iGEM 2018 — Team Kyoto

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:Kyoto/Notebook

* [![](https://static.igem.org/mediawiki/2018/4/42/T--Kyoto--home_simple.png)](https://2018.igem.org/Team%3AKyoto)
* * [Project](https://2018.igem.org/team%3AKyoto/Project)
    + [**Description**](https://2018.igem.org/Team%3AKyoto/Project)
    + [**Design**](https://2018.igem.org/Team%3AKyoto/Design)
    + [**Results**](https://2018.igem.org/Team%3AKyoto/Result)
    + [**Demonstration**](https://2018.igem.org/Team%3AKyoto/Demonstrate)
    + [**Discussion**](https://2018.igem.org/Team%3AKyoto/Discussion)
  * [Modeling](https://2018.igem.org/Team%3AKyoto/Model)
  * [Lab Work](https://2018.igem.org/Team%3AKyoto/Notebook)
    + [**Notebook**](https://2018.igem.org/Team%3AKyoto/Notebook)
    + [**Materials&Methods**](https://2018.igem.org/Team%3AKyoto/MaterialsMethods)
    + [**Special Methods**](https://2018.igem.org/Team%3AKyoto/SpecialMethods)
    + [**Safety**](https://2018.igem.org/Team%3AKyoto/Safety)
  * [Parts](https://2018.igem.org/Team%3AKyoto/Basic_Parts)
    + [**Basic Parts**](https://2018.igem.org/Team%3AKyoto/Basic_Parts)
    + [**Composite Parts**](https://2018.igem.org/Team%3AKyoto/Composite_Parts)
    + [**Improving parts**](https://2018.igem.org/Team%3AKyoto/Improve)
    + [**Parts Collection**](https://2018.igem.org/Team%3AKyoto/Parts_Collection)
  * [Human Practices](https://2018.igem.org/Team%3AKyoto/Human_Practices)
    + [**Human Practice/Silver**](https://2018.igem.org/Team%3AKyoto/Human_Practices)
    + [**Integrated Human Practice/Gold**](https://2018.igem.org/Team%3AKyoto/Integrated_Human_Practices)
    + [**Collaborations**](https://2018.igem.org/Team%3AKyoto/Collaborations)
  * [Team](https://2018.igem.org/Team%3AKyoto/Team)
    + [**Members**](https://2018.igem.org/Team%3AKyoto/Team)
    + [**Attributions**](https://2018.igem.org/Team%3AKyoto/Attributions)

[![](https://static.igem.org/mediawiki/2018/1/11/T--Kyoto--upbotton.jpg)](#wrapper)

# ![](https://static.igem.org/mediawiki/2018/d/dc/T--Kyoto--notebook.png)

| July | | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| 1 | 2 | [3](#0703) | 4 | [5](#0705) | [6](#0706) | 7 |
| 8 | 9 | [10](#0710) | 11 | [12](#0712) | 13 | 14 |
| 15 | 16 | 17 | 18 | 19 | 20 | 21 |
| 22 | 23 | 24 | 25 | 26 | 27 | 28 |
| 29 | 30 | [31](#0731) |  |  |  |  |

| August | | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
|  |  |  | [1](#0801) | 2 | 3 | 4 |
| 5 | 6 | [7](#0807) | [8](#0808) | 9 | 10 | 11 |
| 12 | [13](#0813) | [14](#0814) | 15 | 16 | 17 | [18](#0818) |
| [19](#0819) | [20](#0820) | [21](#0821) | 22 | 23 | 24 | [25](#0825) |
| [26](#0826) | [27](#0827) | [28](#0828) | [29](#0829) | [30](#0830) | [31](#0831) |  |

| September | | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  |  | [1](#0901) |
| [2](#0902) | [3](#0903) | [4](#0904) | [5](#0905) | [6](#0906) | [7](#0907) | [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](#0921) | [22](#0922) |
| [23](#0923) | [24](#0924) | [25](#0925) | [26](#0926) | [27](#0927) | [28](#0928) | [29](#0929) |
| [30](#0930) |  |  |  |  |  |  |

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

### 7/3

#### Restriction Enzyme Digestion

Tong, Tamukai

| component | volume(μl) |
| --- | --- |
| BSA | 0.5 |
| buffer(10xH) | 5 |
| EcoR1 | 0.5 |
| Pst1 | 0.5 |
| Dpn1 | 0.5 |
| dH2O | 18 |

add 4μl linearized plasmid backbone (25ng/μl)
add 4μl Enzyme Master Mix
digest 37℃, 30min
add 8μl NaOAc (pH5.2,3M)
add 16μl 99.5% EtOh
Vortex 2sec
add 184μl 99.5% EtOH
add 72μl DW

### 7/5

#### OE-PCR

Nambu, Shimazoe

| sample | Primer Forward | volume (ul) | Primer Reverse | volume (ul) | template DNA | volume (ul) | Q5 HF 2xMasterMix (ul) | DW (ul) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | Z007 | 1.25 | Z009 | 1.25 | Yeast genome | 1.0 | 1.0 | 9.0 |
| 2 | Z008 | 1.25 | Z011 | 1.25 | GFP | 1.0 | 1.0 | 9.0 |
| 3 | Z010 | 1.25 | Z012 | 1.25 | Yeast genome | 1.0 | 1.0 | 9.0 |
| 4 | Z007 | 1.25 | Z014 | 1.25 | Yeast genome | 1.0 | 1.0 | 9.0 |
| 5 | Z013 | 1.25 | Z016 | 1.25 | RFP | 1.0 | 1.0 | 9.0 |
| 6 | Z015 | 1.25 | Z012 | 1.25 | Yeast genome | 1.0 | 1.0 | 9.0 |

### 7/6

#### OE-PCR

Nambu, Shimazoe

| sample | Primer Forward | volume (ul) | Primer Reverse | volume (ul) | Template DNA | volume (ul) | Q5 HF2x Master Mix (ul) | DW (ul) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | Z007 | 1.25 | Z009 | 1.25 | Yeast genome | 1.0 | 12.5 | 9.0 |
| 2 | Z010 | 1.25 | Z012 | 1.25 | Yeast genome | 1.0 | 12.5 | 9.0 |
| 3 | Z007 | 1.25 | Z014 | 1.25 | Yeast genome | 1.0 | 12.5 | 9.0 |
| 4 | Z015 | 1.25 | Z012 | 1.25 | Yeast genome | 1.0 | 12.5 | 9.0 |
| 5\* | Z007 | 1.25 | Z009 | 1.25 | p079 | 1.0 | 12.5 | 9.0 |
| 6\* | Z010 | 1.25 | Z012 | 1.25 | p079 | 1.0 | 12.5 | 9.0 |
| 7\* | Z007 | 1.25 | Z014 | 1.25 | p079 | 1.0 | 12.5 | 9.0 |
| 8\* | Z015 | 1.25 | Z012 | 1.25 | p079 | 1.0 | 12.5 | 9.0 |

\*sample5~8 x 2set

### 7/6

#### colony\_PCR

| Mangrin colony | DW (ul) | Q5 HF2x MasterMix (ul) | m13 Forward (ul) | m13 Reverse (ul) |
| --- | --- | --- | --- | --- |
| 1 | 2.0 | 2.5 | 0.25 | 0.25 |
| 2 | 2.0 | 2.5 | 0.25 | 0.25 |
| 3 | 2.0 | 2.5 | 0.25 | 0.25 |
| 4 | 2.0 | 2.5 | 0.25 | 0.25 |
| 5 | 2.0 | 2.5 | 0.25 | 0.25 |

Store PCR product at 4℃.

### 7/10

#### Restriction Enzyme Digestion

Tong, Tamukai

| component | volume(μl) |
| --- | --- |
| BSA | 0.5 |
| buffer(10xH) | 5 |
| EcoR1 | 0.5 |
| Pst1 | 0.5 |
| Dpn1 | 0.5 |
| dH2O | 18 |

add 4, 8 μl (add two by two to sample)
add 4, 8μl Enzyme master mix (add two by two to sample)digest 37℃ 30min
70℃ 20min
add 1,1.6 μl NaOAc pH5.2, 3M(add two by two to sample)
20, 40μl (add two by two to sample) 99.5% EtOHVortex
preserve in -20℃

### 7/12

#### OE-PCR

Nambu

| sample | KOD one PCR master mix (ul) | TDH promoter | volume (ul) | ORF | volume (ul) | CYC terminator | volume (ul) | DW (ul) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| A | 12.5 | 5 | 1.5 | GFP | 1.5 | 6 | 1.5 | 8.0 |
| B | 12.5 | 7 | 1.5 | RFP | 1.5 | 8 | 1.5 | 8.0 |
| C | 12.5 | 5 | 1.5 | GFP | 1.5 | 6 | 1.5 | 8.0 |
| D | 12.5 | 7 | 1.5 | RFP | 1.5 | 8 | 1.5 | 8.0 |

PCR
98℃　0:30
98℃\* 0:10
45℃\* 0:05
68℃\* 0:05
68℃ 2:00
10℃
(\* x 30 cycle)

### 7/31

#### Transformation & Blue-White Selection

Shimazoe

Mix 10μl of Competent Cell and 2μl G,R Ligation Product on ice
On ice for 30min
Heat Shock 42℃ for 45sec
 On ice for 1,2 min
Add 100μl SOC medium and culture in 37℃ for 1h
 Spread on a plate (50μl X-gal added)

### 8/1

#### Colony PCR

Tong

| Component | Volume(μl) |
| --- | --- |
| DW | 30 |
| Q5 | 37.5 |
| m13f | 3.75 |
| m13r | 3.75 |
| colony | Poke |
| Total | 75 |

Add 0.6μl 10x Buffer to all sample (1~14)
Culture in 10mL

### 8/7

#### YPD

Tong

| Component | (1L) | (900mL) | (600mL) |
| --- | --- | --- | --- |
| Peptone | 20g | 18g | 12g |
| Yeast Extract | 10g | 9g | 6g |
| Agar | 15g | 13.5g | 9g |
| DW | xmL | 810ml | 480mL |
| Glucose+DW | 20g+ymL (x+y=1000) | 18g | 12g |

fluid culture
cultured 9 yeast strains in liquid YPD.
Put 15 ml of YPD medium in a 50 ml sterile tube
push the yeast with a white chip, put it in the tube
loosen the lid and put it in the shaker

### 8/8

#### OD measure

Tong

| A | OD |
| --- | --- |
| 562 | 0.38 |
| 561 | 0.40 |
| 564 | 0.36 |
| 554 | 0.33 |
| 555 | 0.38 |
| 547 | 0.30 |
| 557 | 0.32 |
| 559 | 0.39 |

For each 8 samples, transferred to a 1.5 ml tube　1mL each.
For soy sauce yeast only 15 ml was centrifuged, decantation was repeated and scaled down to 1 ml.

### 8/13

#### Ligation

Nambu

| Number | 1 | 2 | 3 | 4 | 5 | 6 |
| --- | --- | --- | --- | --- | --- | --- |
| vector | 426 | 316 | 426 | 316 | 426 | 316 |
| insert | Mangrin | Mangrin | ZrFPS1-2 | ZrFPS1-2 | McHKT2 | McHKT2 |

Mix the following material (Contamination are shown in the table above)

Plasmid(vector): 0.5&microL
Insert: 1.0&microl
Ligation High: 1.5&microl

Put in Heatblock

### 8/13

#### YPD culture

Tong

| NaCl 25%(400mL) | null |
| --- | --- |
| Tryptone | 8g |
| Yeast Extract | 4g |
| Agar | 8g |
| Glucose | 8g |
| NaCl | 100g |

| Normal | null |
| --- | --- |
| Tryptone | 4g |
| Yeast Extract | 2g |
| Agar | 4g |
| Glucose | 4g |

Make
25% NaCl YPD solid medium 400 mL
Normal YPD solid medium 200 mL
YPD liquid medium 500 mL

### 8/14

#### Transformation

Tamukai

| Sample Name | Sample Volume (μl) | Competent Cells/(μl) |
| --- | --- | --- |
| Mangrin | 3 | 15 |
| ZrFPS1-2 | 3 | 15 |
| McHKT2 | 3 | 15 |

### 8/18

#### Colony PCR

Nambu

| Component | Amount(μl) |
| --- | --- |
| DW | 112 |
| Q5 | 140 |
| m13f | 14 |
| m13r | 14 |
| Colony | Poke |
| Total | 280 |

![](https://static.igem.org/mediawiki/2018/f/f6/T--Kyoto--0818colonyPCR.png)

PCR　　　　　　　Colony
98℃ 0:30　　　　　 number　　Backbone　　Insert
98℃\* 0:10　　　　　301~306　　SmaCIP　　AtHKT1
57℃\* 0:30　　　　　307~317　　SmaCIP　　SseNHX1
72℃\* 1:00　　　　　318~326　　SmaCIP　　ZrFPs1-1st
72℃ 2:00　　　　　 327~337　　SmaCIP　　AtNHXS1
4℃ ∞　　　　　　　338~356　　SmaCIP　　 FgβF3
 (\*× 30 cycle)

### 8/18

#### Liquid Culture

Nambu

| Material | Volume |
| --- | --- |
| LB | 90ml |
| Amp | 90ul |

mix materials ( Finally concentration Amp 100 ug/ml )
put colony\* into the liquid
(\*colony number: 304,309,310,313,314,315,317,322,325,336,342,343,352,353,354)
put in incubator at 37℃ for 18 hours

### 8/18

#### Liquid Culture

Tong

| Number | Material |
| --- | --- |
| 547 | WT |
| 554 | NHA1Δ |
| 555 | G19, ΔENA1-4 |
| 557 | NHA1Δ, ENA1Δ |
| 559 | ENA1Δ |
| 561 | Z.roxii ATCC42981 |
| 562 | Z.roxii CBS732 |
| 564 | Z.roxii NBRC1876 |

37℃, 130rpm
prepared each two pieces of plates of 0,5,10,15,20,25% salt concentration and observed the yeasts.

### 8/19

#### Ligation

Nambu

| Number | 1 | 2 | 3 | 4 | 5 | 6 |
| --- | --- | --- | --- | --- | --- | --- |
| gBlock | Mangrin | ZrFPS1-2nd | McHKT2 | AtHKT1 | ZrFPS1-1st | AtNHXS1 |

mix the following material

SmaCIP (Backbone) : 1ul
gBlock (Insert) : 1ul
Ligation High : 2ul
(Total : 4ul)

putin HeatBlock overnight at 18℃

### 8/19

#### PCR

Nambu

| sample | content (gBlock) | primer1 | primer2 |
| --- | --- | --- | --- |
| 1 | Mangrin | Z026 | Z027 |
| 2 | ZrFPS1\_2nd | Z026 | Z027 |
| 3 | McHKT2 | Z026 | Z027 |
| 4 | AtHKT1 | Z026 | Z027 |
| 5 | ZrFPS1\_1st | Z026 | Z027 |
| 6 | AtNHXS1 | Z026 | Z027 |
| 7 | SseNHX1 | Z026 | Z027 |
| 8 | AVP1\_1st + AVP1\_2nd | Z026 | Z027 |

![](https://static.igem.org/mediawiki/2018/4/4c/T--Kyoto--0819PCR.png)

94℃ 2:00
98℃\* 0:10
48℃ \*0:30
68℃ \*3:00
68℃ 0:10
10℃
\* x 30 cycle

### 8/20

#### Ligation

Nambu

| number | gBlock (Insert) | volume (ul) | SmaCIP (backbone) (ul) | Ligation High (ul) |
| --- | --- | --- | --- | --- |
| 7 | SdrG | 1 | 1 | 2 |
| 8 | SdrG-Fg overlap | 1 | 1 | 2 |
| 9 | ZrGPD1 | 1 | 1 | 2 |

Put in Incubator overnight at 18℃.

### 8/20

#### Transformation

Nambu

| Sample Name | Sample Volume (μl) | Competent Cells/(μl) | Medium |
| --- | --- | --- | --- |
| Mangrin | 4 | 16 | SOC Medium |
| ZrFPS1\_2nd | 4 | 16 | SOC Medium |
| McHKT2 | 4 | 16 | SOC Medium |
| AtHKT1 | 4 | 16 | SOC Medium |
| ZrFPS1\_1st | 4 | 16 | SOC Medium |
| AtNHXS1 | 4 | 16 | SOC Medium |

### 8/21

#### Restriction Enzyme Digestion

Shimazoe

| plasmid | plasmid volume (ul) | DW (ul) | 10x Fast digest buffer (ul) | EcoR1 (ul) | Pst1 (ul) | Total (ul) |
| --- | --- | --- | --- | --- | --- | --- |
| 17 | 2 | 14 | 2 | 1 | 1 | 20 |
| 19 | 1 | 14 | 2 | 1 | 1 | 20 |
| 26 | 2 | 14 | 2 | 1 | 1 | 20 |
| 110 | 2 | 14 | 2 | 1 | 1 | 20 |
| 120 | 2 | 14 | 2 | 1 | 1 | 20 |

![0821Restriction Enzyme Digestion ](https://static.igem.org/mediawiki/2018/1/16/T--Kyoto--0821RestrictionEnzymeDigestion.png)

### 8/21

#### Digest plasmid

Tong

| plasmid | plasmid volume (ul) | DW (ul) | 10x Fast Digest Buffer (ul) | EcoR1 (ul) | Pst1 (ul) |
| --- | --- | --- | --- | --- | --- |
| 304 | 2 | 14 | 2 | 1 | 1 |
| 309 | 2 | 14 | 2 | 1 | 1 |
| 313 | 2 | 14 | 2 | 1 | 1 |
| 314 | 2 | 14 | 2 | 1 | 1 |
| 317 | 2 | 14 | 2 | 1 | 1 |
| 325 | 2 | 14 | 2 | 1 | 1 |
| 342 | 2 | 14 | 2 | 1 | 1 |
| 322 | 2 | 14 | 2 | 1 | 1 |
| 336 | 2 | 14 | 2 | 1 | 1 |
| 343 | 2 | 14 | 2 | 1 | 1 |
| 352 | 2 | 14 | 2 | 1 | 1 |
| 353 | 2 | 14 | 2 | 1 | 1 |
| 354 | 2 | 14 | 2 | 1 | 1 |

![0821Digest plasmid ](https://static.igem.org/mediawiki/2018/3/30/T--Kyoto--0821Digestplasmid.png)

15min 37℃ incubate

### 8/25

#### OE-PCR

Shimazoe

| plasmid | plasmid volume (ul) | DW (ul) | Q5 (ul) | primer1 | primer1 volume (ul) | primer2 | primer2 volume (ul) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 38 (AVP1\_1st) | 0.5 | 3.5 | 5 | Z064 | 0.5 | Z026(p) | 0.5 |
| 83 (AVP1\_2nd) | 0.5 | 3.5 | 5 | Z065 | 0.5 | Z027(p) | 0.5 |

### 8/25

#### colony\_PCR

Shimazoe

| sample | Q5 (ul) | DW (ul) | primer m13f (ul) | primer m13r (ul) |
| --- | --- | --- | --- | --- |
| 38 | 2.5 | 2.0 | 0.25 | 0.25 |
| 83 | 2.5 | 2.0 | 0.25 | 0.25 |

![0825colonyPCR1](https://static.igem.org/mediawiki/2018/d/df/T--Kyoto--0825colonyPCR1.png)

![0825colonyPCR2](https://static.igem.org/mediawiki/2018/8/8d/T--Kyoto--0825colonyPCR2.png)

![0825colonyPCR3](https://static.igem.org/mediawiki/2018/4/4d/T--Kyoto--0825colonyPCR3.png)

colony PCR x 2 set

### 8/26

#### Liquid Culture

Nambu

| sample | LB (ml) | Amp(100mg/ml) (ul) |
| --- | --- | --- |
| 505 | 50 | 50 |
| 506 | 50 | 50 |
| 507 | 50 | 50 |
| 508 | 50 | 50 |
| 516 | 50 | 50 |
| 522 | 50 | 50 |
| 530 | 50 | 50 |

Put them in incubator at 37℃ for 19 hours.

