Purification Pcon+mamB+His | 20 | 1 | 0 | 1 | 0 | 0.8 | 8(B) | 49.2 | 80 |

#### Liquid Culture

Tatsui

| Name | medium |
| --- | --- |
| 9/18 Transformation Pcon+mamB+His+pSB3T5 Colony\_1 | Plusgrow medium(TET) |
| 9/18 Transformation Pcon+mamB+His+pSB3T5 Colony\_2 | Plusgrow medium(TET) |
| 9/18 Transformation Pcon+mamB+His+pSB3T5 Colony\_3 | Plusgrow medium(TET) |
| 9/18 Transformation Pcon+mamB+His+pSB3T5 Colony\_4 | Plusgrow medium(TET) |
| 9/18 Transformation Pcon+mamB+His+pSB3T5 Colony\_5 | Plusgrow medium(TET) |

#### Colony PCR

Tatsui

| NameⅠ |
| --- |
| 9/18 Transformation Pcon+mamQ+pSB1C3 Colony\_1 |
| 9/18 Transformation Pcon+mamQ+pSB1C3 Colony\_2 |
| 9/18 Transformation Pcon+mamQ+pSB1C3 Colony\_3 |
| 9/18 Transformation Pcon+mamQ+pSB1C3 Colony\_4 |
| 9/18 Transformation Pcon+mamQ+pSB1C3 Colony\_5 |

### 9/20

#### Electrophoresis

Tatsui

| Lane | Colony PCR ProductsⅠ |
| --- | --- |
| 1 | - |
| 2 | 100b ladder |
| 3 | Pcon+mamQ+pSB1C3 Colony\_1 |
| 4 | Pcon+mamQ+pSB1C3 Colony\_2 |
| 5 | Pcon+mamQ+pSB1C3 Colony\_3 |
| 6 | Pcon+mamQ+pSB1C3 Colony\_4 |
| 7 | Pcon+mamQ+pSB1C3 Colony\_5 |

#### Miniprep

Tatsui

| DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
| --- | --- | --- | --- |
| 9/19 Liquid Culture Pcon+mamQ+pSB1C3 Colony\_3 | - | - | - |
| 9/19 Liquid Culture Pcon+mamQ+pSB1C3 Colony\_5 | - | - | - |

#### Colony PCR

Tatsui

| NameⅠ |
| --- |
| Pcon+mamB+His+pSB3T5 Colony\_1(VF+VR) |
| Pcon+mamB+His+pSB3T5 Colony\_1(seq\_4+VR) |

#### Electrophoresis

Yasuda

| Lane | Colony PCR ProductsⅠ |
| --- | --- |
| 1 | - |
| 2 | 100b ladder |
| 3 | 100b ladder |
| 4 | Pcon+mamB+His+pSB3T5 Colony\_1(VF+VR) |
| 5 | Pcon+mamB+His+pSB3T5 Colony\_1(seq\_4+VR) |
| 6 | 100b ladder |

#### Restriction Enzyme Digestion

Honda

| Sample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | BSA | Buffur/(µl) | MilliQ/(µl) | TOTAL/(µl) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 9/18 Miniprep Mutation mamB Colony\_10\_3 | 11.2 | 0 | 2 | 0 | 2 | 0.8 | 8(D) | 56 | 80 |
| 9/18 Miniprep Mutation mamB Colony\_10\_3(Nega) | 1 | 0 | 0 | 0 | 0 | 0 | 1(D) | 8 | 10 |
| 9/18 Miniprep Mutation mamB Colony\_10\_4 | 15.1 | 0 | 2 | 0 | 2 | 0.8 | 8(D) | 56 | 83.9 |
| 9/18 Miniprep Mutation mamB Colony\_10\_4(Nega) | 1 | 1 | 0 | 1 | 0 | 0 | 1(D) | 8 | 10 |

#### Gel Extraction

Tatsui

| Lane | SampleⅠ | Volume/(µl) |
| --- | --- | --- |
| 1 | - | - |
| 2 | 100b ladder | - |
| 3 | Digestion Mutation mamB Colony\_10\_3(X+P) | - |
| 4 | - | - |
| 5 | - | - |
| 6 | - | - |
| 7 | Digestion Mutation mamB Colony\_11\_3(X+P) | - |
| 8 | Digestion Mutation mamB Colony\_11\_3(Nega) | - |
| 9 | 1kb ladder | - |
| 10 | - | - |
| 11 | Digestion Mutation mamB Colony\_11\_3(X+P) | - |