### 8/27

#### OE-PCR

Shimazoe

| content | volume (ul) |
| --- | --- |
| DW | 19 |
| KOD one | 25 |
| primer Z026 | 1.5 |
| prmer Z027 | 1.5 |
| AVP1-1 | 1 |
| AVP1-2 | 2 |

### 8/27

#### Miniprep\_Nanodrop

Nambu

sample:505,506,507,508,516,522,530

### 8/28

#### PCR

Shimazoe

| Component | Amount(μl) |
| --- | --- |
| DW | 8.7 |
| KOD | 10 |
| Z026℗ | 0.6 |
| Z064 | 0.6 |
| 38(template) | 0.1 |
| Total | 20 |

![0828PCR](https://static.igem.org/mediawiki/2018/4/45/T--Kyoto--0828PCR.png)

98℃\* 10s
50℃\* 5s
68℃\* 20s
68℃ 1min
4℃ ∞
　(\*× 40 cycle)

### 8/28

#### PCR

Shimazoe

| Component | Amount(μl) |
| --- | --- |
| DW | 8.7 |
| KOD | 10 |
| Z027℗ | 0.6 |
| Z065 | 0.6 |
| 83(template) | 0.1 |
| Total | 20 |

PCR
98℃\* 10s
50℃\* 5s
68℃\* 20s
68℃ 1min
4℃ ∞
　(\*× 40 cycle)

### 8/28

#### OE-PCR

Shimazoe

| Component | Amount(μl) |
| --- | --- |
| DW | 20 |
| Z026℗ | 1.5 |
| Z027℗ | 1.5 |
| KOD one | 25 |
| AVP1-1st | 1 |
| AVP1-2nd | 1 |
| Total | 50 |

![0828OE-PCR](https://static.igem.org/mediawiki/2018/4/42/T--Kyoto--0828OE-PCR.png)

PCR
98℃\* 10s
65℃\* 5s
68℃\* 30s
68℃ 1min
4℃ ∞
　(\*× 25 cycle )

### 8/28

#### Colony PCR

Shimazoe

| Component | Amount(μl) |
| --- | --- |
| 2× Gflex PCR Buffer | 162.5 |
| primer m13f | 6.5 |
| primer m13r | 6.5 |
| Tks Gflex DNA polymerase | 6.5 |
| DW | 143 |
| Colony | Poke |
| Total | 325 |

![0828ColonyPCR1](https://static.igem.org/mediawiki/2018/a/a7/T--Kyoto--0828ColonyPCR1.png
)

![0828ColonyPCR2](https://static.igem.org/mediawiki/2018/0/0c/T--Kyoto--0828ColonyPCR2.png)

PCR
　　　　　　　　　 　　　　 　　　 number
94℃ 1min　　　　　　 SdrG　　　　 601~611
98℃\* 10s　　　　　　Sdr-Fg　　　　 612~623
55℃\* 15s　　　　　　ZrGPD1　　　 624~631
68℃\* 2min　　　　　 Mangrin　　　 633~638
4℃ ∞ 　　　　　　　Zr-FPS1-2nd 　　 639~642
　(\*× 30 cycle)　　　　McHKT2 　　　643~649
 　　　　　　　　　　ZrFPS1-1st 　　 650~657

### 8/28

#### Liquid Culture

Tong

| Number | Material |
| --- | --- |
| 547 | WT |
| 554 | NHA1Δ |
| 555 | G19, ΔENA1-4 |
| 557 | NHA1Δ, ENA1Δ |
| 559 | ENA1Δ |
| 561 | Z.roxii ATCC42981 |
| 562 | Z.roxii CBS732 |
| 564 | Z.roxii NBRC1876 |

### 8/28

#### YPD culture, LB culture

Tong

|  | 100ml 9% YPD | 50ml YPD | 250ml LB |
| --- | --- | --- | --- |
| Peptone(g) | 2 | 1 | 2.5 |
| Yeast Extract(g) | 1 | 0.5 | 1.25 |
| Agar(g) | 1.5 | 0.75 | 3.75 |
| DW(ml) | 100 | 50 | 250 |
| Glucose(g) | 2 | 1 | × |
| NaCl(g) | 9 | × | 1.25 |

### 8/29

#### PCR

Shimazoe

| Component | Amount(μl) |
| --- | --- |
| 5× Q5 Reaction Buffer | 15 |
| 10mM dNTPs | 1.5 |
| 10uM Z041 | 3.75 |
| 10uM Z042 | 3.75 |
| Template | Poke |
| Q5 DNA Polymerase | 0.75 |
| DW | 47.25 |
| Total | 72 |

Template
AtHKT1(304), SseNHXS1(317), AtNHXS1(336)

PCR
98℃ 30s
98℃\* 10s
65℃\* 30s
72℃\* 1min
72℃ 2min
4℃ ∞
 (\*× 30 cycle)

### 8/29

#### PCR

Shimazoe

| Component | Amount(μl) |
| --- | --- |
| 5× Q5 Reaction Buffer | 5 |
| 10mM dNTPs | 0.5 |
| 10uM Z041 | 1.25 |
| 10uM Z042 | 1.25 |
| Template | Poke |
| Q5 DNA Polymerase | 0.25 |
| DW | 16.75 |
| Total | 24 |

![0829PCR2](https://static.igem.org/mediawiki/2018/e/ef/T--Kyoto--0829PCR2.png)

Template
FgβF3

PCR
98℃ 30s
98℃\* 10s
65℃\* 30s
72℃\* 1min
72℃ 2min
4℃ ∞
 (\*× 30 cycle)

### 8/30

#### Reaction Enzyme Digestion

Shimazoe

| plasmid | plasmid(ul) | 10× T(ul) | 10× BSA(ul) | SmaⅠ(ul) | CIAP(ul) | DW(ul) | Total/(ul) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 426 | 10 | 10 | 10 | 4 | 2 | 64 | 100 |
| 316 | 10 | 10 | 10 | 4 | 2 | 64 | 100 |
| 425 | 10 | 10 | 10 | 4 | 2 | 64 | 100 |
| 315 | 10 | 10 | 10 | 4 | 2 | 64 | 100 |
| 423 | 10 | 10 | 10 | 4 | 2 | 64 | 100 |
| 313 | 10 | 10 | 10 | 4 | 2 | 64 | 100 |

6 sample
37℃ overnight

### 8/30

#### OE-PCR

Shimazoe

|  | sample | volume (ul) | 5× Q5 Reaction Buffer (ul) | 10uM dNTPs (ul) | 10uM Z026℗ (ul) | 10uM Z027℗ (ul) | Z026-Z039 (ul) | Z040-Z027 (ul) | Q5 DNA Polymerase (ul) | DW (ul) | Total (ul) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| A | AtHKT1 | 0.5 | 2 | 0.2 | 0.5 | 0.5 | 0.5 | 0.5 | 0.1 | 5.2 | 10 |
| B | SseNHX1 | 0.5 | 2 | 0.2 | 0.5 | 0.5 | 0.5 | 0.5 | 0.1 | 5.2 | 10 |
| C | AtNHXS1 | 0.5 | 2 | 0.2 | 0.5 | 0.5 | 0.5 | 0.5 | 0.1 | 5.2 | 10 |

PCR

98℃ 30s
98℃\* 10s
72℃\* 30s
72℃\* 1m30s
72℃ 2min
4℃ ∞
 (\*× 25 cycle)

### 8/30

#### PCR

Shimazoe

| template | volume (ul) | primer① | volume (ul) | primer② | volume (ul) | 5× Q5 Reaction Buffer (ul) | 10uM dNTPs (ul) | Q5 DNA Polymerase (ul) | DW (ul) | Total (ul) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| AVP1-1st | 0.5 | 10uM Z026℗ | 0.5 | 10uM Z064 | 0.5 | 2 | 0.2 | 0.1 | 6.2 | 10 |
| AVP1-2nd | 0.5 | 10uM Z027℗ | 0.5 | 10uM Z065 | 0.5 | 2 | 0.2 | 0.1 | 6.2 | 10 |

![0830PCR1](https://static.igem.org/mediawiki/2018/b/b8/T--Kyoto--0830PCR1.png)

PCR
98℃ 30s
98℃\* 10s
67℃\* 30s
72℃\* 1min
72℃ 2min
4℃ ∞
 (\*× 25 cycle)

### 8/30

#### OE-PCR

Shimazoe

| number | template | volume (ul) | 10uM Z026℗ | 10uM Z027℗ | Z026-Z039 (ul) | Z040-Z027 (ul) | 5× Q5 Reaction Buffer (ul) | 10uM dNTPs (ul) | Q5 DNA Polymerase (ul) | DW (ul) | Total (ul) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| A | AtHKT1 | 1 | 0.5 | 0.5 | 1 | 1 | 2 | 0.2 | 0.1 | 3.7 | 10 |
| B | SseNHX1 | 1 | 0.5 | 0.5 | 1 | 1 | 2 | 0.2 | 0.1 | 3.7 | 10 |
| C | AtNHXS1 | 1 | 0.5 | 0.5 | 1 | 1 | 2 | 0.2 | 0.1 | 3.7 | 10 |
| D | GFP | 1 | 0.5 | 0.5 | 1 | 1 | 2 | 0.2 | 0.1 | 3.7 | 10 |
| E | RFP | 1 | 0.5 | 0.5 | 1 | 1 | 2 | 0.2 | 0.1 | 3.7 | 10 |

![0830OE-PCR2](https://static.igem.org/mediawiki/2018/f/fe/T--Kyoto--0830OE-PCR2.png)

PCR
98℃ 30s
98℃\* 10s
55℃\* 30s
72℃\* 1m30s
72℃ 2min
4℃ ∞
 (\*× 30 cycle)

### 8/30

#### PCR

Shimazoe

| template | volume (ul) | 10uM primer① | volume (ul) | 10uM primer② | volume (ul) | 5× Q5 Reaction Buffer (ul) | 10uM dNTPs (ul) | Q5 DNA Polymerase (ul) | DW (ul) | Total (ul) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| AVP1-1st (PCR products) | 1 | Z026℗ | 1.25 | Z064 | 1.25 | 5 | 0.5 | 0.25 | 15.75 | 25 |
| AVP1-1st (Plasmid) | 1 | Z026℗ | 1.25 | Z064 | 1.25 | 5 | 0.5 | 0.25 | 15.75 | 25 |
| AVP1-2nd (PCR products) | 1 | Z027℗ | 1.25 | Z065 | 1.25 | 5 | 0.5 | 0.25 | 15.75 | 25 |
| AVP1-2nd (Plasmid) | 1 | Z027℗ | 1.25 | Z065 | 1.25 | 5 | 0.5 | 0.25 | 15.75 | 25 |

![0830PCR2](https://static.igem.org/mediawiki/2018/6/63/T--Kyoto--0830PCR2.png)

PCR
98℃ 30s
98℃\* 10s
65℃\* 30s
72℃\* 1min
72℃ 2min
4℃ ∞
 (\*× 30 cycle)

### 8/30

#### Yeast Selection

Tong

・add 3ul of cultures to 3ml of nutrient mediums
・measure OD
・concussion culture (30%, 180rpm)

### 8/31

#### OE-PCR

| sample | template | volume (ul) | 5× Q5 Reaction Buffer (ul) | 10uM dNTPs (ul) | 10uM Z026℗ | 10uM Z027℗ | Z026-Z039 (ul) | Z040-Z027 (ul) | Q5 DNA Polymerase (ul) | DW (ul) | Total (ul) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| A | AtHKT1 | 5 | 10 | 1 | 2.5 | 2.5 | 5 | 5 | 0.5 | 18.5 | 50 |
| B | AtNHXS1 | 5 | 10 | 1 | 2.5 | 2.5 | 5 | 5 | 0.5 | 18.5 | 50 |
| C | GFP | 5 | 10 | 1 | 2.5 | 2.5 | 5 | 5 | 0.5 | 18.5 | 50 |
| D | RFP | 5 | 10 | 1 | 2.5 | 2.5 | 5 | 5 | 0.5 | 18.5 | 50 |

![0831OE-PCR1](https://static.igem.org/mediawiki/2018/e/eb/T--Kyoto--0831OE-PCR1.png)

PCR
98℃ 30s
98℃\* 10s
55℃\* 30s
72℃\* 1m30s
72℃ 2min
4℃ ∞
 (\*× 30 cycle)

### 8/31

#### PCR

|  | template | volume (ul) | 10uM primer① | volume (ul) | 10uM primer② | volume (ul) | 5× Q5 Reaction Buffer (ul) | 10uM dNTPs (ul) | Q5 DNA Polymerase (ul) | DW (ul) | Total (ul) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | AVP1-2nd (PCR products) | 1 | Z065 | 1.25 | Z027℗ | 1.25 | 5 | 0.5 | 0.25 | 15.75 | 25 |
| 2 | AVP1-2nd (Plasmid) | 1 | Z065 | 1.25 | Z027℗ | 1.25 | 5 | 0.5 | 0.25 | 15.75 | 25 |
| 3 | GFP (PCR2) | 1 | Z026℗ | 1.25 | Z027℗ | 1.25 | 5 | 0.5 | 0.25 | 15.75 | 25 |
| 4 | RFP (PCR5) | 1 | Z026℗ | 1.25 | Z027℗ | 1.25 | 5 | 0.5 | 0.25 | 15.75 | 25 |

![0831PCR ](https://static.igem.org/mediawiki/2018/8/84/T--Kyoto--0831PCR.png)

PCR
98℃ 30s
98℃\* 10s
55℃\* 30s
72℃\* 1min
72℃ 2min
4℃ ∞
 (\*× 30 cycle)

### 8/31

#### OE-PCR

Shimazoe

| sample | template | volume (ul) | 10uM Z026℗ (ul) | 10uM Z027℗ (ul) | Z026-Z039 (ul) | Z040-Z027 (ul) | KOD one (ul) | DW (ul) | Total (ul) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| A | SseNHX1 | 1 | 0.3 | 0.3 | 1 | 1 | 5 | 1.4 | 10 |
| B | GFP | 1 | 0.3 | 0.3 | 1 | 1 | 5 | 1.4 | 10 |
| C | RFP | 1 | 0.3 | 0.3 | 1 | 1 | 5 | 1.4 | 10 |

![0831OE-PCR2](https://static.igem.org/mediawiki/2018/b/bb/T--Kyoto--0831OE-PCR2.png)

PCR
98℃\* 10s
50℃\* 5s
68℃\* 30s
68℃ 30s
 (\*× 30 cycle)

### 9/1

#### Ligation

Shimazoe

| Number | 1 | 2 | 3 | 4 |
| --- | --- | --- | --- | --- |
| vector | pRS 426 | pRS 316 | pRS 423 | pRS 313 |
| insert | AtHKT1 | AtHKT1 | AtNHXS1 | AtNHXS1 |

Mix the following material (Contamination are shown in the table above)

Plasmid(vector): 1.0ul
Insert: 1.0ul
Ligation High: 2.0ul

Put in Heatblock

### 9/1

#### OE-PCR

Shimazoe

| template | volume (ul) | 10uM Z026℗ (ul) | 10uM Z027℗ (ul) | Z026-Z039 (ul) | Z040-Z027 (ul) | Q5 (ul) | DW (ul) | Total (ul) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| SseNHX1 | 5 | 1.5 | 1.5 | 5 | 5 | 25 | 7 | 50 |

PCR
98℃ 30s
98℃\* 10s
50℃\* 20s
72℃\* 1min30s
72℃ 2min
4℃ ∞
(\*× 30 cycle)

### 9/1

#### OE-PCR

Shimazoe

| template | volume (ul) | 10uM Z026℗ (ul) | 10uM Z027℗ (ul) | 5× Q5 Reaction Buffer (ul) | 10uM dNTPs (ul) | Q5 DNA Polymerase (ul) | DW (ul) | Total (ul) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| AVP1-1st | 1 | 1.25 | 1.25 | 5 | 0.5 | 0.25 | 14.75 | 25 |
| AVP1-2nd | 1 | 1.25 | 1.25 | 5 | 0.5 | 0.25 | 14.75 | 25 |

![0901OE-PCR2](https://static.igem.org/mediawiki/2018/8/85/T--Kyoto--0901OE-PCR2.png)

PCR
98℃ 30s
98℃\* 10s
55℃\* 20s
72℃\* 2min
72℃ 2min
(\*× 30 cycle)

### 9/1

#### Colony PCR

Shimazoe

| Material | Volume |
| --- | --- |
| 2× Emerald amp MAX PCR Master Mix | 375 |
| 10uM m13f | 7.5 |
| 10uM m13r | 7.5 |
| DW | 360 |
| Total | 750 |

![0901ColonyPCR1](https://static.igem.org/mediawiki/2018/9/90/T--Kyoto--0901ColonyPCR1.png)

![0901ColonyPCR2](https://static.igem.org/mediawiki/2018/b/b3/T--Kyoto--0901ColonyPCR2.png)

98℃ 30s
98℃\* 10s
60℃\* 30s
72℃\* 4min
72℃ 4min
4℃ ∞
(\*× 30cycle)

### 9/2

#### Ligation

Tong

| Material | Volume(ul) |
| --- | --- |
| SseNHX1 (template) | 2 |
| Vector (313, 423) | 2 |
| Ligation High | 4 |
| Total | 8 |

### 9/2

#### Transformation

Tong

| Ligation products(ul) | Competent Cells/(ul) | Total(ul) |
| --- | --- | --- |
| 8 (SseNHX1) | 25 | 33 |

on ice 20min

### 9/2

#### PCR

Shimazoe

| Material | Volume |
| --- | --- |
| DW | 6.8 |
| KODone | 10 |
| Z027 | 0.6 |
| Z065 | 0.6 |
| template | 2 |
| Total | 20 |

98℃ 10s
98℃\* 10s
50℃\* 5s
68℃\* 30s
68℃ 30s
(\*× 30cycle)

### 9/2

#### PCR

Shimazoe

| Material | Volume |
| --- | --- |
| 5× Q5 Reaction Buffer | 5 |
| 10uM dNTPs | 0.5 |
| 10uM primer① | 1.25 |
| 10uM primer② | 1.25 |
| template | 1 |
| Q5 DNA Polymerase | 0.25 |
| DW | 15.75 |
| Total | 25 |

primer① (Z026/Z040)
primer② (Z039/Z027)

98℃ 30s
98℃\* 10s
54℃\* 20s
72℃\* 30s
72℃ 2min
4℃ ∞
(\*× 30 cycle)

### 9/2

#### OE-PCR

Shimazoe

| Material | Volume |
| --- | --- |
| 2× Emerald Amp MAX | 12.25 |
| 10uM Z026℗ | 0.25 |
| 10uM Z027℗ | 0.25 |
| AVP1-1st | 1 |
| AVP1-2nd | 1 |
| DW | 10.25 |
| Total | 25 |

98℃ 30s
98℃\* 10s
55℃\* 30s
72℃\* 3m30s
72℃ 3m30s
4℃ ∞
(\*× 30 cycle)

### 9/2

#### OE-PCR

Shimazoe

| Material | Volume |
| --- | --- |
| 5× Q5 Reaction Buffer | 10 |
| 10uM Z026℗ | 2.5 |
| 10uM Z027℗ | 2.5 |
| 10uM dNTPs | 1 |
| template⓪ | 5 |
| template① | 5 |
| template② | 5 |
| Q5 DNA Polymerase | 0.5 |
| DW | 18.5 |
| Total | 50 |

template ⓪(AtHKT1/SseNHX1/AtNHXS1/RFP), ① (Z026-Z039), ② (Z040-Z027)

98℃ 30s
98℃\* 10s
55℃\* 30s
72℃\* 1m30s
72℃ 2min
4℃ ∞
(\*× 30 cycle)

### 9/3

#### Ligation

Tong

| sample | volume(ul) | pRS316(ul) | Ligation High(ul) | Total(ul) |
| --- | --- | --- | --- | --- |
| Mangrin | 1 | 2 | 3 | 6 |
| ZrFPS1-1st | 1 | 2 | 3 | 6 |
| ZrFPS1-2nd | 1 | 2 | 3 | 6 |
| ZrGPD1 | 1 | 2 | 3 | 6 |
| McHKT2 | 1 | 2 | 3 | 6 |
| SdrG | 1 | 2 | 3 | 6 |
| SdrG-Fgβ | 1 | 2 | 3 | 6 |

18℃overnight

### 9/4

#### Transformation

| Ligation products(ul) | Competent Cells/(ul) | Total(ul) |
| --- | --- | --- |
| 6 | 55 | 61 |

on ice　20min
42℃　45sec
on ice　3min
SOC　550
37℃　40min

### 9/4

#### PCR

Tong

| Material | volume (ul) |
| --- | --- |
| DW | 22.8 |
| Template (pIEG-SSS) | 1.6 |
| Primer F (Z067) | 2 |
| Primer R (Z066) | 2 |
| DNA Pol (Q5) | 0.4 |
| Reaction buffer | 8 |
| dNTP mix | 3.2 |
| Total | 40 |

98℃ 30s
98℃\* 10s
48℃\* 30s
72℃\* 3min
72℃ 2min
4℃ ∞
(\*× 30cycle)

### 9/4

#### OE-PCR

Shimazoe

| Material | Volume |
| --- | --- |
| 5× Q5 Reaction Buffer | 10 |
| 10uM Z026℗ | 2.5 |
| 10uM Z027℗ | 2.5 |
| 10uM dNTPs | 1 |
| template⓪ | 5 |
| template① | 5 |
| template② | 5 |
| Q5 DNA Polymerase | 0.5 |
| DW | 18.5 |
| Total | 50 |

template ⓪(AtHKT1/SseNHX1/AtNHXS1/RFP), ① (Z026-Z039), ② (Z040-Z027)

98℃ 30s
98℃\* 10s
55℃\* 30s
72℃\* 1m30s
72℃ 2min
4℃ ∞
(\*× 30 cycle)

### 9/4

#### OE-PCR

Shimazoe

| Material | Volume |
| --- | --- |
| 5× Q5 Reaction Buffer | 10 |
| 10uM Z026 | 2.5 |
| 10uM Z027 | 2.5 |
| 10uM dNTPs | 1 |
| template⓪ | 5 |
| template① | 5 |
| template② | 5 |
| Q5 DNA Polymerase | 0.5 |
| DW | 18.5 |
| Total | 50 |

template ⓪(AtHKT1/SseNHX1/AtNHXS1/RFP), ① (Z026-Z039), ② (Z040-Z027)

98℃ 30s
98℃\* 10s
55℃\* 30s
72℃\* 1m30s
72℃ 2min
4℃ ∞
(\*× 30 cycle)

### 9/4

#### Colony PCR

Shimazoe

| Material | Volume(ul) |
| --- | --- |
| 2× Emerald amp MAX PCR Master Mix | 40 |
| 10uM m13f | 0.8 |
| 10uM m13r | 0.8 |
| DW | 38.4 |
| Total | 80 |

98℃ 30s
98℃\* 10s
60℃\* 30s
72℃\* 4min
72℃ 4min
4℃ ∞
(\*× 30cycle)

### 9/4

#### Ligation

Tong

| number | sample | volume(ul) | vector | volume(ul) | Ligation High(ul) | Total(ul) |
| --- | --- | --- | --- | --- | --- | --- |
| 1 | AtHKT1 | 1 | 316,426 | 2 | 3 | 6 |
| 2 | AtNHXS1 | 1 | 423,313 | 2 | 3 | 6 |
| 3 | SseNHX1 | 1 | 423,313 | 2 | 3 | 6 |

### 9/5

#### PCR

Tong

| Material | volume (ul) |
| --- | --- |
| DW | 22 |
| Template (pIEG-SSS) | 1 |
| Primer F (Z067) | 1 |
| Primer R (Z066) | 1 |
| PrimeSTAR Max | 25 |
| Total | 50 |

98℃\* 10s
55℃\* 15s
72℃\* 35s
(\*× 30cycle)

### 9/5

#### OE-PCR

Shimazoe

| Material | Volume |
| --- | --- |
| 5× Q5 Reaction Buffer | 12 |
| 10uM Z026℗ | 3 |
| 10uM Z027℗ | 3 |
| 10uM dNTPs | 1.2 |
| template⓪ | 6 |
| template① | 6 |
| template② | 6 |
| Q5 DNA Polymerase | 0.6 |
| DW | 22.2 |
| Total | 60 |

template ⓪(AtHKT1/SseNHX1/AtNHXS1/RFP), ① (Z026-Z039), ② (Z040-Z027)

98℃ 30s
98℃\* 10s
55℃\* 30s
72℃\* 1m30s
72℃ 2min
4℃ ∞
(\*× 30 cycle)

### 9/5

#### Transformation

Shimazoe

|  | 1 | 2 | 3 | 4 | 5 | 6 |
| --- | --- | --- | --- | --- | --- | --- |
| sample | AtHKT1 | AtHKT1 | AtNHXS1 | AtNHXS1 | SseNHX1 | SseNHX1 |
| vector | 426 | 316 | 423 | 313 | 423 | 313 |
| competent cell (ul) | 20 | 30 | 20 | 30 | 20 | 30 |

### 9/5

#### PCR

Shimazoe

| template | volume (ul) | 10uM primer① (ul) | volume (ul) | 10uM primer② (ul) | volume (ul) | 5× Q5 Reaction Buffer (ul) | 10uM dNTPs (ul) | Q5 DNA Polymerase (ul) | DW (ul) | Total (ul) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| AVP1-1st | 1 | Z026 | 1.25 | Z071 | 1.25 | 5 | 0.5 | 0.25 | 15.75 | 25 |
| AVP1-2nd | 1 | Z027 | 1.25 | Z072 | 1.25 | 5 | 0.5 | 0.25 | 15.75 | 25 |
| AVP1-1st | 1 | Z026 | 1.25 | Z070 | 1.25 | 5 | 0.5 | 0.25 | 15.75 | 25 |

98℃ 30s
98℃\* 10s
55℃\* 30s
72℃\* 1min
72℃ 2min
4℃ ∞
(\*× 30 cycle)

### 9/5

#### OE-PCR

Shimazoe

| Material | Volume |
| --- | --- |
| 5× Q5 Reaction Buffer | 10 |
| 10uM Z026℗ | 2.5 |
| 10uM Z027℗ | 2.5 |
| AVP1-1st | 2 |
| AVP1-2nd | 2 |
| 10uM dNTPs | 1 |
| Q5 DNA Polymerase | 0.5 |
| DW | 24.5 |
| Total | 50 |

template ⓪(AtHKT1/SseNHX1/AtNHXS1/RFP), ① (Z026-Z039), ② (Z040-Z027)

98℃ 30s
98℃\* 10s
55℃\* 30s
72℃\* 1m30s
72℃ 2min
4℃ ∞
(\*× 30 cycle)

### 9/5

#### Colony PCR

Tamukai

| Material | Volume(ul) |
| --- | --- |
| 2× Emerald amp MAX PCR Master Mix | 180 |
| 10uM m13f | 7.2 |
| 10uM m13r | 7.2 |
| DW | 180 |
| Total | 373.4 |

![0905ColonyPCR1](https://static.igem.org/mediawiki/2018/d/dd/T--Kyoto--0905ColonyPCR1.png)

![0905ColonyPCR2](https://static.igem.org/mediawiki/2018/c/c2/T--Kyoto--0905ColonyPCR2.png)

![0905ColonyPCR3](https://static.igem.org/mediawiki/2018/9/95/T--Kyoto--0905ColonyPCR3.png)

![0905ColonyPCR4](https://static.igem.org/mediawiki/2018/1/11/T--Kyoto--0905ColonyPCR4.png)

98℃ 30s
98℃\* 10s
60℃\* 30s
72℃\* 4min
72℃ 4min
4℃ ∞
(\*× 30cycle)

### 9/5

#### Genome extracion

Tamukai

| Material | volume (ul) |
| --- | --- |
| Proteinase K | 1.2ul |
| Extraction Reagent | 1.2ml |

・Seperate into 17 microtube 150uL each
・incubate in 70℃ for 10min
・on ice for 5min
・Add Precipation Reagent 90ul, vortex, on ice 10min, remove supreme
・Add 80ul Phend-chloroform, vortex, max rpm (15,000rpm) 10min, remove supreme
・ -30℃

### 9/5

#### OE-PCR

Shimazoe

| Material | Volume(ul) |
| --- | --- |
| 2× Gflex PCR Buffer | 25 |
| 10uM Z026℗ | 1 |
| 10uM Z027℗ | 1 |
| template⓪ | 1 |
| template① | 1 |
| template② | 1 |
| Tks Gflex DNA Polymerase | 1 |
| DW | 19 |
| Total | 50 |

⓪(SseNHX1/RFP)
94℃ 1min
98℃\* 10s
55℃\* 15s
68℃\* 1m30s
68℃ 2min
(\*× 30 cycle)

### 9/5

#### Ligation

Shimazoe

| insert | volume(ul) | vector | volume(ul) | Ligation High(ul) | Total(ul) |
| --- | --- | --- | --- | --- | --- |
| AtHKT1 | 2 | 316 | 2 | 4 | 8 |
| AtHKT1 | 2 | 426 | 2 | 4 | 8 |
| AtNHXS1 | 2 | 423 | 2 | 4 | 8 |
| AtNHXS1 | 2 | 313 | 2 | 4 | 8 |
| SseNHX1 | 2 | 423 | 2 | 4 | 8 |
| SseNHX1 | 2 | 313 | 2 | 4 | 8 |

### 9/6

#### Transformation

Shimazoe

| Ligation products(ul) | Competent Cells/(ul) | Total(ul) |
| --- | --- | --- |
| 8 | 30 | 38 |

### 9/6

#### Colony PCR

Shimazoe

| Material | Volume(ul) |
| --- | --- |
| 2× Emerald amp MAX PCR Master Mix | 125 |
| 10uM m13f | 2.5 |
| 10uM m13r | 2.5 |
| DW | 120 |
| Total | 250 |

98℃ 30s
98℃\* 10s
60℃\* 30s
72℃\* 2min
72℃ 2min
4℃ ∞
(\*× 30cycle)

### 9/6

#### OE-PCR

Shimazoe

| Material | Volume(ul) |
| --- | --- |
| 2× Gflex PCR Buffer | 25 |
| 10uM Z026℗ | 1 |
| 10uM Z027℗ | 1 |
| AVP1-1st | 1 |
| AVP1-2nd | 1 |
| Tks Gflex DNA Polymerase | 1 |
| DW | 20 |
| Total | 50 |

94℃ 1min
98℃\* 10s
55℃\* 15s
68℃\* 2min
68℃ 2min
(\*× 30 cycle)

### 9/6

#### PCR

Shimazoe

| Material | Volume |
| --- | --- |
| 5× Q5 Reaction Buffer | 5 |
| 10uM primer① | 1.25 |
| 10uM primer② | 1.25 |
| 10uM dNTPs | 0.5 |
| template | 1 |
| Q5 DNA Polymerase | 0.25 |
| DW | 15.75 |
| Total | 25 |

ZrGPD1(primer①②:Z041,Z042 template:506), SdrG(primer①②:Z044,Z045 template:752cp)

98℃ 30s
98℃\* 10s
55℃\* 30s
72℃\* 1min
72℃ 2min
4℃ ∞
(\*× 30 cycle)

### 9/7

#### Restriction Enzyme Digestion

Tong

| plasmid | plasmid(ul) | 10×K buffer(ul) | 10×H buffer(ul) | 10× BSA(ul) | EcoRⅠ(ul) | NcoⅠ(ul) | PstⅠ(ul) | DpnⅠ(ul) | DW(ul) | Total/(ul) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 38 | 10 | 4 | × | 4 | 2 | 2 | × | × | 28 | 40 |
| 83 | 10 | 4 | × | 4 | × | 2 | 2 | × | 28 | 40 |
| pSB1C3 | 20 | × | 4 | × | 2 | × | 2 | 2 | 10 | 40 |
| YCplac111 | 2 | × | 4 | × | 2 | × | 2 | × | 30 | 40 |

### 9/8

#### Restriction Enzyme Digestion

Tong

|  | volume(ul) | 10×H buffer(ul) | EcoRⅠ(ul) | PstⅠ(ul) | DW(ul) | Total/(ul) |
| --- | --- | --- | --- | --- | --- | --- |
| DNA 404 | 5 | 4 | 2 | 2 | 27 | 40 |

### 9/8

#### Ligation

Tong

| sample | volume(ul) | vector | volume(ul) | Ligation High(ul) | Total(ul) |
| --- | --- | --- | --- | --- | --- |
| AtHKT1 | 2 | 316 | 2 | 4 | 8 |
| AtHKT1 | 2 | 426 | 2 | 4 | 8 |
| AtNHXS1 | 2 | 423 | 2 | 4 | 8 |
| AtNHXS1 | 2 | 313 | 2 | 4 | 8 |
| SseNHX1 | 2 | 423 | 2 | 4 | 8 |
| SseNHX1 | 2 | 313 | 2 | 4 | 8 |

### 9/8

#### OE-PCR

Tong

| template | volume (ul) | 10uM Z026℗ (ul) | 10uM Z027℗ (ul) | 5× Q5 Reaction Buffer (ul) | 10uM dNTPs (ul) | promoter(ul) | terminator(ul) | Q5 DNA Polymerase (ul) | DW (ul) | Total (ul) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| ZrGPD1 ORF | 5 | 2.5 | 2.5 | 10 | 1 | 5 | 5 | 0.5 | 18.5 | 50 |

![0908OE-PCR1](https://static.igem.org/mediawiki/2018/6/68/T--Kyoto--0908OE-PCR1.png)

98℃ 30s
98℃\* 10s
55℃\* 30s
72℃\* 1min30s
72℃ 2min
4℃ ∞
(\*× 30cycle)

### 9/8

#### Transformation

Tong

| Ligation products(ul) | Competent Cells/(ul) | Total(ul) |
| --- | --- | --- |
| 8 | 30 | 38 |

・on ice 10min
・42℃ 45sec
・+SOC 300ul
・37℃ 40min

### 9/8

#### OE-PCR

Tong

| template① | volume (ul) | template② | volume (ul) | 10uM Z026℗ (ul) | 10uM Z027℗ (ul) | 2× Prime Star (ul) | DW (ul) | Total (ul) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| AVP1-1st | 0.5 | AVP1-2nd | 0.5 | 1 | 1 | 10 | 7 | 20 |

98℃\* 10s
55℃\* 15s
72℃\* 20s
(\*× 30cycle)

### 9/8

#### PCR

Tong

| Material | volume (ul) |
| --- | --- |
| DW | 22 |
| Template (pIEG-SSS) | 1 |
| Primer F (Z067) | 1 |
| Primer R (Z066) | 1 |
| PrimeSTAR Max | 25 |
| Total | 50 |

98℃\* 10s
55℃\* 15s
72℃\* 35s
(\*× 30cycle)

### 9/8

#### Restriction Enzyme Digestion

Tong

|  | volume(ul) | 10×M buffer(ul) | EcoRⅠ(ul) | XbaⅠ(ul) | DW(ul) | Total/(ul) |
| --- | --- | --- | --- | --- | --- | --- |
| AVP1-1st (38) | 20 | 4 | 2 | 2 | 12 | 40 |
| YCplac111 | 2 | 4 | 2 | 2 | 30 | 40 |

### 9/8

#### Ligation

Tong

| material | volume(ul) |
| --- | --- |
| AVP1-1st | 1.5 |
| AVP1-2nd | 1.5 |
| vector | 1.0 |
| Ligation High | 4.5 |
| Total | 9.0 |

### 9/9

#### OE-PCR

Tong

| template | volume (ul) | Stalk (ul) | Z044 (ul) | Z047 (ul) | 2× Prime Star (ul) | DW (ul) | Total (ul) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| SdrG, Fgβ | 1 | 1 | 1 | 1 | 20 | 16 | 40 |

98℃\* 10s
55℃\* 5s
72℃\* 20s
(\*× 30cycle)

### 9/9

#### OE-PCR

Tong

| template | volume (ul) | 10uM Z026℗ (ul) | 10uM Z027℗ (ul) | promoter(ul) | terminator(ul) | 2× Prime Star (ul) | DW (ul) | Total (ul) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| ZrGPD1 | 0.5 | 1 | 1 | 0.5 | 0.5 | 10 | 6.5 | 20 |

### 9/9

#### OE-PCR

Tong

| template① | volume (ul) | template② | volume (ul) | 10uM Z026℗ (ul) | 10uM Z027℗ (ul) | 2× Prime Star (ul) | DW (ul) | Total (ul) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| AVP1-1st | 0.5 | AVP1-2nd | 0.5 | 0.5 | 0.5 | 10 | 8 | 20 |

98℃\* 10s
55℃\* 5s
72℃\* 15s
(\*× 30cycle)

### 9/9

#### Ligation

Tong

| Material | volume (ul) |
| --- | --- |
| AVP1-1st | 3 |
| YCplac111 | 3 |
| Ligation high | 6 |
| Total | 12 |

### 9/10

#### Transformation

Tong

| Ligation products(ul) | Competent Cells/(ul) | Total(ul) |
| --- | --- | --- |
| 12 | 36 | 48 |

on ice 20min

### 9/10

#### Colony PCR

Tong

| Material | Volume(ul) |
| --- | --- |
| 2× Emerald amp MAX PCR Master Mix | 70 |
| 10uM m13f | 1.4 |
| 10uM m13r | 1.4 |
| DW | 67.2 |
| Total | 140 |

![0910ColonyPCR1-1](https://static.igem.org/mediawiki/2018/d/df/T--Kyoto--0910ColonyPCR1-1.png)

![0910ColonyPCR1-2](https://static.igem.org/mediawiki/2018/3/35/T--Kyoto--0910ColonyPCR1-2.png)

98℃ 30s
98℃\* 10s
60℃\* 30s
72℃\* 4min
72℃ 4min
4℃ ∞
(\*× 30cycle)

### 9/10

#### Colony PCR

Tamukai

| Material | Volume(ul) |
| --- | --- |
| 2× Emerald amp MAX PCR Master Mix | 400 |
| 10uM m13f | 8 |
| 10uM m13r | 8 |
| DW | 384 |
| Total | 800 |

98℃ 30s
98℃\* 10s
60℃\* 30s
72℃\* 2min
72℃ 2min
4℃ ∞
(\*× 30cycle)

### 9/10

#### OE-PCR

Shimazoe

| Material | Volume(ul) |
| --- | --- |
| 2× Gflex PCR Buffer | 25 |
| 10uM Z026℗ | 1 |
| 10uM Z027℗ | 1 |
| template⓪ | 1 |
| template① | 1 |
| template② | 1 |
| Tks Gflex DNA Polymerase | 1 |
| DW | 19 |
| Total | 50 |

ZrGPD1
94℃ 1min
98℃\* 10s
55℃\* 15s
68℃\* 1m30s
68℃ 2min
4℃ ∞
(\*× 30 cycle)

### 9/10

#### Colony PCR

Nambu

| Material | Volume(ul) |
| --- | --- |
| 2× Emerald amp MAX PCR Master Mix | 35 |
| 10uM m13f | 0.6 |
| 10uM m13r | 0.6 |
| DW | 33.6 |
| Total | 70 |

![0910ColonyPCR3](https://static.igem.org/mediawiki/2018/c/cc/T--Kyoto--0910ColonyPCR3.png)

98℃ 30s
98℃\* 10s
58℃\* 30s
72℃\* 4min
72℃ 4min
4℃ ∞
(\*× 30cycle)

### 9/10

#### Restriction Enzyme Digestion

Shimazoe

| material | volume(μl) |
| --- | --- |
| EcoT14Ⅰ | 25 |
| 10×H buffer | 50 |
| λDNA | 71 |
| DW | 354 |
| Total | 500 |

### 9/11

#### Restriction Enzyme Digestion

Nambu

| material | volume(μl) |
| --- | --- |
| Mangrin(pRS425) | 10 |
| BSA | 4 |
| 10×H buffer(10xH) | 4 |
| Not1 | 2 |
| TritonX-100 | 4 |
| DW | 16 |
| Total | 40 |

### 9/11

#### Restriction Enzyme Digestion

Nambu

| material | volume(μl) |
| --- | --- |
| AVP1 | 5 |
| BSA | 2 |
| 10×H buffer(10xH) | 2 |
| EcoR1 | 1 |
| Nco1 | 1 |
| Pst1 | 1 |
| DW | 8 |
| Total | 20 |

### 9/11

#### OE-PCR

Shimazoe

| Material | Volume(ul) |
| --- | --- |
| 2× Gflex PCR Buffer | 30 |
| 10uM Z026℗ | 1.2 |
| 10uM Z027℗ | 1.2 |
| template⓪ | 1.2 |
| template① | 1.2 |
| template② | 1.2 |
| Tks Gflex DNA Polymerase | 1.2 |
| DW | 22.8 |
| Total | 60 |

ZrGPD1
94℃ 1min
98℃\* 10s
55℃\* 15s
68℃\* 1m30s
68℃ 2min
4℃ ∞
(\*× 30 cycle)

### 9/11

#### Ligation

Shimazoe

| insert | volume(ul) | vector | volume(ul) | Ligation High(ul) | Total(ul) |
| --- | --- | --- | --- | --- | --- |
| GFP | 1 | 426 | 1 | 2 | 4 |
| GFP | 1 | 316 | 1 | 2 | 4 |
| RFP | 1 | 426 | 1 | 2 | 4 |
| RFP | 1 | 316 | 1 | 2 | 4 |

### 9/11

#### PCR

Tamukai

| Material | Volume(ul) |
| --- | --- |
| 2×Emerald Amp buffer | 25 |
| Z003 | 0.5 |
| Z004 | 0.5 |
| DW | 24 |
| Total | 50 |