| Lane | SampleⅡ | Volume/(µl) |
| --- | --- | --- |
| 1 | - | - |
| 2 | 100b ladder | - |
| 3 | 9/19 Digestion pSB3T5(Nega) | - |
| 4 | 9/19 Digestion pSB3T5(E+S) | - |

| Lane | SampleⅢ | Volume/(µl) |
| --- | --- | --- |
| 1 | - | - |
| 2 | 100b ladder | - |
| 3 | Digestion Mutation mamB Colony\_11\_4(X+P) | - |
| 4 | Digestion Mutation mamB Colony\_11\_4(Nega) | - |

| DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
| --- | --- | --- | --- |
| mamB Colony\_10\_3(X+P) | 60 | - | - |
| mamB Colony\_11\_3(X+P) | 85 | 1.38 | 2.04 |
| mamB Colony\_11\_4(X+P) | 60 | 0.58 | 2.32 |
| pSB3T5(E+S) | 70 | 0.44 | 2.04 |

#### DNA Purification

Tatsui

| DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
| --- | --- | --- | --- |
| Pcon+mamL+mamQ(S+P) | - | - | - |

#### Ligation

Tatsui

| Insert/(µL) | | Vector/(µL) | | T4 Ligase/(µl) | 2\*Buffer/(µl) | MilliQ/(µl) | Total/(µl) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| mamB Colony\_10\_3(X+P) | 1.7 | Pcon+mamL+mamQ(S+P) | 2.3 | 1 | 5 | 0 | 10 |
| mamB Colony\_10\_4(X+P) | 1.7 | Pcon+mamL+mamQ(S+P) | 2.3 | 1 | 5 | 0 | 10 |
| mamB Colony\_11\_3(X+P) | 1.7 | Pcon+mamL+mamQ(S+P) | 2.3 | 1 | 5 | 0 | 10 |
| mamB Colony\_11\_4(X+P) | 1.7 | Pcon+mamL+mamQ(S+P) | 2.3 | 1 | 5 | 0 | 10 |
| pSB3T5(E+S) | 1.4 | Pcon+mamB+His(E+S) | 2.6 | 1 | 5 | 0 | 10 |

#### Transformation

Tatsui

| Sample/(µL) | | CompetentCells/(µl) | Total/(µl) | Medium |
| --- | --- | --- | --- | --- |
| 9/20 Ligation Pcon+mamL+mamQ+mamB Colony\_10\_3 | 1 | 10 | 11 | LB(CP) |
| 9/20 Ligation Pcon+mamL+mamQ+mamB Colony\_10\_4 | 1 | 10 | 11 | LB(CP) |
| 9/20 Ligation Pcon+mamL+mamQ+mamB Colony\_11\_3 | 1 | 10 | 11 | LB(CP) |
| 9/20 Ligation Pcon+mamL+mamQ+mamB Colony\_11\_4 | 1 | 10 | 11 | LB(CP) |
| 9/20 Ligation Pcon+mamB+His | 1 | 10 | 11 | LB(TET) |

### 9/22

#### Colony PCR

Tatsui

| NameⅠ | NameⅡ | NameⅢ | NameⅣ |
| --- | --- | --- | --- |
| 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_10\_3\_1 | 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_10\_4\_1 | 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_11\_3\_1 | 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_11\_4\_1 |
| 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_10\_3\_2 | 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_10\_4\_2 | 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_11\_3\_2 | 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_11\_4\_2 |
| 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_10\_3\_3 | 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_10\_4\_3 | 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_11\_3\_3 | 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_11\_4\_3 |
| 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_10\_3\_4 | 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_10\_4\_4 | 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_11\_3\_4 | 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_11\_4\_4 |
| 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_10\_3\_5 | 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_10\_4\_5 | 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_11\_3\_5 | 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_11\_4\_5 |
| 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_10\_3\_6 | 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_10\_4\_6 | 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_11\_3\_6 | 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_11\_4\_6 |
| 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_10\_3\_7 | 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_10\_4\_7 | 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_11\_3\_7 | 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_11\_4\_7 |
| 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_10\_3\_8 | 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_10\_4\_8 | 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_11\_3\_8 | 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_11\_4\_8 |

| NameⅤ | NameⅥ |
| --- | --- |
| 9/20 Transformation Pcon+mamB+His Colony\_1 | 9/19 Transformation Pcon+mamB+His Colony\_1 |
| 9/20 Transformation Pcon+mamB+His Colony\_2 | 9/19 Transformation Pcon+mamB+His Colony\_2 |
| 9/20 Transformation Pcon+mamB+His Colony\_3 | 9/19 Transformation Pcon+mamB+His Colony\_3 |
| 9/20 Transformation Pcon+mamB+His Colony\_4 | - |
| 9/20 Transformation Pcon+mamB+His Colony\_5 | - |

#### Electrophoresis

Tatsui

| Lane | Colony PCR ProductsⅠ Colony PCR ProductsⅡ | |
| --- | --- | --- |
| 1 | Pcon+mamL+mamQ+mamB Colony\_10\_3\_1 | Pcon+mamL+mamQ+mamB Colony\_11\_3\_1 |
| 2 | Pcon+mamL+mamQ+mamB Colony\_10\_3\_2 | Pcon+mamL+mamQ+mamB Colony\_11\_3\_2 |
| 3 | Pcon+mamL+mamQ+mamB Colony\_10\_3\_3 | Pcon+mamL+mamQ+mamB Colony\_11\_3\_3 |
| 4 | Pcon+mamL+mamQ+mamB Colony\_10\_3\_4 | Pcon+mamL+mamQ+mamB Colony\_11\_3\_4 |
| 5 | Pcon+mamL+mamQ+mamB Colony\_10\_3\_5 | Pcon+mamL+mamQ+mamB Colony\_11\_3\_5 |
| 6 | Pcon+mamL+mamQ+mamB Colony\_10\_3\_6 | Pcon+mamL+mamQ+mamB Colony\_11\_3\_6 |
| 7 | Pcon+mamL+mamQ+mamB Colony\_10\_3\_7 | Pcon+mamL+mamQ+mamB Colony\_11\_3\_7 |
| 8 | Pcon+mamL+mamQ+mamB Colony\_10\_3\_8 | Pcon+mamL+mamQ+mamB Colony\_11\_3\_8 |
| 9 | 100b ladder | 100b ladder |
| 10 | Pcon+mamL+mamQ+mamB Colony\_10\_4\_1 | Pcon+mamL+mamQ+mamB Colony\_11\_4\_1 |
| 11 | Pcon+mamL+mamQ+mamB Colony\_10\_4\_2 | Pcon+mamL+mamQ+mamB Colony\_11\_4\_2 |
| 12 | Pcon+mamL+mamQ+mamB Colony\_10\_4\_3 | Pcon+mamL+mamQ+mamB Colony\_11\_4\_3 |
| 13 | Pcon+mamL+mamQ+mamB Colony\_10\_4\_4 | Pcon+mamL+mamQ+mamB Colony\_11\_4\_4 |
| 14 | Pcon+mamL+mamQ+mamB Colony\_10\_4\_5 | Pcon+mamL+mamQ+mamB Colony\_11\_4\_5 |
| 15 | Pcon+mamL+mamQ+mamB Colony\_10\_4\_6 | Pcon+mamL+mamQ+mamB Colony\_11\_4\_6 |
| 16 | Pcon+mamL+mamQ+mamB Colony\_10\_4\_7 | Pcon+mamL+mamQ+mamB Colony\_11\_4\_7 |
| 17 | Pcon+mamL+mamQ+mamB Colony\_10\_4\_8 | Pcon+mamL+mamQ+mamB Colony\_11\_4\_8 |

#### Ligation

No Name

| Insert/(µL) | | Vector/(µL) | | T4 Ligase/(µl) | 10\*Buffer/(µl) | MilliQ/(µl) | Total/(µl) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| mamB Colony\_10\_3(X+P) | 1 | Pcon+mamL+mamQ(S+P) | 7.5 | 0.5 | 1 | 0 | 10 |

#### Restriction Enzyme Digestion

Tatsui

| Sample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | BSA | Buffur/(µl) | CLAP/(µl) | MilliQ/(µl) | TOTAL/(µl) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 9/19 Miniprep Pcon+mamL+mamQ+pSB1C3 | 34.8 | 0 | 0 | 2 | 2 | 0.8 | 8(B) | 1 | 31.4 | 80 |

#### Colony PCR

Honda

| NameⅠ | NameⅡ | NameⅢ | NameⅣ |
| --- | --- | --- | --- |
| 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_10\_3\_1 | 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_10\_4\_1 | 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_11\_3\_1 | 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_11\_4\_1 |
| 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_10\_3\_2 | 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_10\_4\_2 | 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_11\_3\_2 | 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_11\_4\_2 |
| 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_10\_3\_3 | 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_10\_4\_3 | 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_11\_3\_3 | 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_11\_4\_3 |
| 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_10\_3\_4 | 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_10\_4\_4 | 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_11\_3\_4 | 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_11\_4\_4 |
| 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_10\_3\_5 | 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_10\_4\_5 | 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_11\_3\_5 | 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_11\_4\_5 |
| 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_10\_3\_6 | 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_10\_4\_6 | 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_11\_3\_6 | 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_11\_4\_6 |
| 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_10\_3\_7 | 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_10\_4\_7 | 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_11\_3\_7 | 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_11\_4\_7 |
| 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_10\_3\_8 | 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_10\_4\_8 | 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_11\_3\_8 | 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_11\_4\_8 |

| NameⅤ |
| --- |
| 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_11\_3\_2(VF+VR) |
| 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_11\_3\_2(seq\_4+VR) |
| 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_11\_3\_2(L\_F+B\_R) |
| 9/20 Transformation Pcon+mamL+mamQ+mamB Colony\_11\_3\_2(Q\_F+B\_R) |