NHA1
98℃ 2min
98℃\* 30s
48℃\* 30s
68℃\* 3m30s
68℃ 5min
4℃ ∞
(\*× 30 cycle)

### 9/11

#### PCR

Tamukai

| material | volume (ul) |
| --- | --- |
| DW | 47.8 |
| 5× Q5 Reaction Buffer | 16 |
| 10uM dNTPs | 6.4 |
| Q5 DNA Polymerase | 0.8 |
| 10uM Z005 | 4 |
| 10uM Z028 | 4 |
| template(pFA6a-hphMX) | 1 |
| Total | 80 |

ENA1Δ1,2,5

### 9/11

#### PCR

Tamukai

| material | volume (ul) |
| --- | --- |
| DW | 45.8 |
| 5× Q5 Reaction Buffer | 16 |
| 10uM dNTPs | 6.4 |
| Q5 DNA Polymerase | 0.8 |
| 10uM Z005 | 4 |
| 10uM Z028 | 4 |
| template(yeast genome) | 1 |
| Total | 80 |

NHA1

### 9/11

#### Ligation

Shimazoe

| insert | volume(ul) | vector | volume(ul) | Ligation High(ul) | Total(ul) |
| --- | --- | --- | --- | --- | --- |
| AtHKT1 | 1 | 316 | 1 | 2 | 4 |
| AtHKT1 | 1 | 426 | 1 | 2 | 4 |
| AtNHXS1 | 1 | 423 | 1 | 2 | 4 |
| AtNHXS1 | 1 | 313 | 1 | 2 | 4 |
| SseNHX1 | 1 | 423 | 1 | 2 | 4 |
| SseNHX1 | 1 | 313 | 1 | 2 | 4 |

### 9/12

#### Restriction Enzyme Digestion

Nambu

| material | volume(μl) |
| --- | --- |
| CIAP | 1 |
| pRS426 or pRS425 | 2 |
| BSA | 4 |
| 10×H buffer(10xH) | 4 |
| Not1 | 2 |
| TritonX-100 | 4 |
| DW | 23 |
| Total | 40 |

| material | volume(μl) |
| --- | --- |
| AVP1(on pSB1C3) | 5 |
| BSA | 2 |
| 10×H buffer(10xH) | 2 |
| Not1 | 1 |
| TritonX-100 | 4 |
| DW | 8 |
| Total | 20 |

| material | volume(μl) |
| --- | --- |
| AVP1(on pSB1C3) | 5 |
| BSA | 2 |
| 10×H buffer | 2 |
| Nco1 | 1 |
| EcoR1 | 1 |
| Pst1 | 1 |
| DW | 8 |
| Total | 20 |

4sample(3,6,7,11)

### 9/12

#### Ligation

Shimazoe

| Material | Volume(ul) |
| --- | --- |
| Ligation high | 4 |
| pRS425 Not1 CIAP | 2 |
| AVP1 Not1 ③ | 2 |
| Total | 8 |

### 9/13

#### Transformation

Shimazoe

| Ligation products(ul) | Competent Cells/(ul) | Total(ul) |
| --- | --- | --- |
| 8 | 30 | 38 |

### 9/13

#### OE-PCR

Shimazoe

| Material | Volume(ul) |
| --- | --- |
| 2× Gflex PCR Buffer | 30 |
| 10uM Z017℗ | 1.2 |
| 10uM Z018℗ | 1.2 |
| template⓪ | 6 |
| template① | 6 |
| template② | 6 |
| Tks Gflex DNA Polymerase | 1.2 |
| DW | 8.4 |
| Total | 60 |

![0913OE-PCR1](https://static.igem.org/mediawiki/2018/0/0f/T--Kyoto--0913OE-PCR1.png)

ZrGPD1
94℃ 1min
98℃\* 10s
55℃\* 15s
68℃\* 1m30s
68℃ 2min
4℃ ∞
(\*× 30 cycle)

### 9/13

#### OE-PCR

Shimazoe

| Material | Volume(ul) |
| --- | --- |
| 2× Prime Star Max | 5 |
| Z047 | 1 |
| Z074 or Z048 | 1 |
| template | 1 |
| DW | 2 |
| Total | 10 |

98℃\* 10s
55℃\* 15s
68℃\* 10s
(\*× 35 cycle)

### 9/13

#### PCR

Shimazoe

| material | volume (ul) |
| --- | --- |
| 2× Prime Star Max | 5 |
| 10uM Z047 | 1 |
| 10uM Z074 | 1 |
| template | 0.5 |
| DW | 2.5 |
| Total | 10 |

### 9/13

#### Colony PCR

Shimazoe

| Material | Volume(ul) |
| --- | --- |
| 2× Emerald amp MAX PCR Master Mix | 10 |
| 10uM m13f | 0.2 |
| 10uM m13r | 0.2 |
| DW | 9.6 |
| Total | 20 |

98℃ 30s
98℃\* 10s
60℃\* 30s
72℃\* 2min
72℃ 2min
4℃ ∞
(\*× 30cycle)

### 9/13

#### OE-PCR

Shimazoe

| Material | Volume(ul) |
| --- | --- |
| 2× Gflex PCR Buffer | 5 |
| 10uM Z044 | 0.2 |
| 10uM Z047 | 0.2 |
| template① | 0.5 |
| template② | 0.5 |
| Tks Gflex DNA Polymerase | 0.1 |
| DW | 3.5 |
| Total | 10 |

Stalk-SdrG, Stalk-FgβF3
94℃ 1min
98℃\* 10s
55℃\* 15s
68℃\* 2min
68℃ 2min
4℃ ∞
(\*× 30 cycle)

### 9/14

#### Ligation

Nambu

| Material | Volume(ul) |
| --- | --- |
| Ligation high | 3 |
| pRS425 | 1 |
| AVP1(3,7) | 2 |
| Total | 6 |

### 9/14

#### Restriction Enzyme Digestion

Nambu

| material | volume(μl) |
| --- | --- |
| sample(404/910/938) | 10/5/3 |
| EcoR1 | 2 |
| 10×H buffer | 4 |
| Pst1 | 2 |
| DW | 22/27/32 |
| Total | 40 |

### 9/14

#### Colony PCR

Shimazoe

| Material | Volume(ul) |
| --- | --- |
| 2× Emerald amp MAX PCR Master Mix | 62.5 |
| 10uM m13f | 1.25 |
| 10uM m13r | 1.25 |
| DW | 60 |
| Total | 125 |

![0914ColonyPCR ](https://static.igem.org/mediawiki/2018/0/03/T--Kyoto--0914ColonyPCR.png)

SseNHX1/AVP1
98℃ 30s
98℃\* 10s
60℃\* 30s
72℃\* 2min
72℃ 2min
4℃ ∞
(\*× 30cycle)

### 9/14

#### Restriction Enzyme Digestion

Nambu

| sample | 404 | 910 | 938 |
| --- | --- | --- | --- |
| volume(ul) | 10 | 5 | 3 |
| 10×H buffer(10xH) | 4 | 4 | 4 |
| EcoR1 | 2 | 2 | 2 |
| Not1 | 2 | 2 | 2 |
| DW | 22 | 27 | 32 |
| Total | 40 | 40 | 43 |

![0914restriction](https://static.igem.org/mediawiki/2018/9/97/T--Kyoto--0914restriction.jpg)

### 9/15

#### OE-PCR

Tong

| Material | Volume(ul) |
| --- | --- |
| 2× Gflex PCR Buffer | 30 |
| 10uM Z017℗ | 1.2 |
| 10uM Z018℗ | 1.2 |
| template pro | 6 |
| template term | 6 |
| template ORF | 6 |
| Tks Gflex DNA Polymerase | 1.2 |
| DW | 8.4 |
| Total | 60 |

ZrGPD1
94℃ 1min
98℃\* 10s
55℃\* 15s
68℃\* 1m30s
68℃ 2min
4℃ ∞
(\*× 30 cycle)

### 9/15

#### OE-PCR

Tong

| Material | Volume(ul) |
| --- | --- |
| 2× Prime Star Max | 20 |
| Z047 | 4 |
| Z074 | 4 |
| template | 4 |
| DW | 8 |
| Total | 40 |

98℃\* 10s
55℃\* 15s
72℃\* 10s
(\*× 35 cycle)

### 9/15

#### OE-PCR

Tong

| number | gene(plasmid) | strain | marker |
| --- | --- | --- | --- |
| 1 | ×(pRS426) | WT | URA3 |
| 2 | AtHKT1(pRS426) | WT | URA3 |
| 3 | Mangrin(pRS316) | G19 | URA3 |
| 4 | ×(pRS316) | G19 | URA3 |
| 5 | ×(pRS316) | WT | URA3 |
| 6 | Mangrin(pRS316) | WT | URA3 |

### 9/15

#### OE-PCR

Shimazoe

| Material | Volume(ul) |
| --- | --- |
| 2× Gflex PCR Buffer | 25 |
| 10uM Z017℗ | 1 |
| 10uM Z043 | 1 |
| template① | 3 |
| template② | 3 |
| Tks Gflex DNA Polymerase | 1 |
| DW | 16 |
| Total | 50 |

ZrGPD1
98℃ 30s
98℃\* 10s
55℃\* 30s
72℃\* 1m30s
72℃ 2min
4℃ ∞
(\*× 30 cycle)

### 9/15

#### Restriction Enzyme Digestion

Nambu

| material | volume(μl) |
| --- | --- |
| ZrFPS1-1st | 7 |
| EcoR1 | 0.5 |
| Nco1 | 0.5 |
| 10×K buffer | 1 |
| BSA | 1 |
| Total | 10 |

| material | volume(μl) |
| --- | --- |
| ZrFPS1-2nd (718) | 4 |
| Spe1 | 2 |
| Nco1 | 2 |
| 10×K buffer | 4 |
| BSA | 4 |
| DW | 24 |
| Total | 40 |

| material | volume(μl) |
| --- | --- |
| pRS426 | 2 |
| EcoR1 | 2 |
| Spe1 | 2 |
| 10×H buffer | 4 |
| CIAP | 1 |
| DW | 29 |
| Total | 40 |

| material | volume(μl) |
| --- | --- |
| pRS316 | 2 |
| EcoR1 | 2 |
| Spe1 | 2 |
| 10×H buffer | 4 |
| CIAP | 1 |
| DW | 29 |
| Total | 40 |

![0915RestrictionEnzymeDigestion ](https://static.igem.org/mediawiki/2018/a/ad/T--Kyoto--0915RestrictionEnzymeDigestion.png)

### 9/16

#### PCR

Tong

| material | volume (ul) |
| --- | --- |
| 2× Prime Star Max | 20 |
| 10uM Z047 | 4 |
| 10uM Z074 | 4 |
| template | 2 |
| DW | 10 |
| Total | 40 |

Stalk
98℃\* 10s
55℃\* 15s
72℃\* 10s
4℃ ∞
(\*× 30 cycle)

### 9/16

#### PCR

Tong

| Material | Volume(ul) |
| --- | --- |
| 2× Gflex PCR Buffer | 30 |
| 10uM Z017℗ | 1.2 |
| 10uM Z018℗ | 1.2 |
| template⓪ | 6 |
| template① | 6 |
| template② | 6 |
| Tks Gflex DNA Polymerase | 1.2 |
| DW | 8.4 |
| Total | 60 |

ZrGPD1
94℃ 1min
98℃\* 10s
55℃\* 15s
68℃\* 1m30s
68℃ 2min
4℃ ∞
(\*× 35 cycle)

### 9/16

#### PCR

Tong

| material | volume (ul) |
| --- | --- |
| 2× Prime Star Max | 5 |
| 10uM Z047 | 1 |
| 10uM Z074 | 1 |
| template | 0.5 |
| DW | 2.5 |
| Total | 10 |

Stalk

### 9/16

#### PCR

Shimazoe

| Material | Volume(ul) |
| --- | --- |
| 2× Gflex PCR Buffer | 25 |
| 10uM primer① | 1 |
| 10uM primer② | 1 |
| template | 1 |
| Tks Gflex DNA Polymerase | 1 |
| DW | 21 |
| Total | 50 |

![0916PCR4](https://static.igem.org/mediawiki/2018/e/e3/T--Kyoto--0916PCR4.png)

ZrGPD1
94℃ 1min
98℃\* 10s
55℃\* 15s
68℃\* 1m30s
68℃ 2min
4℃ ∞
(\*× 30 cycle)

### 9/16

#### OE-PCR

Tong

| material | volume (ul) |
| --- | --- |
| 2× Prime Star Max | 20 |
| 10uM Z017℗ | 1 |
| 10uM Z018℗ | 1 |
| promoter | 3 |
| SseNHX1 | 3 |
| terminator | 3 |
| DW | 9 |
| Total | 40 |

### 9/16

#### Restriction Enzyme Digestion

Nambu

| sample | 1203(RFP) | 1204(GFP) |
| --- | --- | --- |
| volume(ul) | 2 | 1 |
| 10×H buffer(10xH) | 2 | 2 |
| EcoR1 | 1 | 1 |
| Not1 | 1 | 1 |
| DW | 14 | 15 |
| Total | 20 | 20 |

### 9/17

#### OE-PCR

Shimazoe

| Material | Volume(ul) |
| --- | --- |
| 2× Gflex PCR Buffer | 30 |
| 10uM Z017℗ | 1.2 |
| 10uM Z018℗ | 1.2 |
| template⓪ | 6 |
| template① | 6 |
| template② | 6 |
| Tks Gflex DNA Polymerase | 1.2 |
| DW | 8.4 |
| Total | 60 |

![0917OE-PCR ](https://static.igem.org/mediawiki/2018/d/de/T--Kyoto--0917OE-PCR.png)

ZrGPD1
94℃ 1min
98℃\* 10s
55℃\* 15s
68℃\* 1m30s
68℃ 2min
4℃ ∞
(\*× 30 cycle)

### 9/17

#### PCR

Shimazoe

| material | volume (ul) |
| --- | --- |
| 5× Q5 Reaction Buffer | 5 |
| 10uM dNTPs | 0.5 |
| Q5 DNA Polymerase | 0.25 |
| DW | 15.75 |
| 10uM primer① | 1.25 |
| 10uM primer② | 1.25 |
| template | 1 |
| Total | 25 |

pro/term/Sse
Z017,Z039/Z040,Z018/Z041,Z042 (primer)
P079/P079/317 (template)
98℃ 30s
98℃\* 10s
55℃\* 30s
72℃\* 1min
72℃ 2min
4℃ ∞
(\*× 30 cycle)

### 9/17

#### PCR

Shimazoe

| Material | Volume(ul) |
| --- | --- |
| 2× Gflex PCR Buffer | 12.5 |
| 10uM primer① | 1 |
| 10uM primer② | 1 |
| template | 1 |
| Tks Gflex DNA Polymerase | 1 |
| DW | 8.5 |
| Total | 25 |

![0917PCR2](https://static.igem.org/mediawiki/2018/d/d9/T--Kyoto--0917PCR2.png)

pro,term,Sse ORF
94℃ 1min
98℃\* 10s
55℃\* 15s
68℃\* 1m30s
68℃ 2min
4℃ ∞
(\*× 30 cycle)

### 9/17

#### Transformation

Nambu

|  | volume(ul) | Competent Cells(ul) | Total(ul) |
| --- | --- | --- | --- |
| DNA J04450 | 1 | 10 | 11 |

|  | volume(ul) | Competent Cells(ul) | Total(ul) |
| --- | --- | --- | --- |
| DNA | 0.5 | 10 | 10.5 |

DNA J04450…2017 Kit Plate 1 24A

### 9/18

#### OE-PCR

Shimazoe

| Material | Volume(ul) |
| --- | --- |
| 2× Gflex PCR Buffer | 30 |
| 10uM Z017℗ | 1.2 |
| 10uM Z018℗ | 1.2 |
| template⓪ | 3 |
| template① | 3 |
| template② | 3 |
| Tks Gflex DNA Polymerase | 1.2 |
| DW | 17.4 |
| Total | 60 |

ZrGPD1,SseNHX1
94℃ 1min
98℃\* 10s
55℃\* 15s
68℃\* 1m30s
68℃ 2min
4℃ ∞
(\*× 30 cycle)

### 9/18

#### Ligation

Tong

| Material | Volume(ul) |
| --- | --- |
| Ligation high | 2 |
| pRS425 | 1 |
| AVP1(3) | 1 |
| Total | 4 |

| Material | Volume(ul) |
| --- | --- |
| Ligation high | 3.5 |
| pRS425 | 0.5 |
| AVP1(3) | 3 |
| Total | 7 |

### 9/18

#### Inverse PCR

Shimazoe

| material | volume (ul) |
| --- | --- |
| 2× Prime Star Max | 25 |
| 10uM Z039 | 1 |
| 10uM Z040 | 1 |
| template(938) | 0.5 |
| DW | 22.5 |
| Total | 50 |

98℃\* 10s
55℃\* 5s
72℃\* 40s
(\*× 30 cycle)

### 9/18

#### SLiCE

Shimazoe

| Material | Volume(ul) |
| --- | --- |
| vector | 0.5 |
| insert | 1 |
| 10× SLiCE Buffer | 1 |
| SLiCE | 1 |
| DW | 6.5 |
| Total | 10 |

SseNHX1/GPD1

| Material | Volume(ul) |
| --- | --- |
| vector | 0.5 |
| insert① | 1 |
| insert② | 1 |
| 10× SLiCE Buffer | 1 |
| SLiCE | 1 |
| DW | 5.5 |
| Total | 10 |

![0918SLiCE ](https://static.igem.org/mediawiki/2018/4/46/T--Kyoto--0918SLiCE.png)

Fgβ/SdrG

### 9/18

#### Transformation

Shimazoe

| Ligation products | volume(ul) | Competent Cells/(ul) | Total(ul) |
| --- | --- | --- | --- |
| SseNHX1/GPD1 | 10 | 50 | 60 |
| Fgβ/SdrG | 10 | 50 | 60 |

### 9/19

#### OE-PCR

Tong

| Material | Volume(ul) |
| --- | --- |
| 2× Gflex buffer | 12.5 |
| Z044 | 0.5 |
| Z047 | 0.5 |
| SdrG or Fgβ | 1 |
| Stalk | 1 |
| Gflex DNA polymerase | 0.5 |
| DW | 9 |
| Total | 25 |

### 9/19

#### Colony PCR

Tong

| Material | Volume(ul) |
| --- | --- |
| 2× Emerald amp MAX PCR Master Mix | 82.5 |
| 10uM m13f | 1.65 |
| 10uM m13r | 1.65 |
| DW | 79.2 |
| Total | 165 |

98℃ 30s
98℃\* 10s
60℃\* 30s
72℃\* 4min
72℃ 4min
4℃ ∞
(\*× 30cycle)

### 9/19

#### Inverse PCR

Tong

| material | volume (ul) |
| --- | --- |
| 2× Prime Star Max | 25 |
| 10uM Z039 | 1 |
| 10uM Z040 | 1 |
| template(938) | 0.6 |
| DW | 22.5 |
| Total | 50 |

98℃\* 10s
55℃\* 5s
72℃\* 40s
(\*× 30cycle)

### 9/19

#### PCR

Shimazoe

| Material | Volume(ul) |
| --- | --- |
| 2× Gflex PCR Buffer | 25 |
| 10uM primer① | 1 |
| 10uM primer② | 1 |
| template | 1 |
| Tks Gflex DNA Polymerase | 1 |
| DW | 21 |
| Total | 50 |

template(SseNHX1/ZrGPD1/Stalk)
primer(Z041,Z042/Z041,Z042/Z047,Z074)

94℃ 1min
98℃\* 10s
55℃\* 15s
68℃\* 1m30s
68℃ 2min
4℃ ∞
(\*× 30 cycle)

### 9/19

#### Colony PCR

Tong

| Material | Volume(ul) |
| --- | --- |
| 2× Emerald amp MAX PCR Master Mix | 17.5 |
| 10uM Z047 | 0.35 |
| 10uM Z074 | 0.35 |
| DW | 16.8 |
| Total | 35 |

98℃ 30s
98℃\* 10s
60℃\* 30s
72℃\* 3min
72℃ 2min
4℃ ∞
(\*× 30cycle)

### 9/19

#### Restriction Enzyme Digestion

Tong

| material | volume (ul) |
| --- | --- |
| PCR products | 42 |
| Dpn1 | 2 |
| Total | 44 |

![0919Dpn1](https://static.igem.org/mediawiki/2018/d/db/T--Kyoto--0919Dpn1.png)