#### Electrophoresis

Tatsui

| Lane | Colony PCR ProductsⅠ Colony PCR ProductsⅡ Colony PCR ProductsⅢ | | |
| --- | --- | --- | --- |
| 1 | Pcon+mamL+mamQ+mamB Colony\_10\_3\_1 | Pcon+mamL+mamQ+mamB Colony\_11\_3\_1 | - |
| 2 | Pcon+mamL+mamQ+mamB Colony\_10\_3\_2 | Pcon+mamL+mamQ+mamB Colony\_11\_3\_2 | - |
| 3 | Pcon+mamL+mamQ+mamB Colony\_10\_3\_3 | Pcon+mamL+mamQ+mamB Colony\_11\_3\_3 | - |
| 4 | Pcon+mamL+mamQ+mamB Colony\_10\_3\_4 | Pcon+mamL+mamQ+mamB Colony\_11\_3\_4 | - |
| 5 | Pcon+mamL+mamQ+mamB Colony\_10\_3\_5 | Pcon+mamL+mamQ+mamB Colony\_11\_3\_5 | - |
| 6 | Pcon+mamL+mamQ+mamB Colony\_10\_3\_6 | Pcon+mamL+mamQ+mamB Colony\_11\_3\_6 | - |
| 7 | Pcon+mamL+mamQ+mamB Colony\_10\_3\_7 | Pcon+mamL+mamQ+mamB Colony\_11\_3\_7 | - |
| 8 | Pcon+mamL+mamQ+mamB Colony\_10\_3\_8 | Pcon+mamL+mamQ+mamB Colony\_11\_3\_8 | - |
| 9 | 100b ladder | 100b ladder | 100b ladder |
| 10 | Pcon+mamL+mamQ+mamB Colony\_10\_4\_1 | Pcon+mamL+mamQ+mamB Colony\_11\_4\_1 | 9/19 Transformation Pcon+mamB+His Colony\_1 |
| 11 | Pcon+mamL+mamQ+mamB Colony\_10\_4\_2 | Pcon+mamL+mamQ+mamB Colony\_11\_4\_2 | 9/19 Transformation Pcon+mamB+His Colony\_2 |
| 12 | Pcon+mamL+mamQ+mamB Colony\_10\_4\_3 | Pcon+mamL+mamQ+mamB Colony\_11\_4\_3 | 9/19 Transformation Pcon+mamB+His Colony\_3 |
| 13 | Pcon+mamL+mamQ+mamB Colony\_10\_4\_4 | Pcon+mamL+mamQ+mamB Colony\_11\_4\_4 | Pcon+mamL+mamQ+mamB Colony\_11\_3\_2(VF+VR) |
| 14 | Pcon+mamL+mamQ+mamB Colony\_10\_4\_5 | Pcon+mamL+mamQ+mamB Colony\_11\_4\_5 | Pcon+mamL+mamQ+mamB Colony\_11\_3\_2(seq\_4+VR) |
| 15 | Pcon+mamL+mamQ+mamB Colony\_10\_4\_6 | Pcon+mamL+mamQ+mamB Colony\_11\_4\_6 | Pcon+mamL+mamQ+mamB Colony\_11\_3\_2(L\_F+B\_R) |
| 16 | Pcon+mamL+mamQ+mamB Colony\_10\_4\_7 | Pcon+mamL+mamQ+mamB Colony\_11\_4\_7 | Pcon+mamL+mamQ+mamB Colony\_11\_3\_2(Q\_F+B\_R) |
| 17 | Pcon+mamL+mamQ+mamB Colony\_10\_4\_8 | Pcon+mamL+mamQ+mamB Colony\_11\_4\_8 | - |

#### Miniprep

Tatsui

| DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
| --- | --- | --- | --- |
| Pcon+mamQ+pSB1C3 Colony\_3 | 85 | 1.51 | 0.79 |
| Pcon+mamQ+pSB1C3 Colony\_5 | 60 | 1.94 | 1.12 |

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