37℃/1h

### 9/20

#### Inverse PCR

Tong

| material | volume (ul) |
| --- | --- |
| 2× Prime Star Max | 25 |
| 10uM Z039 | 1 |
| 10uM Z040 | 1 |
| template(938) 1/200 | 0.6 |
| DW | 22.4 |
| Total | 50 |

![0920InversePCR ](https://static.igem.org/mediawiki/2018/9/96/T--Kyoto--0920InversePCR.png)

98℃\* 10s
55℃\* 5s
72℃\* 40s
(\*× 30cycle)

### 9/20

#### Restriction Enzyme Digestion

Tong

Dpn1 digestion

### 9/20

#### PCR

Tong

| material | volume (ul) |
| --- | --- |
| 2× Prime Star Max | 25 |
| 10uM Z047 | 1 |
| 10uM Z074 | 1 |
| template①/② | 1/1 |
| DW | 22 |
| Total | 50 |

98℃\* 10s
55℃\* 15s
72℃\* 10s
4℃ ∞
(\*× 30cycle)

### 9/20

#### Ligation

Shimazoe

| Material | Volume(ul) |
| --- | --- |
| Ligation high | 2 |
| vector | 1 |
| insert | 1 |
| Total | 4 |

Mangrin : pRS426
AVP1 : pRS425

### 9/20

#### Nanodrop

Nambu

| 1 | 2 | 3 | 4 |
| --- | --- | --- | --- |
| 222.6 ng/ul | 181.2 ng/ul | 232.1 ng/ul | 128.7 ng/ul |

### 9/20

#### Restriction Enzyme Digestion

Nambu

| sample | 1 | 2 | 3 | 4 |
| --- | --- | --- | --- | --- |
| volume(ul) | 6 | 6 | 6 | 9 |
| 10×H buffer(10xH) | 3 | 3 | 3 | 3 |
| EcoR1 | 1.5 | 1.5 | 1.5 | 1.5 |
| Pst1 | 1.5 | 1.5 | 1.5 | 1.5 |
| DW | 18 | 18 | 18 | 15 |
| Total | 30 | 30 | 30 | 30 |

![0920restriction](https://static.igem.org/mediawiki/2018/4/42/T--Kyoto--0920restriction.jpg)

put in incubator at 37℃

### 9/21

#### Qubit^tm dsDNA HS Assay Kit

Shimazoe

| Material | Volume(ul) |
| --- | --- |
| dsDNA buffer | 200 |
| dsDNA reagent | 1 |
| dsDNA solution | 1 |
| Total | 202 |

### 9/21

#### Ligation

Shimazoe

| Material | Volume(ul) |
| --- | --- |
| Ligation high | 6 |
| vector 426/316 | 2/2 |
| ZrFPS1-1st | 2 |
| ZrFPS1-2nd | 2 |
| Total | 12 |

### 9/21

#### SLiCE

Shimazoe

| Material | Volume(ul) |
| --- | --- |
| DW | 5.5 |
| pIEG-SSS 1/20 | 1 |
| FgβF3 1/10 | 1 |
| Stalk | 0.5 |
| 10× SLiCE Buffer | 1 |
| SLiCE | 1 |
| Total | 10 |

| Material | Volume(ul) |
| --- | --- |
| DW | 6.1 |
| pIEG-SSS 1/20 | 1 |
| SdrG | 0.4 |
| Stalk | 0.5 |
| 10× SLiCE Buffer | 1 |
| SLiCE | 1 |
| Total | 10 |

### 9/21

#### PCR

Shimazoe

| material | volume (ul) |
| --- | --- |
| 2× Prime Star Max | 12.5 |
| 10uM Z041 | 1 |
| 10uM Z042 | 1 |
| template 1/200 | 1 |
| DW | 9.5 |
| Total | 25 |

template : 317(SseNHX1), 506(ZrGPD1)
98℃\* 10s
55℃\* 5s
72℃\* 10s
(\*× 30cycle)

### 9/21

#### PCR

Shimazoe

| material | volume (ul) |
| --- | --- |
| 2× Prime Star Max | 25 |
| 10uM Z041 | 2 |
| 10uM Z042 | 2 |
| template 1/200 | 1 |
| DW | 20 |
| Total | 50 |

template : 317(SseNHX1), 506(ZrGPD1)
98℃\* 10s
55℃\* 5s
72℃\* 10s
(\*× 30cycle)

### 9/21

#### Transformation

Shimazoe

| Material | Volume(ul) |
| --- | --- |
| SLiCE products(①～④) | 4 |
| competent cell | 50 |
| Total | 54 |

template(SseNHX1/ZrGPD1/Stalk)
on ice 30min
42℃45s
on ice 2min
SOC 500ul
37℃ 40min

### 9/21

#### Transformation

Shimazoe

| Material | Volume(ul) |
| --- | --- |
| In-Fusion products(⑤，⑥) | 4 |
| competent cell | 50 |
| Total | 54 |

### 9/21

#### Transformation

Tong

|  | yeast | plasmid |
| --- | --- | --- |
| ⑦ | WT(547) | ・AVP1(YCplac111) ・AtNHXS1(pRS423)(938) ・empty(pRS316) |
| ⑧ | WT | ・AVP1(YCplac111) ・AtNHXS1(pRS313)(938) ・empty(pRS316) |
| ⑨ | WT | ・AVP1(YCplac111) ・empty(pRS313) ・empty(pRS316) |
| ⑩ | WT | ・AtNHXS1(pRS423)(938) ・Mangrin(pRS316) |
| ⑪ | WT | ・empty(YCplac111) ・AtNHXS1(pRS313) ・empty(pRS316) |
| ⑫ | ΔENA1(559) | ・AVP1(YCplac111) ・AtNHXS1(pRS423) ・empty(pRS316) |

competent cell : 10ul
plasmid : 0.5ul
Solution3 : 100ul
vortex 30s
37℃/35min

### 9/22

#### SLiCE

Shimazoe

| Material | Volume(ul) |
| --- | --- |
| DW | 5.5 |
| pIEG-SSS 1/20 | 1 |
| FgβF3 1/10 | 1 |
| Stalk | 0.5 |
| 10× SLiCE Buffer | 1 |
| SLiCE | 1 |
| Total | 10 |

| Material | Volume(ul) |
| --- | --- |
| DW | 5 |
| pIEG-SSS 1/20 | 1 |
| SdrG 1/4 | 1 |
| Stalk 1/2 | 1 |
| 10× SLiCE Buffer | 1 |
| SLiCE | 1 |
| Total | 10 |

### 9/22

#### In Fusion

Shimazoe

| Material | Volume(ul) |
| --- | --- |
| DW | 5 |
| pIEG-SSS 1/20 | 1 |
| FgβF3 1/10 | 1 |
| Stalk | 0.5 |
| 5× In-Fusion HD Enzyme Premix | 2 |
| Total | 10 |

| Material | Volume(ul) |
| --- | --- |
| DW | 5 |
| pIEG-SSS 1/20 | 1 |
| SdrG 1/4 | 1 |
| Stalk 1/2 | 1 |
| 5× In-Fusion HD Enzyme Premix | 2 |
| Total | 10 |

### 9/22

#### Colony PCR

Shimazoe

| Material | Volume(ul) |
| --- | --- |
| 2× Emerald amp MAX PCR Master Mix | 25 |
| 10uM m13f | 0.5 |
| 10uM m13r | 0.5 |
| DW | 24 |
| Total | 50 |

| Material | Volume(ul) |
| --- | --- |
| 2× Emerald amp MAX PCR Master Mix | 15 |
| 10uM Z044 | 0.3 |
| 10uM Z047 | 0.3 |
| DW | 14.4 |
| Total | 30 |

![0922ColonyPCR ](https://static.igem.org/mediawiki/2018/a/aa/T--Kyoto--0922ColonyPCR.png)

98℃ 30s
98℃\* 10s
60℃\* 30s
72℃\* 3min
72℃ 2min
4℃ ∞
(\*× 30cycle)

### 9/22

#### PCR

Tong

| Material | Volume |
| --- | --- |
| 5× Q5 Reaction Buffer | 8 |
| 10uM dNTPs | 3.2 |
| 10uM Z005 | 1 |
| 10uM Z028 | 1 |
| template | 1 |
| enzyme | 0.4 |
| DW | 25.4 |
| Total | 40 |

Cassette for ΔENA1,2,5
98℃\* 10s
55℃\* 15s
72℃\* 2min
(\*× 30 cycle)

### 9/22

#### Spot

Tong

| Number | Material< /th> |
| --- | --- |
| ① | 547 WT |
| ② | 554 ΔNHA1 |
| ③ | 555 G19 |
| ④ | 557 ΔNHA1,ΔENA1 |
| ⑤ | 559 ΔENA1 |
| ⑥ | ΔENA1.2.5, ΔNHA1 |
| ⑦ | 564 Z.roxii |

Cassette for ΔENA1,2,5
98℃\* 10s
55℃\* 15s
72℃\* 2min
(\*× 30 cycle)

### 9/22

#### Restriction Enzyme Digestion

Nambu

| sample | 1087(AtNHXS1) | 910(AtHKT1) |
| --- | --- | --- |
| volume(ul) | 20 | 20 |
| 10×H buffer(10xH) | 4 | 4 |
| EcoR1 | 1 | 1 |
| Not1 | 1 | 1 |
| DW | 14 | 14 |
| Total | 40 | 40 |

enzyme inactivation 60℃ 15min

### 9/22

#### Restriction Enzyme Digestion

Nambu

| sample | pSB1C3 |
| --- | --- |
| volume(ul) | 20 |
| 10×H buffer(10xH) | 4 |
| EcoR1 | 2 |
| Not1 | 2 |
| Dpu1 | 2 |
| DW | 10 |
| Total | 40 |

enzyme inactivation 80℃ 20min

### 9/22

#### Transformation

Nambu

|  | volume(ul) | Competent Cells(ul) | Total(ul) |
| --- | --- | --- | --- |
| Mangrin 404 | 2 | 10 | 12 |

### 9/23

#### Colony PCR

Shimazoe

| Material | Volume(ul) |
| --- | --- |
| 2× Emerald amp MAX PCR Master Mix | 35 |
| 10uM m13f | 0.7 |
| 10uM m13r | 0.7 |
| DW | 33.6 |
| Total | 70 |

| Material | Volume(ul) |
| --- | --- |
| 2× Emerald amp MAX PCR Master Mix | 85 |
| 10uM Z044 | 1.7 |
| 10uM Z047 | 1.7 |
| DW | 81.6 |
| Total | 170 |

![0923ColonyPCR ](https://static.igem.org/mediawiki/2018/1/17/T--Kyoto--0923ColonyPCR.png)

98℃ 30s
98℃\* 10s
60℃\* 30s
72℃\* 3min
72℃ 2min
4℃ ∞
(\*× 30cycle)

### 9/23

#### PCR

Tong

| Material | Volume |
| --- | --- |
| 5× Q5 Reaction Buffer | 8 |
| 10uM dNTPs | 3.2 |
| 10uM Z003 | 1 |
| 10uM Z004 | 1 |
| Yeast Genome | 1 |
| enzyme | 0.4 |
| DW | 25.4 |
| Total | 40 |

Cassette for ΔENA1,2,5
98℃\* 10s
55℃\* 15s
72℃\* 4min
(\*× 35 cycle)

### 9/23

#### Restriction Enzyme Digestion

Tong

| material | volume (ul) |
| --- | --- |
| Plasmid(404,716,910,3) | 10 |
| 10×H buffer | 2 |
| BSA | 2 |
| Triton X-100 | 2 |
| Not1 | 1 |
| DW | 3 |
| Total | 20 |

404,716: Mangrin(pRS716)
910: AtHKT1(pRS426)
3: AVP1(pSB1C3)

### 9/23

#### Colony PCR

Nambu

| Material | Volume(ul) |
| --- | --- |
| 2× Emerald amp MAX PCR Master Mix | 18.75 |
| 10uM m13f | 0.375 |
| 10uM m13r | 0.375 |
| DW | 18 |
| Total | 37.5 |

![0923colonyPCR101](https://static.igem.org/mediawiki/2018/8/8b/T--Kyoto--0923colonyPCR101.jpg)

98℃ 30s
98℃\* 10s
60℃\* 30s
72℃\* 3min
72℃ 2min
4℃ ∞
(\*× 30cycle)

### 9/24

#### Colony PCR

Shimazoe

| Material | Volume(ul) |
| --- | --- |
| 2× Emerald amp MAX PCR Master Mix | 120 |
| 10uM Z044 | 2.4 |
| 10uM Z047 | 2.4 |
| DW | 115.2 |
| Total | 240 |

![0924ColonyPCR ](https://static.igem.org/mediawiki/2018/4/4e/T--Kyoto--0924ColonyPCR.png)

### 9/24

#### Ligation

Shimazoe

| Material | Volume(ul) |
| --- | --- |
| Ligation high | 4 |
| vector 426/316 | 1/1 |
| McHKT2 | 3 |
| Total | 8 |

### 9/24

#### Ligation

Nambu

| number | insert | volume(ul) | vector | volume(ul) | Ligation High(ul) | Total(ul) |
| --- | --- | --- | --- | --- | --- | --- |
| 1.2.1 | 910(heat) | 1.5 | 1.2 | 0.5 | 2 | 4 |
| 1.2.2 | 910(column) | 1.5 | 1.2 | 0.5 | 2 | 4 |
| 1.2.3 | 1087(heat) | 1.5 | 1.2 | 0.5 | 2 | 4 |
| 1.2.4 | 1087(column) | 1.5 | 1.2 | 0.5 | 2 | 4 |
| PL1 | 910(heat) | 1.5 | pSB1C3 | 0.5 | 2 | 4 |
| PL2 | 910(column) | 1.5 | pSB1C3 | 0.5 | 2 | 4 |
| PL3 | 1087(heat) | 1.5 | pSB1C3 | 0.5 | 2 | 4 |
| PL4 | 1087(column) | 1.5 | pSB1C3 | 0.5 | 2 | 4 |

### 9/24

#### Transformation

Nambu

|  | volume(ul) | Competent Cells(ul) | Total(ul) |
| --- | --- | --- | --- |
| DNA | 2 | 10 | 12 |

DNA(1.2.1/1.2.2/1.2.3/1.2.4/PL1/PL2/PL3/PL4)

### 9/25

#### Ligation

Shimazoe

| Material | Volume(ul) |
| --- | --- |
| Ligation high | 2 |
| insert | 1 |
| vector | 1 |
| Total | 4 |

|  | ① | ② | ③ | ④ | ⑤ | ⑥ | ⑦ | ⑧ | ⑨ |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| insert | 404 | 716 | 1304 | 1306 | 1310 | 1118 | 1301 | 1302 | 1303 |
| vector | 426 | 426 | 313 | 313 | 313 | 425 | 313 | 313 | 313 |

### 9/25

#### Colony PCR

Nambu

| Material | Volume(ul) |
| --- | --- |
| 2× Emerald amp MAX PCR Master Mix | 75 |
| 10uM m13f | 1.5 |
| 10uM m13r | 1.5 |
| DW | 72 |
| Total | 150 |

AtNHXS1(pSB1C3)/AtHKT1(pRS426)/McHKT2(pRS426,pRS316)

98℃ 30s
98℃\* 10s
60℃\* 30s
72℃\* 3min
72℃ 2min
4℃ ∞
(\*× 30cycle)

### 9/25

#### Restriction Enzyme Digestion

Nambu

| sample | DNA(pSB1C3 Linear) |
| --- | --- |
| volume(ul) | 5 |
| 10×M buffer | 2 |
| Xba1 | 1 |
| Pst1 | 1 |
| Dpn1 | 1 |
| DW | 10 |
| Total | 20 |

enzyme inactivation 80℃ 20min

### 9/25

#### Restriction Enzyme Digestion

Nambu

| sample | 1301 | 1302 | 1303 | 1304 | 1306 | 1310 |
| --- | --- | --- | --- | --- | --- | --- |
| volume(ul) | 10 | 10 | 10 | 10 | 10 | 2 |
| 10×H buffer | 2 | 2 | 2 | 2 | 2 | 2 |
| EcoR1 | 1 | 1 | 1 | 1 | 1 | 1 |
| Pst1 | 1 | 1 | 1 | 1 | 1 | 1 |
| DW | 6 | 6 | 6 | 6 | 6 | 14 |
| Total | 20 | 20 | 20 | 20 | 20 | 20 |

enzyme inactivation 60℃ 15min

### 9/26

#### Colony PCR

Shimazoe

| Material | Volume |
| --- | --- |
| 5× Q5 Reaction Buffer | 36 |
| 10uM dNTPs | 3.6 |
| 10uM m13r | 9 |
| 10uM m13f | 9 |
| Q5 DNA Polymerase | 1.8 |
| DW | 120.5 |
| Total | 180 |

98℃ 30s
98℃\* 10s
55℃\* 30s
72℃\* 1m30s
72℃ 2min
4℃ ∞
(\*× 30cycle)

### 9/26

#### PCR

Shimazoe

| Material | Volume(ul) |
| --- | --- |
| 2× Gflex PCR Buffer | 12.5 |
| 10uM primer① | 0.5 |
| 10uM primer② | 0.5 |
| template | 0.5 |
| Tks Gflex DNA Polymerase | 0.5 |
| DW | 10.5 |
| Total | 25 |

|  | ZrFPS1-1st | ZrFPS1-2nd |
| --- | --- | --- |
| primer | Z041, Z043 | Z077, Z042 |
| template | 927 | 718 |

![0926PCR ](https://static.igem.org/mediawiki/2018/f/fd/T--Kyoto--0926PCR.jpg)

94℃ 1min
98℃\* 10s
55℃\* 15s
68℃\* 1min
68℃ 2min
4℃ ∞
(\*× 30 cycle)

### 9/26

#### Restriction Enzyme Digestion

Nambu

| sample | Plasmid(AVP1.pSB1C3) |
| --- | --- |
| volume(ul) | 20 |
| 10×H buffer | 4 |
| EcoR1 | 1 |
| Pst1 | 1 |
| CIAP | 1 |
| DW | 13 |
| Total | 40 |

### 9/26

#### Restriction Enzyme Digestion

Nambu

| sample | λDNA |
| --- | --- |
| volume(ul) | 71 |
| 10×H buffer | 50 |
| Sty1 | 3 |
| DW | 376 |
| Total | 500 |

### 9/27

#### Transformation(yeast)

Tong

|  | yeast | plasmid |
| --- | --- | --- |
| 34 | WT | ・empty(pRS425) ・empty(pRS316)  ・Mangrin(pRS316) |
| 35 | WT | ・AVP1(YCplac111) ・SseNHX1(pRS423) ・empty(pRS316) |
| 36 | WT | ・AVP1(YCplac111) ・AtNHXS1 (pRS423)  ・Mangrin(pRS316) |
| 37 | WT | ・AVP1(YCplac111) ・AtNHXS1(pRS313) ・Mangrin(pRS316) |
| 38 | WT | ・AVP1(YCplac111) ・(1301) SseNHX1(pRS423) ・Mangrin(pRS316) |
| 39 | WT | ・empty(pRS425)  ・(1432)SdrG(pIEG-SSS:HIS) ・(1204)GFP(pRS316) |
| 40 | WT | ・empty(pRS425)  ・(1444)SdrG ・(1204)GFP |
| 41 | WT | ・empty(pRS425)  ・(1448)SdrG ・(1204)GFP(pRS316) |

### 9/28

#### OD measure

Tong

| number | OD |  | number | OD |
| --- | --- | --- | --- | --- |
| 7 | 4.8 |  | 20 | 6.6 |
| 8 | 5.8 |  | 21 | 6.2 |
| 9 | 6.4 |  | 22 | 5.9 |
| 10 | 6.8 |  | 23 | × |
| 11 | 6.6 |  | 24 | × |
| 12 | 6.0 |  | 25 | × |
| 13 | 6.6 |  | 26 | × |
| 14 | 6.4 |  | 27 | × |
| 15 | × |  | 28 | 4.1 |
| 16 | 6.6 |  | 29 | 4.7 |
| 17 | 6.9 |  | 30 | 3.8 |
| 18 | 6.6 |  | 31 | /RFP |
| 19 | 6.4 |  | 32 | /GFP |

### 9/28

#### PCR

Shimazoe

| material | volume (ul) |
| --- | --- |
| 2× Prime Star Max | 12.5 |
| 10uM primer① | 1 |
| 10uM primer② | 1 |
| template | 0.5 |
| DW | 10 |
| Total | 25 |

|  | Fgβ | SdrG | stalk for Fgβ | stalk for SdrG |
| --- | --- | --- | --- | --- |
| template | 342, 1/300 | 752, 1/100 | gBlock | gBlock |
| primer | Z086 Z084 | Z086 Z045 | Z046 Z087 | Z074 Z087 |

### 9/28

#### Inverse PCR

Shimazoe

| material | volume (ul) |
| --- | --- |
| 2× Prime Star Max | 25 |
| 10uM Z085 | 2 |
| 10uM Z088 | 2 |
| template(pIEG-SSS) | 1 |
| DW | 20 |
| Total | 50 |

98℃\* 10s
55℃\* 5s
72℃\* 40s
(\*× 30 cycle)

### 9/28

#### OE-PCR

Shimazoe

| Material | Volume(ul) |
| --- | --- |
| 2× Gflex PCR Buffer | 25 |
| 10uM Z041 | 1 |
| 10uM Z042 | 1 |
| ZrFPS1-1st | 1 |
| ZrFPS1-2nd | 1 |
| Tks Gflex DNA Polymerase | 1 |
| DW | 20 |
| Total | 50 |

![0928OE-PCR](https://static.igem.org/mediawiki/2018/6/60/T--Kyoto--0928OE-PCR.jpg)

94℃ 1min
98℃\* 10s
55℃\* 15s
68℃\* 2min
68℃ 2min
4℃ ∞
(\*× 30 cycle)

### 9/28

#### PCR

Shimazoe

| Material | Volume(ul) |
| --- | --- |
| 2× Gflex PCR Buffer | 25 |
| 10uM primer① | 1 |
| 10uM primer② | 1 |
| template | 1 |
| Tks Gflex DNA Polymerase | 1 |
| DW | 21 |
| Total | 50 |

![0928PCR2](https://static.igem.org/mediawiki/2018/d/dc/T--Kyoto--0928PCR2.jpg)

94℃ 1min
98℃\* 10s
55℃\* 15s
68℃\* 2min
68℃ 2min
4℃ ∞
(\*× 30 cycle)

### 9/28

#### Restriction Enzyme Digestion

Nambu

| sample | Mangrin | AVP1・pSB1C3 |
| --- | --- | --- |
| volume(ul) | 20 | 20 |
| 10×H buffer | 4 | 4 |
| EcoR1 | 1 | 1 |
| Pst1 | 1 | 1 |
| CIAP | × | 1 |
| DW | 14 | 13 |
| Total | 40 | 40 |

![0928restriction1](https://static.igem.org/mediawiki/2018/9/9b/T--Kyoto--0928restriction1.jpg)

### 9/28

#### PCR

Nambu

|  | Stalk | FgβF3 | SdrG | AtHKT1(910) |
| --- | --- | --- | --- | --- |
| 2× Prime Star Max (ul) | 20 | 20 | 20 | 20 |
| F.primer | 1(Z082) | 1(Z080) | 1(Z078) | 1(Z094) |
| R.primer | 1(Z083) | 1(Z081) | 1(Z079) | 1(Z095) |
| template | 1(gBlock) | × | 0.5(752,1/100) | 1(910,1/100) |
| DW | 17 | 18 | 17.5 | 17 |
| Total | 40 | 40 | 40 | 40 |

![0928PCR101](https://static.igem.org/mediawiki/2018/b/bf/T--Kyoto--0928PCR101.jpg)

### 9/28

#### Restriction Enzyme Digestion

Nambu

| sample | 910-1 | 910-2 |
| --- | --- | --- |
| volume(ul) | 20 | 20 |
| 10×M buffer | 4 | 4 |
| Xba1 | 1 | 1 |
| Pst1 | 1 | 1 |
| DW | 14 | 14 |
| Total | 40 | 40 |

### 9/29

#### Restriction Enzyme Digestion

Tong

| material | volume (ul) |
| --- | --- |
| plasmid(716/1301/1304/938) | 10/15/15/15 |
| 10×H buffer | 4 |
| EcoR1 | 0.5 |
| Pst1 | 0.5 |
| DW | 25/20/20/20 |
| Total | 40 |

insert

| material | volume (ul) |
| --- | --- |
| plasmid(6) | 20 |
| SAP | 1 |
| 10×H buffer | 8 |
| EcoR1 | 1 |
| Pst1 | 1 |
| DW | 49 |
| Total | 80 |

Backbone

| material | volume (ul) |
| --- | --- |
| plasmid(910) | 15 |
| 10×M buffer | 4 |
| Xba1 | 0.5 |
| Pst1 | 0.5 |
| DW | 20 |
| Total | 40 |

insert

| material | volume (ul) |
| --- | --- |
| plasmid(6) | 10 |
| SAP | 0.5 |
| 10×M buffer | 4 |
| Xba1 | 0.5 |
| Pst1 | 0.5 |
| DW | 24.5 |
| Total | 40 |

Backbone

### 9/29

#### PCR

Tong

| Material | Volume |
| --- | --- |
| 5× Q5 Reaction Buffer | 8 |
| 10uM dNTPs | 3.2 |
| 10uM primer① | 1 |
| 10uM primer② | 1 |
| template | 1 |
| enzyme | 0.4 |
| DW | 25.4 |
| Total | 40 |

|  | SdrG | Fgβ | Stalk for Fgβ | Stalk　for SdrG |
| --- | --- | --- | --- | --- |
| template | 752, 1/100 | 342, 1/300 | gBlock | gBlock |
| primer | Z045, Z086 | Z084, Z086 | Z046, Z087 | Z074, Z087 |

### 9/29

#### OD measure

Tong

| number | OD |  | number | OD |  | number | OD |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 7-A | 1.13 |  | 11-A | 1.59 |  | 16-A | 0.54 |
| 7-B | 1.12 |  | 11-B | 1.57 |  | 16-B | 0.58 |
| 8-A | 1.24 |  | 12-A | 0.81 |  | 17-A | 1.49 |
| 8-B | 1.19 |  | 12-B | 0.80 |  | 17-B | 1.55 |
| 9-A | 1.11 |  | 13-A | 1.48 |  | 20-A | 0.87 |
| 9-B | 1.16 |  | 13-B | 1.44 |  | 20-B | 0.72 |
| 10-A | 1.59 |  | 14-A | 1.53 |  | 28-A | 1.13 |
| 10-B | 1.61 |  | 14-B | 1.52 |  | 28-B | 1.15 |

### 9/29

#### PCR

Shimazoe

| Material | Volume(ul) |
| --- | --- |
| 2× Gflex PCR Buffer | 20 |
| 10uM primer① | 1 |
| 10uM primer② | 1 |
| template | 1 |
| Tks Gflex DNA Polymerase | 1 |
| DW | 16 |
| Total | 40 |

|  | Fgβ | SdrG |
| --- | --- | --- |
| template | 342 | 752 |
| primer | Z089, Z090 | Z091, Z092 |

94℃ 1min
98℃\* 10s
55℃\* 15s
68℃\* 2min
68℃ 2min
4℃ ∞
(\*× 30 cycle)

### 9/29

#### PCR

Shimazoe

| material | volume (ul) |
| --- | --- |
| 2× Prime Star Max | 12.5 |
| 10uM primer① | 1 |
| 10uM primer② | 1 |
| template | 0.5 |
| DW | 10 |
| Total | 25 |

|  | Stalk  for Fgβ | Stalk   for SdrG | Fgβ  for OE-PCR | SdrG  for OE-PCR |
| --- | --- | --- | --- | --- |
| template | gBlock | gBlock | 342 | gBlock |
| primer | Z086 Z084 | Z074 Z087 | Z086 Z045 | Z093 Z087 |

![0929PCR1](https://static.igem.org/mediawiki/2018/9/9a/T--Kyoto--0929PCR1.jpg)

98℃\* 10s
55℃\* 15s
72℃\* 10s
(\*× 30 cycle)

### 9/29

#### SLiCE

Shimazoe

| Material | Volume(ul) |
| --- | --- |
| DW | 7 |
| pRS423 | 0.5 |
| ZrFPS1-OE | 0.5 |
| 10× SLiCE Buffer | 1 |
| SLiCE | 1 |
| Total | 10 |

### 9/29

#### Restriction Enzyme Digestion

Nambu

| sample | SdrG | Fgβ |
| --- | --- | --- |
| volume(ul) | 34 | 34 |
| 10×H buffer | 4 | 4 |
| EcoR1 | 1 | 1 |
| Pst1 | 1 | 1 |
| Total | 40 | 40 |

37℃ 3h

### 9/29

#### PCR

Nambu

| Material | Volume(ul) |
| --- | --- |
| 2× Prime Star Max (ul) | 12.5 |
| F.primer (Z082) | 1 |
| R.primer (Z083) | 1 |
| template (gBlock) | 1 |
| DW | 9.5 |
| Total | 25 |

![0929PCR101](https://static.igem.org/mediawiki/2018/9/9a/T--Kyoto--0929PCR101.jpg)

98℃\* 10s
55℃\* 5s
72℃\* 11s
(\*× 30cycle)

### 9/29

#### Ligation

Nambu

| insert | volume(ul) | vector | volume(ul) | Ligation High(ul) | Total(ul) |
| --- | --- | --- | --- | --- | --- |
| Mangrin | 2 | AVP1・pSB1C3(E,P cut) | 1 | 2.5 | 5.5 |
| ZrGPD1(1304) | 2 | AVP1・pSB1C3(E,P cut) | 1 | 2.5 | 5.5 |
| SseNHX1(1302) | 2 | AVP1・pSB1C3(E,P cut) | 1 | 2.5 | 5.5 |
| Fgβ | 2 | AVP1・pSB1C3(E,P cut) | 1 | 2.5 | 5.5 |
| SdrG | 2 | AVP1・pSB1C3(E,P cut) | 1 | 2.5 | 5.5 |
| AtNHXS1(1087) | 2 | AVP1・pSB1C3(E,P cut) | 1 | 2.5 | 5.5 |
| Control(MilliQ) | 2 | AVP1・pSB1C3(E,P cut) | 1 | 2.5 | 5.5 |

### 9/29

#### Transformation

Nambu

|  | volume(ul) | Competent Cells(ul) | Total(ul) |
| --- | --- | --- | --- |
| Mangrin | 2 | 10 | 12 |
| ZrGPD1(1304) | 2 | 10 | 12 |
| SseNHX1(1302) | 2 | 10 | 12 |
| Fgβ | 2 | 10 | 12 |
| SdrG | 2 | 10 | 12 |
| AtNHXS1(1087) | 2 | 10 | 12 |
| Control(MilliQ) | 2 | 10 | 12 |

20℃ 40h

### 9/29

#### PCR

Tong

| Material | Volume |
| --- | --- |
| 5× Q5 Reaction Buffer | 8 |
| 10uM dNTPs | 3.2 |
| 10uM primer① | 1 |
| 10uM primer② | 1 |
| template | 1 |
| enzyme | 0.4 |
| DW | 25.4 |
| Total | 40 |

|  | Fgβ | Stalk for Fgβ | Stalk for Fgβ |
| --- | --- | --- | --- |
| template | 342, 1/300 | gBlock,1/10 | gBlock,1/10 | |
| primer | Z045, Z086 | Z046, Z087 | Z093, Z087 |

|  | SdrG | Fgβ | Stalk　for SdrG |
| --- | --- | --- | --- |
| template | 752, 1/100 | 342, 1/300 | gBlock |
| primer | Z045, Z086 | Z084, Z086 | Z074, Z087 |

### 9/29

#### Ligation

Tong

| insert | volume(ul) | vector | volume(ul) | Ligation High(ul) | Total(ul) |
| --- | --- | --- | --- | --- | --- |
| AVP1・pSB1C3(E,P cut) | 1 | 1301 | 1 | 2 | 4 |
| AVP1・pSB1C3(E,P cut) | 1 | 1304 | 1 | 2 | 4 |
| AVP1・pSB1C3(E,P cut) | 1 | 716 | 1 | 2 | 4 |
| AVP1・pSB1C3(E,P cut) | 1 | 938 | 1 | 2 | 4 |

| insert | volume(ul) | vector | volume(ul) | Ligation High(ul) | Total(ul) |
| --- | --- | --- | --- | --- | --- |
| AVP1・pSB1C3(E,P cut) | 1 | 910 | 3 | 4 | 8 |

### 9/29

#### Transformation

Tong

| number | plasmid | Yeast Type |
| --- | --- | --- |
| 42 | pRS425 pRS423 (404)Mangrin(pRS316) | WT |

### 9/30

#### PCR

Tong

| Material | Volume |
| --- | --- |
| 5× Q5 Reaction Buffer | 8 |
| 10uM dNTPs | 3.2 |
| 10uM primer① | 1 |
| 10uM primer② | 1 |
| template | 1 |
| enzyme | 0.4 |
| DW | 25.4 |
| Total | 40 |

| number | ① | ② | ③ | ④ | ⑤ | ⑥ | ⑦ |
| --- | --- | --- | --- | --- | --- | --- | --- |
| template | 342, 1/300 | Stalk gBlock 1/10 | Stalk gBlock 1/10 | 13 gBlock | 13 gBlock | 13 1/50 | 13 1/50 |
| primer | Z045, Z086 | Z046, Z087 | Z093, Z087 | Z046, Z087 | Z093, Z087 | Z046, Z087 | Z093, Z087 |

### 9/30

#### OE-PCR

Tong

| Material | Volume |
| --- | --- |
| 5× Q5 Reaction Buffer | 8 |
| 10uM dNTPs | 3.2 |
| 10uM primerF | 1 |
| 10uM primerR | 1 |
| template 1 | 1 |
| template 2 | 1 |
| enzyme | 0.4 |
| DW | 24.6 |
| Total | 40 |

| number | Template1 | Template2 | Primer |
| --- | --- | --- | --- |
| 1 | Z045, Z086 | Z074, Z087 | Z086, Z087 |
| 2 | Z084, Z086 | Z074, Z087 | Z086, Z087 |

98℃\* 10s
55℃\* 5s
72℃\* 2m30s
(\*× 30cycle)

### 9/30

#### OE-PCR

Tong

| Material | Volume |
| --- | --- |
| 5× Q5 Reaction Buffer | 8 |
| 10uM dNTPs | 3.2 |
| 10uM primer① | 1 |
| 10uM primer② | 1 |
| template 1 | 1 |
| template 2 | 1 |
| enzyme | 0.4 |
| DW | 24.4 |
| Total | 40 |

template2: ①Z046-Z087, ②Z093-Z087

### 9/30

#### Transformation

Tong

|  | volume(ul) | Competent Cells(ul) | Total(ul) |
| --- | --- | --- | --- |
| plasmid(1432/1444) | 1 | 10 | 11 |

on ice 15min/42℃ 45s/on ice 1min

### 10/1

#### Colony PCR

Shimazoe

| Material | Volume(ul) |
| --- | --- |
| 2× Emerald amp MAX PCR Master Mix | 90 |
| 10uM m13f | 1.8 |
| 10uM m13r | 1.8 |
| DW | 86.4 |
| Total | 180 |

### 10/1

#### Restriction Enzyme Digestion

Nambu

| sample | Stalk | AVP1・pSB1C3 |
| --- | --- | --- |
| volume(ul) | 19 | 10 |
| 10×H buffer | 3 | × |
| 10×M buffer | × | 2 |
| Xba1 | × | 1 |
| EcoR1 | 1 | × |
| Pst1 | 1 | 1 |
| DW | 6 | 7 |
| Total | 30 | 21 |

### 10/1

#### Colony PCR

Nambu

| Material | Volume(ul) |
| --- | --- |
| 2× Emerald amp MAX PCR Master Mix | 162.5 |
| 10uM m13f | 3.25 |
| 10uM m13r | 3.25 |
| DW | 156 |
| Total | 325 |

![1001colonyPCR101](https://static.igem.org/mediawiki/2018/4/4b/T--Kyoto--1001colonyPCR101.jpg)

![1001colonyPCR102](https://static.igem.org/mediawiki/2018/e/ea/T--Kyoto--1001colonyPCR102.jpg)

98℃ 30s
98℃\* 10s
55℃\* 30s
72℃\* 3min
72℃ 2min
15℃ ∞
(\*× 30cycle)

### 10/1

#### Colony PCR

Tong

| Material | Volume(ul) |
| --- | --- |
| 2× Emerald amp MAX PCR Master Mix | 2.5 |
| VF2 | 0.1 |
| VR | 0.1 |
| DW | 2.3 |
| Total | 5 |

| Material | Volume(ul) |
| --- | --- |
| 2× Emerald amp MAX PCR Master Mix | 2.5 |
| VF2 | 0.1 |
| Z027 | 0.1 |
| DW | 2.3 |
| Total | 5 |

### 10/1

#### PCR

Tong

| Material | Volume |
| --- | --- |
| 5× Q5 Reaction Buffer | 8 |
| 10uM dNTPs | 3.2 |
| Z086 | 1 |
| Z087 | 1 |
| template 1 | 1 |
| template 2 | 1 |
| enzyme | 0.4 |
| DW | 24.4 |
| Total | 40 |

| number | 1 | 2 | 3 | 4 |
| --- | --- | --- | --- | --- |
| template 1 | Z045, Z086 | Z045, Z086 | Z084, Z086 | Z084, Z086 |
| template 2 | Z046, Z087 | Z093, Z087 | Z046, Z087 | Z093, Z087 |

### 10/1

#### In Fusion

Shimazoe

| Material | Volume(ul) |
| --- | --- |
| DW | 5 |
| pIEG-SSS 1/3 | 1 |
| Fgβ 1/10 | 1 |
| Stalk for Fgβ1/2 | 1 |
| 5× In-Fusion HD Enzyme Premix | 2 |
| Total | 10 |

| Material | Volume(ul) |
| --- | --- |
| DW | 5 |
| pIEG-SSS 1/3 | 1 |
| SdrG 1/4 | 1 |
| Stalk for SdrG | 1 |
| 5× In-Fusion HD Enzyme Premix | 2 |
| Total | 10 |

### 10/1

#### Colony PCR

Shimazoe

| Material | Volume(ul) |
| --- | --- |
| 2× Emerald amp MAX PCR Master Mix | 160 |
| 10uM Z086 | 3.2 |
| 10uM Z087 | 3.2 |
| DW | 153.6 |
| Total | 320 |

![0922ColonyPCR ]()

98℃ 10s
98℃\* 10s
60℃\* 30s
72℃\* 3min
72℃ 3min
(\*× 30cycle)

### 10/2

#### Colony PCR

Nambu

| Material | Volume(ul) |
| --- | --- |
| 2× Emerald amp MAX PCR Master Mix | 18.75 |
| 10uM m13f | 0.375 |
| 10uM m13r | 0.375 |
| DW | 18 |
| Total | 37.5 |

![1002colonyPCR101](https://static.igem.org/mediawiki/2018/8/81/T--Kyoto--1002colonyPCR101.jpg)

98℃ 30s
98℃\* 10s
55℃\* 30s
72℃\* 1m45s
72℃ 2min
15℃ ∞
(\*× 30cycle)

### 10/2

#### Colony PCR

Nambu

| Material | Volume(ul) |
| --- | --- |
| 2× Emerald amp MAX PCR Master Mix | 150 |
| 10uM m13f | 3 |
| 10uM m13r | 3 |
| DW | 144 |
| Total | 300 |

![1002colonyPCR102](https://static.igem.org/mediawiki/2018/9/97/T--Kyoto--1002colonyPCR102.jpg)

4(Mangrin)/12(ZrGPD1)/32(AtNHXS1)/33(Fgβ)/44(SdrG)

98℃ 30s
98℃\* 10s
55℃\* 30s
72℃\* 3min
72℃ 2min
15℃ ∞
(\*× 30cycle)

### 10/3

#### Nanodrop

Nambu

| Mangrin(4) | ZrGPD1(12) | AtNHXS1(32) | Fgβ(33) | SdrG(44) | AtHKT1(910) |
| --- | --- | --- | --- | --- | --- |
| 267.7 ng/ul | 211.6 ng/ul | 243.5 ng/ul | 222.1 ng/ul | 250.8 ng/ul | 23.7 ng/ul |

### 10/2

#### Colony PCR

Tong

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| 1～14 | 716(Mangrin) | pRS316 | 1516 | 3,8,14 |
| 15～26 | 1304(ZrGPD1) | pRS423 | 2501 | 19,20,22,24,26 |
| 27～42 | 938(AtNHXS1) | pRS423 | 2024 | 30,31,33 |
| 43～54,66,71 | 1301(SseNHX1) | pRS423 | 2957(VR,VF2)/2811(VF2,Z027) | 40,41,44,47 |
| 55～65,67,(68),69,70 | 910(AtHKT1) | pRS423 | 2670(VF2,Z027)/2816(VR,VF) | 55,63,67,68,69,70 |

![1002colonyPCR102](https://static.igem.org/mediawiki/2018/9/97/T--Kyoto--1002colonyPCR102.jpg)

4(Mangrin)/12(ZrGPD1)/32(AtNHXS1)/33(Fgβ)/44(SdrG)

98℃ 30s
98℃\* 10s
55℃\* 30s
72℃\* 3min
72℃ 2min
15℃ ∞
(\*× 30cycle)

### 10/2

#### PCR

Tong

| Material | Volume |
| --- | --- |
| 5× Q5 Reaction Buffer | 8 |
| 10uM dNTPs | 3.2 |
| 10uM primerF 1 | | |
| 10uM primerR | 1 |
| template | 1 |
| enzyme | 0.4 |
| DW | 25.4 |
| Total | 40 |

|  | ① | ② |
| --- | --- | --- |
| template | 752, 1/100 | gBlock, 1/10 |
| primer | Z078, Z079 | Z082, Z083 |

SdrG, Stalk

### 10/2

#### PCR

Tong

| Material | Volume |
| --- | --- |
| 5× Q5 Reaction Buffer | 8 |
| 10uM dNTPs | 3.2 |
| 10uM primerF(Z080) | 1 |
| 10uM primerR(Z081) | 1 |
| enzyme | 0.4 |
| DW | 26.4 |
| Total | 40 |

Fgβ

### 10/2

#### Restriction Enzyme Digestion

Tong

| sample | 3/8/14/19/20/22/24/26/30/31/33/40/41/44/47 |
| --- | --- |
| volume(ul) | 10 |
| 10×H buffer | 4 |
| EcoR1 | 0.5 |
| Pst1 | 0.5 |
| DW | 25 |
| Total | 40 |

### 10/2

#### Ligation

Shimazoe

| Material | Volume(ul) |
| --- | --- |
| Ligation high | 3 |
| vector | 1 |
| insert | 2 |
| Total | 6 |

### 10/2

#### PCR

Shimazoe

| Material | Volume |
| --- | --- |
| 5× Q5 Reaction Buffer | 4 |
| 10uM dNTPs | 1.6 |
| 10uM Z094 | 0.5 |
| 10uM Z039 | 0.5 |
| template(p079) | 0.5 |
| DW | 12.7 |
| PrimeSTAR HS | 0.2 |
| Total | 20 |

| Material | Volume |
| --- | --- |
| 5× Q5 Reaction Buffer | 4 |
| 10uM dNTPs | 1.6 |
| 10uM Z095 | 0.5 |
| 10uM Z077 | 0.5 |
| template(718) | 0.5 |
| DW | 12.7 |
| PrimeSTAR HS | 0.2 |
| Total | 20 |

Fgβ

### 10/3

#### Restriction Enzyme Digestion

Nambu

| sample | Mangrin(4) | ZrGPD1(12) | AtNHXS1(32) | Fgβ(33) | SdrG(44) |
| --- | --- | --- | --- | --- | --- |
| volume(ul) | 5 | 5 | 5 | 5 | 5 |
| 10×H buffer | 2 | 2 | 2 | 2 | 2 |
| EcoR1 | 1 | 1 | 1 | 1 | 1 |
| Pst1 | 1 | 1 | 1 | 1 | 1 |
| DW | 11 | 11 | 11 | 11 | 11 |
| Total | 20 | 20 | 20 | 20 | 20 |

| sample | AtHKT1 | AVP1・pSB1C3 |
| --- | --- | --- |
| volume(ul) | 30 | 20 |
| 10×M buffer | 4 | 3 |
| Xba1 | 1 | 1 |
| Pst1 | 1 | 1 |
| CIAP | × | 1 |
| DW | 4 | 4 |
| Total | 40 | 30 |

### 10/3

#### Liquid Culture

Tong

21,28,53,57 Amp

### 10/3

#### Ligation

Tong

| insert | volume(ul) | vector | volume(ul) | Ligation High(ul) | Total(ul) |
| --- | --- | --- | --- | --- | --- |
| gBlock | 1 | pRS426(Sma1 cut)/326 | 1 | 2 | 4 |

### 10/3

#### Ligation

Tong

|  | volume(ul) | vector | volume(ul) | Ligation High(ul) | Total(ul) |
| --- | --- | --- | --- | --- | --- |
| Z082-Z83/DW | 1 | pSB1C3 | 1 | 2 | 4 |

### 10/3

#### Restriction Enzyme Digestion

Tong

| sample | volume(ul) |
| --- | --- |
| gBlock(McHKT2) | 20 |
| 10×H buffer | 10 |
| EcoR1 | 1 |
| Pst1 | 1 |
| DW | 68 |
| Total | 100 |

### 10/3

#### PCR

Tong

| Material | Volume |
| --- | --- |
| 5× Q5 Reaction Buffer | 8 |
| 10uM dNTPs | 3.2 |
| 10uM Z094 | 1 |
| 10uM Z095 | 1 |
| template① | 1 |
| template② | 1 |
| template③ | 1 |
| DW | 23.4 |
| PrimeSTAR HS | 0.4 |
| Total | 40 |

### 10/3

#### PCR

Shimazoe

| Material | Volume |
| --- | --- |
| 5× Q5 Reaction Buffer | 8 |
| 10uM dNTPs | 3.2 |
| 10uM Z080 | 1 |
| 10uM Z083 | 1 |
| template | 1 |
| Polymerase | 0.4 |
| DW | 25.4 |
| Total | 40 |

| Material | Volume |
| --- | --- |
| 5× Q5 Reaction Buffer | 8 |
| 10uM dNTPs | 3.2 |
| 10uM Z078 | 1 |
| 10uM Z083 | 1 |
| template | 1 |
| Polymerase | 0.4 |
| DW | 25.4 |
| Total | 40 |

Fgβ-Stalk, SdrG-Stalk

### 10/4

#### Nanodrop

Nambu

| sample | 1 | 2 | 9 | 12 | 17 | 20 | 25 | 33 | 40 | 46 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| concentration(ng/ul) | 182.6 | 199.9 | 323.0 | 246.3 | 264.2 | 283.5 | 192.9 | 187.5 | 212.5 | 199.8 |

### 10/4

#### Restriction Enzyme Digestion

Nambu

| sample | 1/2/9/12/17/20/25/33/40/46 |
| --- | --- |
| volume(ul) | 5 |
| 10×H buffer | 2 |
| EcoR1 | 1 |
| Pst1 | 1 |
| DW | 11 |
| Total | 20 |

### 10/4

#### Colony PCR

Nambu

| Material | Volume(ul) |
| --- | --- |
| 2× Emerald amp MAX PCR Master Mix | 42.5 |
| 10uM VF2 | 0.85 |
| 10uM Z018 | 0.85 |
| DW | 40.8 |
| Total | 85 |

98℃ 30s
98℃\* 10s
55℃\* 30s
72℃\* 3min
72℃ 3min
15℃ ∞
(\*× 30cycle)

### 10/4

#### Liquid Culture

Nambu

AtNHXS1 “2” “3”

### 10/4

#### Ligation

Shimazoe

| Material | Volume(ul) |
| --- | --- |
| Ligation high | 2 |
| vector | 1 |
| insert | 1 |
| Total | 4 |

|  | C1/L | C2/L | C3/L | C4/L | C5/L | C2/H | C3/H |
| --- | --- | --- | --- | --- | --- | --- | --- |
| backbone | pSB1C3 | pRS316 | pRS316 | pSB1C3 | pSB1C3 | YCplac195 | YCplac195 |
| insert | McHKT2 | ZrFPS1 | ZrFPS1 | SdrG | Fgβ | ZrFPS1 | ZrFPS1 |

### 10/4

#### Restriction Enzyme Digestion

Shimazoe

| material | volume(ul) |
| --- | --- |
| DNA | 15 |
| 10×H buffer | 8 |
| EcoR1 | 1 |
| Pst1 | 1 |
| DW | 55 |
| Total | 80 |

### 10/4

#### Ligation

Shimazoe

| Material | Volume(ul) |
| --- | --- |
| Ligation high | 2 |
| vector | 1 |
| insert | 1 |
| Total | 4 |

### 10/5

#### Liquid Culture

Nambu

Fgβ　34,35,36,37,38,39

### 10/5

#### Colony PCR

Nambu

| Material | Volume(ul) |
| --- | --- |
| 2× Emerald amp MAX PCR Master Mix | 87.5 |
| 10uM m13f | 1.75 |
| 10uM m13r | 1.75 |
| DW | 84.0 |
| Total | 175.0 |

Stalk
98℃ 30s
98℃\* 10s
55℃\* 30s
72℃\* 2m30s
72℃ 3min
15℃ ∞
(\*× 30cycle)

### 10/5

#### Restriction Enzyme Digestion

Shimazoe

| sample | volume(ul) |
| --- | --- |
| DNA | 10 |
| 10×H buffer | 2 |
| Sca1 | 1 |
| DW | 7 |
| Total | 20 |

| R | R | R | R | R | R | R | R |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Fgβ | Fgβ | Fgβ | Fgβ | SdrG | SdrG | SdrG | SdrG |
| pIEG | pIEG | 21 | 28 | pIEG | pIEG | 53 | 57 |

### 10/5

#### Transformation

Tong

| Labeling | Yeast type | Gene |
| --- | --- | --- |
| 43 | ΔENA1 | empty(pRS425:Leu)  empty(pRS423)  404 Mangrin(pRS326) |
| 44 | ΔENA1 | empty (pRS425:Leu)  empty (pRS423)  910 AtHKT1(pRS426) |
| 45 | ΔENA1 | 1118 AVP1(YCplac:Leu)  empty (pRS423)  empty (pRS426) |
| 46 | ΔENA1 | empty (pRS425:Leu)  938 AtNHXS1(pRS423)  empty (pRS426) |
| 47 | ΔENA1 | empty (pRS425:Leu)  1087 AtNHXS1(pRS313)  empty (pRS426) |
| 48 | ΔENA1 | empty (pRS425:Leu)  1301 SseNHXS1(pRS423)  empty (pRS426) |
| 49 | ΔENA1 | empty (pRS425:Leu)  1304 ZrGPD1(pRS423)  empty (pRS426) |
| 50 | ΔENA1 | empty (pRS425:Leu)  empty (pRS423)  empty (pRS426) |
| 51 | 564(Zysosaccharomyces rouxii) | empty (pRS425:Leu)  empty (pRS423)  404 Mangrin(pRS326) |
| 52 | 564(Zysosaccharomyces rouxii) | empty (pRS425:Leu)  empty (pRS423)  910 AtHKT1(pRS426) |
| 53 | 564(Zysosaccharomyces rouxii) | 1118 AVP1(YCplac:Leu)  empty (pRS423)  empty (pRS426) |
| 54 | 564(Zysosaccharomyces rouxii) | empty (pRS425:Leu)  938 AtNHXS1(pRS423)  empty (pRS426) |
| 55 | 564(Zysosaccharomyces rouxii) | empty (pRS425:Leu)  1087 AtNHXS1(pRS313)  empty (pRS426) |
| 56 | 564(Zysosaccharomyces rouxii) | empty (pRS425:Leu)  1301 SseNHXS1(pRS423)  empty (pRS426) |
| 57 | 564(Zysosaccharomyces rouxii) | empty (pRS425:Leu)  empty (pRS423)  empty (pRS426) |

### 10/5,10/6

#### Immuno Precipitation

Shimazoe

・SDS-PAGE
・Western Blotting

### 10/7

#### Colony PCR

Shimazoe

| Material | Volume(ul) |
| --- | --- |
| 2× Emerald amp MAX PCR Master Mix | 120 |
| 10uM VR | 2.4 |
| 10uM VF | 2.4 |
| DW | 115.2 |
| Total | 240 |

| Material | Volume(ul) |
| --- | --- |
| 2× Emerald amp MAX PCR Master Mix | 100 |
| 10uM m13r | 2 |
| 10uM m13f | 2 |
| DW | 96 |
| Total | 200 |

### 10/10

#### Transformation

Tong

| Labeling | Yeast type | URA | LEU | HIS |
| --- | --- | --- | --- | --- |
| 73 | ΔENA1 | 910AtHKT1(426) | AVP1-1(425) | 938 AtNHXS1(423) |
| 74 | ΔENA1 | 911 AtHKT1(426) | AVP1-1(425) | 1301 SseNHX1(423) |
| 75 | ΔENA1 | empty, pRS426 | AVP1-1(425) | 938 AtNHXS1(423) |
| 76 | ΔENA1 | empty, pRS426 | AVP1-1(425) | 1301 SseNHX1(423) |
| 77 | ΔENA1 | 910 AtHKT1(426) | empty, pRS425 | 938 AtNHXS1(423) |
| 78 | ΔENA1 | 911 AtHKT1(426) | empty, pRS425 | 1301 SseNHX1(423) |
| 79 | ΔENA1 | 910 AtHKT1(426) | AVP1-1(425) | empty, pRS423 |
| 80 | ΔENA1 | 911 AtHKT1(426) | AVP1-1(425) | empty, pRS423 |
| 81 | ΔENA1 | empty, pRS426 | AVP1-1(425) | empty, pRS423 |
| 82 | ΔENA1 | empty, pRS426 | empty, pRS425 | 938 AtNHXS1(423) |
| 83 | ΔENA1 | empty, pRS426 | empty, pRS425 | 1301 SseNHX1(423) |
| 84 | ΔENA1 | empty, pRS426 | empty, pRS425 | empty, pRS423 |
| 85 | ΔENA1,2,5ΔNHA1 | 910 AtHKT1(426) | AVP1-1(425) | 938 AtNHXS1(423) |
| 86 | ΔENA1,2,5ΔNHA1 | 911 AtHKT1(426) | AVP1-1(425) | 1301 SseNHX1(423) |
| 87 | ΔENA1,2,5ΔNHA1 | empty, pRS426 | AVP1-1(425) | 938 AtNHXS1(423) |
| 88 | ΔENA1,2,5ΔNHA1 | empty, pRS426 | AVP1-1(425) | 1301 SseNHX1(423) |
| 89 | ΔENA1,2,5ΔNHA1 | 910 AtHKT1(426) | empty, pRS425 | 938 AtNHXS1(423) |
| 90 | ΔENA1,2,5ΔNHA1 | 911 AtHKT1(426) | empty, pRS425 | 1301 SseNHX1(423) |
| 91 | ΔENA1,2,5ΔNHA1 | 910 AtHKT1(426) | AVP1-1(425) | empty, pRS423 |
| 92 | ΔENA1,2,5ΔNHA1 | 911 AtHKT1(426) | AVP1-1(425) | empty, pRS423 |
| 93 | ΔENA1,2,5ΔNHA1 | empty, pRS426 | AVP1-1(425) | empty, pRS423 |
| 94 | ΔENA1,2,5ΔNHA1 | empty, pRS426 | empty, pRS425 | 938 AtNHXS1(423) |
| 95 | ΔENA1,2,5ΔNHA1 | empty, pRS426 | empty, pRS425 | 1301 SseNHX1(423) |
| 96 | ΔENA1,2,5ΔNHA1 | empty, pRS426 | empty, pRS425 | empty, pRS423 |
| 97 | ΔENA1,2,5ΔNHA1 | NHA1 | AVP1-1(425) | 938 AtNHXS1(423) |
| 98 | ΔENA1,2,5ΔNHA1 | NHA1 | AVP1-1(425) | 1301 SseNHX1(423) |
| 99 | ΔENA1,2,5ΔNHA1 | NHA1 | empty, pRS425 | 938 AtNHXS1(423) |
| 100 | ΔENA1,2,5ΔNHA1 | NHA1 | empty, pRS425 | 1301 SseNHX1(423) |
| 101 | ΔENA1 | 910 AtHKT1(426) | AVP1(YCplac111) | 938 AtNHXS1(423) |
| 102 | ΔENA1 | 911 AtHKT1(426) | AVP1(YCplac111) | 1301 SseNHX1(423) |
| 103 | ΔENA1 | empty, pRS426 | AVP1(YCplac111) | 938 AtNHXS1(423) |
| 104 | ΔENA1 | empty, pRS426 | AVP1(YCplac111) | 1301 SseNHX1(423) |
| 105 | ΔENA1 | 910 AtHKT1(426) | AVP1(YCplac111) | empty, pRS423 |
| 106 | ΔENA1 | 911 AtHKT1(426) | AVP1(YCplac111) | empty, pRS423 |
| 107 | ΔENA1 | empty, pRS426 | AVP1(YCplac111) | empty, pRS423 |
| 108 | ΔENA1,2,5ΔNHA1 | 910 AtHKT1(426) | AVP1(YCplac111) | 938 AtNHXS1(423) |
| 109 | ΔENA1,2,5ΔNHA1 | 911 AtHKT1(426) | AVP1(YCplac111) | 1301 SseNHX1(423) |
| 110 | ΔENA1,2,5ΔNHA1 | empty, pRS426 | AVP1(YCplac111) | 938 AtNHXS1(423) |
| 111 | ΔENA1,2,5ΔNHA1 | empty, pRS426 | AVP1(YCplac111) | 1301 SseNHX1(423) |
| 112 | ΔENA1,2,5ΔNHA1 | 910 AtHKT1(426) | AVP1(YCplac111) | empty, pRS423 |
| 113 | ΔENA1,2,5ΔNHA1 | 911 AtHKT1(426) | AVP1(YCplac111) | empty, pRS423 |
| 114 | ΔENA1,2,5ΔNHA1 | empty, pRS426 | AVP1(YCplac111) | empty, pRS423 |
| 115 | ΔENA1,2,5ΔNHA1 | NHA1 | AVP1(YCplac111) | 938 AtNHXS1(423) |
| 116 | ΔENA1,2,5ΔNHA1 | NHA1 | AVP1(YCplac111) | 1301 SseNHX1(423) |
| 117 | ΔENA1,2,5ΔNHA1 | NHA1 | AVP1(YCplac111) | 938 AtNHXS1(423) |
| 118 | ΔENA1,2,5ΔNHA1 | NHA1 | AVP1(YCplac111) | 1301 SseNHX1(423) |
| 119 | ΔENA1,2,5ΔNHA1 | NHA1 | AVP1(YCplac111) | empty, pRS423 |
| 120 | ΔENA1,2,5ΔNHA1 | NHA1 | AVP1(YCplac111) | empty, pRS423 |
| 121 | ΔENA1,2,5ΔNHA1 | NHA1 | AVP1(YCplac111) | empty, pRS423 |


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# Team:Kyoto/Experiments

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# Kuwasiku(未)

締め切り：　、原稿担当：

##### Table of contents

* [1) Observe that *B. xylophilus* feeds on yeast](#res1)
* [2) Identify *B. xylophilus* which feeds yeast by fluorescence](#res2)
* [3) Choose dsRNA](#res3)
* [4) Conduct feeding RNAi in yeast](#res4)
* [5) Observe that *B. xylophilus* feeds yeast expressing dsRNA](#res5)
* [6) Improve transition of mRNA to cytosol](#res6)

##### 1) Observe that *B. xylophilus* feeds on yeast

Dr. Y Takeuchi, a researcher of *B. xylophilus*, gave us a strain that was bred in the laboratory. These nematodes were cultivated with gray mold. It was also possible to grow nematodes on the plate growing gray mold.

After we washed nematodes cultured in mold with lactic acid to remove mold, we placed them with budding yeast on a new plate. At first we observed nematodes by microscope, but we could not observe them eating yeast. Although they tried to pierce yeast with their stylet, it seemed to be difficult for them to penetrate it well because the yeast was not fixed on the plate then moved, and also originally the yeast was too small.

So we thought it would be easier to pierce with a stylet if the yeast was bigger, and we modified the conditions and used diploid yeast instead of haploid yeast, which had been used thus far. Visually, diploid yeast is different from haploid with respect to shape and size, where diploid is bigger than haploid (fig 1-a). Therefore, we decided to continue observation with diploid yeast.

![](https://static.igem.org/mediawiki/2017/3/3d/Kyoto_fig1a.jpeg)

**Figure 1-a** *S. cerevisiae* cells.
Diploid (light blue) and haploid (orange) cells were mixed and photographed to compare their sizes.

We also improved the filming conditions. We stopped observing the nematodes on the medium directly, and changed to a method of sandwiching the medium and yeast between the slide glass and the cover glass and injecting nematodes from the side. By doing so, the yeast was fixed firmly on the medium and we could solve the problem that the yeast moved when nematodes were trying to pierce it with their stylet.

Shown in the movie below is the moment *B. xylophilus* preyed on budding yeast (Movie 1). This was filmed for the first time in the world. The needle-shaped structure found near the nematode's mouth is a stylet. The nematode pushed the stylet against the yeast and inserted it in the yeast. The yeast started to shrink rapidly sometime after the nematode pierced it, and in the end its shape almost disappeared. Yeast has a robust cell wall, but it seems to be crushed with a strong force.

[](https://static.igem.org/mediawiki/2017/e/e9/B.xylophilus.mp4)
Movie 1 The moment *B. xylophilus* prey on *S. cerevisiae*.

The results of experiments on whether or not *B. xylophilus* that ate *S. cerevisiae* would grow was shown in the figure (Figure 1-b).

![](https://static.igem.org/mediawiki/2017/1/16/Kyoto_fig1b.png)

**Figure 1-b** Survival rate of *B. xylophilus* with different foods.
Diploid or haploid cells were grown in SD liquid culture and spread on agar plates. 100 nematodes were grown on each plate and the number of survivors were counted at the indicated time points. (n=3)

In the case of haploid or no food, the survival rate decreases at Day 4, but when using diploids the survival rate increases. The large cell diploid yeast seems easier to ingest for *B. xylophilus*.

##### 2) Identify *B. xylophilus* which ate yeast by fluorescence

In order to confirm the effect of RNAi, we needed a marker to distinguish nematodes that preyed on yeast from the others within the experimental period. Cultured nematodes included many growth stages, so it was anticipated that they included some nematodes at the stages where they could not eat yeast. In order to distinguish only the individuals which ate yeast and identify the effect, we decided to repeat the feeding experiment using yeast which fluoresces with eGFP.

The results of observation of nematodes which ate yeast with a fluorescence microscope are shown in the figure (Figure 2-a~Figure 2-d).

The intestines of *B. xylophilus* were highlighted by green fluorescence penetrating through the center throughout the entire body. The mouth is on the left side and the anus is on the right side. eGFP seemed to be very stable in the intestine, and the entire intestine fluoresced uniformly. There was some fluorescence discontinued in the center, and this was correspondent with the part where *B. xylophilus* gonads crossed over the intestine.

From the above results, it was found that by expressing eGFP, it became possible to clearly distinguish nematodes that preyed on yeast from the others. By expressing dsRNA simultaneously with eGFP, it should be possible to measure the effect of dsRNA efficiently. As can be seen from this photograph, feeding nematodes on yeast expressing GFP also made it possible to clearly observe the structure of *B. xylophilus* intestine. This method seemed to be effective for closely observing abnormalities occurring in an intestine of *B. xylophilus*, like gastroscopy using barium in humans.

![](https://static.igem.org/mediawiki/2017/2/27/Kyoto_fig2a2b.png)

![](https://static.igem.org/mediawiki/2017/b/b6/Kyoto_fig2c2d.png)

**Figure 2-a** eGFP expression cassette.
**Figure 2-b** Yeast cells expressing eGFP.
eGFP expression cassette was cloned into a plasmid and introduced into wild type *S. cerevisiae* strain.
Images were recorded by fluorescence microscopy.
**Figure 2-c** Nematodes with GFP signal.
Nematodes fed on Yeast were visualized by fluorescence microscopy. Fed on eGFP(+) yeast (left) and EGFP(-) yeast (right) are shown. Scale bar: 50μm
**Figure 2-d** Nematodes with GFP signal 2.
(top) Nematode with GFP fluorescence was inspected by confocal microscopy. XY plane and Z plane (cross section) are shown, respectively.
(bottom) Negative control. Yeast cells with no eGFP plasmid were used. Scale bar: 25μm

Among the nematodes being cultured, how many nematodes were thought to surely eat yeast? We collected nematodes on the 3rd and 7th day from the start of the culture by eGFP yeast and observed what proportion of them were fluorescent using a fluorescence microscope.
The results are shown in the figure (Figure 2-e).

In observation immediately after giving yeast, nematodes emitting fluorescence could not be observed, but about 30% of nematodes emitted fluorescence at Day 3. From this, it was found that at least 30% of nematodes at day 3 were ingesting yeast by this time (Figure 2-e). Interestingly, keeping the plate warm as it was continued observation, it was observed that the number of fluorescent nematodes was reduced to about half (15% of the total) in Day 7. (p = 0.001) Yeasts applied to nutrient-free water agar may die due to starvation or dryness by this time, and the supply of new GFP to nematodes has stopped, which may be the cause of this decrease. The fact that the proportion of fluorescent nematodes declines means that GFP in the intestine of nematodes is digested and degraded over time.

![](https://static.igem.org/mediawiki/2017/8/8e/Flo2.png)

**Figure 2-e** Time course of the rate of eGFP(+) nematodes.
Nematodes were grown on eGFP(+) yeast and examined by fluorescence microscopy at the indicated time. (n=18)

##### 3) Choose dsRNA

In order to kill *B. xylophilus*, it is necessary to efficiently knock-down genes essential for growth. Examining the literature, we found a paper that succeeded in knocking down essential genes and reducing the survival rate by submerging *B. xylophilus* in high concentrations of dsRNA for a certain period of time (soaking RNAi) [1]. In this paper, the target was mRNA of arginine kinase AK1 which was an essential gene expressed in the intestines and RNAi of AK1 showed a fatal effect also in C. elegans. AK1 is an invertebrate-specific key enzyme of energy metabolism so it is often used as a target for development of invertebrate-specific inhibitors. It was a promising candidate for dsRNA expressed in yeast.
In order to select the most ideal target, we prepared dsRNA of several target mRNAs including AK1 in vitro and tried soaking RNAi. We obtained target sequence from a public database, designed oligos, and cloned genes by RT-PCR. At this time, we put the T7 promoter on both ends of the DNA so that dsRNA was synthesized by in vitro transcription. After transcription, association of dsRNA was induced by an annealing operation, and the template was removed with DNase. We confirmed the dsRNA finally obtained by electrophoresis (Figure 3-a).

![](https://static.igem.org/mediawiki/2017/7/79/Kyoto_fig3a.png)

**Figure 3-a** In vitro synthesized dsRNAs for soaking RNAi experiments.
M DNA size marker, λSty I
1 dsAK-2 (692-bp)
2 dsEef-1g (528-bp)
3 dsAK-1 (449-bp)
4 dsAsb (559-bp)
5 ds14-3-3zeta (534-bp)
6 dsTropomyosin (532-bp)
7 ds14-3-3 protein (610-bp)
8 dsGFP (649-bp)

We prepared these RNAs, adjusted to a concentration of 2 μg /μL, and tried soaking RNAi The results is shown in the figure (Figure 3-b). As is clearly shown, we could not see the phenotype due to the introduction of dsRNA which was inconsistent with the previously reported example. As a result of contacting several *B. xylophilus* researchers and gathering information, it turned out that even several Japanese researchers have attempted to reproduce *B. xylophilus* soaking RNAi, but no group was able to observe a clear effect. The reason may be that soaking RNAi of *B. xylophilus* contains technically unstable steps. Alternatively, since *B. xylophilus* used this time is derived from wild nematodes collected from the field, there may be a difference between the strain we used and nematodes in the publication where soaking RNAi was effective.

Although the effect of soaking RNAi was not observed, we decided to target the AK1 gene because there is already the report[1], and constructed the expression system of dsRNA in yeast.

![](https://static.igem.org/mediawiki/2017/8/83/Kyoto_fig3b.png)

**Figure 3-b** Effects of soaking RNAi
*B. xylophilus* were soaked into 2 mg/mL dsRNAs shown in Figure 3-a. After 4h incubation, nematodes were washed and incubated on M9 buffer plate (time=0h). Plates were examined for mortality of the nematodes up to 24h. The method of soaking RNAi was based on reference[1].

##### 4) Conduct feeding RNAi in yeast

In order to express AK1-dsRNA, we placed inverted repeat derived from AK1 ORF downstream of the Gal1 promoter of the part ([BBa\_K517000](http://parts.igem.org/wiki/index.php/Part%3ABBa_K517000)), and inserted a small loop sequence of 67-nt between repeats. There was a report that this loop sequence was effective when *S. cerevisiae* expressed long dsRNA[2]. Since *S. cerevisiae* has no Dicer homolog, dsRNA is not processed into siRNA. However, overexpression of dsRNA may be toxic to *S. cerevisiae*, so we adopted the Gal1 conditional promoter. When *S.cerevisiae* is cultured in the presence of glucose, this promoter is inactive, and many mRNAs are expressed when the carbon source of the medium is replaced with galactose. At the same time, we also used the GPD promoter ([BBa\_K517001](http://parts.igem.org/Part%3ABBa_K517001)) which is a constitutive expression type promoter. dsGFP with a sequence specific to GFP and was designed as a negative control. Outline of construction is shown below (Figure 4-a).

![](https://static.igem.org/mediawiki/2017/2/28/%E3%82%A2%E3%82%BB%E3%83%83%E3%83%88_14%404x.png)

**Figure 4-a** dsRNA expression vectors we used.
Left: Design of each dsRNA cassette. Plus strand and minus strand are tandemly transcribed as a single strand RNA.
 To enhance dsRNA formation, a short hairpin loop sequence was inserted in between.
Right: Our plasmids used in this study. Gal1 promoter or GPD promoter was fused to dsAK1 or dsGFP (negative control) cassettes. The cassetes were cloned in YEPlac195 plasmid (2-micron, high-copy number plasmid).

We cultured plasmid-containing yeasts in several media, collected RNA, and quantified by qRT-PCR with the "loop" part as a target.Moreover, it is known that various viruses of dsRNA type exist in *S. cerevisiae*. As a factor closely related to the life cycle of such a virus, Ski gene group is known. Many of these are now revealing detailed functions. The Ski complex binds to the 3 'end of RNA and serves as a cofactor for RNA exosome, which is an exonuclease complex that degrades RNA in the 3-5 direction. By binding to the 3 'end to disband the higher-order structure of RNA, it makes the recognition of substrate by exosome efficient. Since the dsRNA virus is known to proliferate in the ski2Δ strain [3], it was hoped that the use of this strain would greatly increase the yield of the target dsRNA.

The following is the result of qRT-PCR (Figure 4-c). First, expression of dsRNA was successfully detected when wild-type yeast into which Gal1 promoter-dsAK1 was introduced was induced by galactose. Almost the same values ​​are obtained even when the target of the primer set used for qRT-PCR is set to the loop portion or set within the AK1 gene. On the other hand, expression was suppressed as expected when replacing the medium with Glucose. From this, it was demonstrated that it is possible to conditionally induce long hairpin RNA expression using our plasmid.
Interestingly, the expression in Ski2Δ strain is higher than that in WT strain (p <0.05). This indicates that in wild type yeast, Ski complex is degrading targeting foreign dsRNA in addition to RNA virus as expected. From these results, it was found that it is possible to raise the intracellular concentration of exogenous dsRNA by using yeast mutant strain.
In GPD promoter ([BBa\_K517001](http://parts.igem.org/Part%3ABBa_K517001)), dsRNA could not be expressed. This part is composed of only the 112 bp sequence near the center out of the TDH 3 promoter (588 bp). Strong expression was confirmed when the 588 bp full-length promoter ([BBa\_K 530008](http://parts.igem.org/Part%3ABBa_K530008)) was used for eGFP expression experiments (Figure 2-a, 2-b), so we believe that there is a high probability that this part is defective.

![](https://static.igem.org/mediawiki/2017/1/12/Kyoto_fig4b.png)

**Figure 4-b** Quantification of dsRNA in *S. cerevisiae*
Dr. Makoto Kitabatake (Institute for frontier life and medical science, Kyoto University) performed qRT-PCR using purified yeast total RNAs. The indicated strains were grown in SD-Glucose or SD-Galactose and harvested at the mid to late log phase. Total RNAs were isolated by MasterPure Yeast RNA purification kit (Lucigen) and analyzed by SuperScript III Platinum SYBR qRT-PCR kit (Thermo) after DNase treatment. The primers used in this assay are shown. Quantification of dsRNA was normalized by 25S rRNA. (n=3)

##### 5) Observe that *B. xylophilus* feeds on yeast expressing dsRNA

We let *B. xylophilus* prey on the yeast prepared as described above and recorded the survival rate and behavior of nematodes as follows.

We counted the number of surviving nematodes which fed on dsRNA / eGFP expressing yeast every other day. We also confirmed the survival rate among nematodes that showed fluorescence of eGFP.

![](https://static.igem.org/mediawiki/2017/9/9c/Kyoto_fig5abc.png)

**Figure 5-a** Survival rate of *B. xylophilus* fed by yeast expressing dsAK1
Diploid yeast strain carrying the both dsAK1 and eGFP plasmids were grown in SD-Glucose (glu) or SD-Galactose (gal) medium and spread on new plates. ~100 nematodes were grown on each plate and examined by microscopy. (n=3)

**Figure 5-b** Mortality of GFP(+) nematodes
Nematodes fed by diploid yeast carrying dsAK1 and eGFP plasmids were examied by fluorescence microscopy. Gal, yeast culture was prepared by SD-Galactose medium (dsRNA induction). Glu, the same yeast strain was grown in SD-Glucose medium (dsRNA repression). (n=3)

**Figure 5-c** Survival rate of *B. xylophilus* fed by Ski2Δ strain
Feeding RNAi experiments were performed as in Figure 5-a. Ski2Δ strain instead of WT strain was used as prey. (n=1)

From the above results, the number of nematodes did not decline predominantly even when using yeast whose expression of dsAK1 was confirmed (Figure 5-a). According to previous experiments, when the nematodes cultured by eGFP(+) yeast, the rate of eGFP(+) labbeled nematodes was only about 30% (Figure 2-d). For this reason, even if dsRNA taken in kills nematodes, since the proportion of nematodes ingesting a sufficient number of yeasts to obtain the effect is not so high, there is a possibility that the effect given by dsRNA has been diluted. We focused only on nematodes that fed yeast and confirmed the mortality of fluorescent nematodes to evaluate the effect of dsRNA (Figure 5-b).
Even in this case, we could not confirm the effect of dsRNA as expected.
Moreover, the survival rate of nematodes is lower when using yeast cultured in +glu SD medium which should suppress the expression of dsRNA.
The results were the same even when using ski2Δ yeast in which the expression level of dsRNA was increased (Figure 5-c).
This seemingly contradictory result will be discussed later in the discussion.
We thought that there might be some obstacle before dsRNA was taken up by nematodes and sought out the cause.

##### 6) Improve transport of mRNA to cytosol

As shown in the figure (Figure 6-a), the diameter of the stylet is very small, about a fraction of a single cell of yeast. For this reason, *B. xylophilus* seemed to draw out the cytoplasmic fraction, but large cellular componentns such as the nucleus may not be efficiently consumed by *B. xylophilus.*

![](https://static.igem.org/mediawiki/2017/f/fd/Stylet.jpeg)

**Figure 6-a** Nematode’s stylet, diploid yeast, and haploid yeast. Scale bar : 10μm

A number of studies have been done on the nuclear export of mRNA, and the basic mechanism has been elucidated. It is known that various RNAs such as mRNA, rRNA, tRNA, etc. are recognized by transporters specific to each type and pass through the nuclear pore complex[4]. However, since the dsRNA as prepared this time does not exist in nature, it is not known whether there is a transport factor that recognizes this RNA or whether it is efficiently transported out of nucleus.

In order to prepare remedies for this problem, we tried experiments utilizing the REV factor of HIV-1 RNA, which is known to have the function of improving the efficiency of nuclear export of RNA.

As shown in the figure, REV plays the role of carrying an unspliced RNA genome to the cytoplasm in the life cycle of HIV-1 (Figure 6-b). In the case of ordinary mRNA, there is a retention mechanism that prevent molecules retaining introns from transferring out of the nucleus, thus preventing the transport of immature mRNA. REV binds to a specific part (RRE: Rev responsive element) of the intron on the HIV-1 RNA genome and binds itself to the nuclear export factor CRM1, and overcomes such a retention mechanism and transports RNA to the cytoplasm[5]. Even if the dsRNA is not recognized as a nuclear export factor or even if it is retained in the nuclear retention factor, we thought that it is possible that the efficiency of nuclear export can be improved by inserting REV-RRE system, and provide these new parts to the iGEM community ([BBa\_K2403000](http://parts.igem.org/Part%3ABBa_K2403000),
 [BBa\_K2403002](http://parts.igem.org/Part%3ABBa_K2403002))

![](https://static.igem.org/mediawiki/2017/d/de/Kyoto_fig6b.jpeg)

**Figure 6-b** Rev protein induces nuclear export of RRE-containing RNAs.

The results of microinjection of RI-labeled dsRNA with RRE into the nucleus of Xenopus oocytes are shown in the figure (Figure6c ~ Figure6f). Nuclear and cytoplasm were separated after a certain period of time following injection, RNA was recovered from each and analyzed .

![](https://static.igem.org/mediawiki/2017/8/86/Kyoto_fig6c.png)

![](https://static.igem.org/mediawiki/2017/7/75/Kyoto_fig6d.png)

![](https://static.igem.org/mediawiki/2017/6/6f/Kyoto_fig6fe.png)

\* Dr. Taniguchi, Institute for fronter life and medical science, Kyoto university, conducted an experiment on the process of treating RI on and after in vitro transcription.

**Figure 6-c** Outline of Xenopus oocyte microinjection

**Figure 6-d** RNAs produced by in vitro transcription
U6 and U6-RRE are used as controls.

**Figure 6-e** Microinjection of dsGFP with or without RRE into Xenopus oocyte
Indicated RNAs with or without Rev protein were injected into Xenopus oocyte nucleus. The oocytes were dissected at the indicated time points (t=0, 60 min). The nuclear RNAs (N) and the cytoplasmic RNAs (C) were extracted and analyzed by PAGE. The left pannel shows dsGFP with RRE (GFP-RRE + GFPrev), the right pannel shows dsGFP without RRE (GFPfwd + GFPrev).

**Figure 6-f** Long exposure

As expected, when U6-RRE is injected together with buffer without Rev, U6-RRE remains in the nucleus, whereas U6 - RRE is injected with buffer containing Rev, U6-RRE was remarkably transported outside the nucleus. This result demonstrated that nuclear export of RNA is promoted depending on both the RRE sequence and the Rev protein in the case of U6 RNA originally staying in the nucleus. These effects indicate that these parts are promising as devices for efficiently transporting highly structured RNA, which is often used in synthetic biology, to the cytoplasm.([BBa\_K2403000](http://parts.igem.org/Part%3ABBa_K2403000)
[BBa\_K2403002](http://parts.igem.org/Part%3ABBa_K2403002))
Unfortunately, transcription products of GFP-RRE were too thin to understand whether they responded to Rev. However, from the figure on the right of Figure 6-f, it was also found that dsRNA remained in the nucleus a lot. It is suggested that implementation of a system to promote nucleocytoplasmic transport is effective.
Compared to the signal at T = 0, since many signals are lost at 60 minutes after injection, there may be a mechanism for degrading dsRNA in cells. In addition to promoting transportation efficiency, there will be room for improvement to improve stability.

This is the Result obtained in this project.
We would like to discuss Discussion on interpretation of Result and Future plan.

###### Reference

* [1] X. rong Wang, X. Cheng, Y. dong Li, J. ai Zhang, Z. fen Zhang, and H. rong Wu, “Cloning arginine kinase gene and its RNAi in *Bursaphelenchus xylophilus* causing pine wilt disease,” Eur. J. Plant Pathol., vol. 134, no. 3, pp. 521–532, 2012.* * [2] A. Sigova, N. Rhind, and P. D. Zamore, “A single Argonaute protein mediates both transcriptional and posttranscriptional silencing in Schizosaccharomyces pombe,” genes Dev., 2004.
    * [3] R. Esteban and R. B. Wickner, “A new non-mendelian genetic element of yeast that increases cytopathology produced by M1 double-stranded RNA in ski strains.,” Genetics, 1987.
    * [4] M. T. B. Sloan, Katherine E, Pierre-Emmanuel Gleizes, “Nucleocytoplasmic Transport of RNAs and RNA–Protein Complexes,” J. Mol. Biol., vol. 428, no. 10, pp. 2040–2059, 2016.
    * [5] V. W. Pollard and M. H. Malim, “the Hiv-1 Rev Protein,” Annu. Rev. Microbiol., vol. 52, no. 1, pp. 491–532, 1998.

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.

### What should this page contain?

* Protocols
* Experiments
* Documentation of the development of your project

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:Kyoto/Results

# Results

Here you can describe the results of your project and your future plans.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember all measurement and characterization data must be on part pages in the Registry.
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2014 TU Darmstadt](https://2014.igem.org/Team%3ATU_Darmstadt/Results/Pathway)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Results)
* [2014 Paris Bettencourt](https://2014.igem.org/Team%3AParis_Bettencourt/Results)