iGEM 2014 — Team Gifu

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# Team:Gifu/Notebook

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[Protocols 1](https://2014.igem.org/Team%3AGifu/Protocol)
[Protocols 2](https://2014.igem.org/Gifu/protocols2)
[Calender](https://2014.igem.org/Team%3AGifu/Notebook)

# Calender

Click on a date to see our research activities on that day.

| August | | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| Mon | Tue | Wed | Thu | Fri | Sat | Sun |
|  |  |  |  | 1 | 2 | 3 |
| 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| [11](#0811) | [12](#0812) | [13](#0813) | 14 | 15 | [16](#0816) | 17 |
| [18](#0818) | [19](#0819) | [20](#0820) | [21](#0821) | [22](#0822) | [23](#0823) | [24](#0824) |
| [25](#0825) | [26](#0826) | [27](#0827) | [28](#0828) | [29](#0829) | [30](#0830) | [31](#0831) |

| September | | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| Mon | Tue | Wed | Thu | Fri | Sat | Sun |
| [1](#0901) | 2 | [3](#0903) | [4](#0904) | [5](#0905) | 6 | 7 |
| [8](#0908) | [9](#0909) | [10](#0910) | [11](#0911) | [12](#0912) | [13](#0913) | [14](#0914) |
| [15](#0915) | [16](#0916) | [17](#0917) | [18](#0918) | [19](#0919) | [20](#0920) | [21](#0921) |
| [22](#0922) | [23](#0923) | [24](#0924) | [25](#0925) | [26](#0926) | [27](#0927) | [28](#0928) |
| 29 | [30](#0930) |  |  |  |  |  |

| October | | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| Mon | Tue | Wed | Thu | Fri | Sat | Sun |
|  |  | 1 | [2](#1002) | [3](#1003) | [4](#1004) | [5](#1005) |
| [6](#1006) | [7](#1007) | [8](#1008) | [9](#1009) | [10](#1010) | [11](#1011) | [12](#1012) |
| [13](#1013) | [14](#1014) | [15](#1015) | [16](#1016) | [17](#1017) | 18 | 19 |
| 20 | 21 | 22 | 23 | 24 | 25 | 26 |
| 27 | 28 | 29 | 30 | 31 |  |  |

# Glossary

His
:   Histidine tag

GFP
:   Green Fluorescence Protein

RFP
:   Red Fluorescent ProteinThis is [BBa\_E1010.](http://parts.igem.org/Part%3ABBa_E1010)

5'-intron
:   The 5’ side of the intron from td gene of T4 phage

5'-intron M
:   The 5’ side of the intron from td gene of T4 phage without stop codon

3'-intron
:   The 3’ side of the intron from td gene of T4 phage

Lac
:   LacI regulated promoter.This is [BBa\_R0010.](http://parts.igem.org/Part%3ABBa_R0010)

Amp
:   Ampicillin resistance gene

Cm
:   Chloramphenicol resistance gene.This is [pSB1C3.](http://parts.igem.org/Part%3ApSB1C3)

RBS
:   Ribosome Binding Sites.This is [BBa\_B0034.](http://parts.igem.org/Part%3ABBa_B0034)

DT
:   Double Terminator consisting of BBa\_B0010 and BBa\_B0012.
    This is [BBa\_B0015.](http://parts.igem.org/Part%3ABBa_B0015)

EcoRI
:   Restriction site that is cut by EcoRI or restriction enzyme

XbaI
:   Restriction site that is cut by XbaI or restriction enzyme

SpeI
:   Restriction site that is cut by SpeI or restriction enzyme

PstI
:   Restriction site that is cut by PstI or restriction enzyme

TTHA0715
:   Cold shock protein (attached amino acid sequence cut by thrombin)

SmtA
:   Metallothionein that can catch heavy metal ions such as Zn2+.
    This is [BBa\_K519010](http://parts.igem.org/Part%3ABBa_K519010).

# Prologue -before August-

### Parts amplification with PCR

1. Lac
2. RBS
3. RFP
4. DT
5. Cm (pSB1C3)
6. pSB1A3
7. pSB1K3

### T4 phage Multiplication with *E. coli*

### DNA Extraction from T4 phage

# August

## August 11th

### Restriction Enzyme Digestion

| sample | restriction enzymes |
| --- | --- |
| His-RFP(with stop codon) | SpeI,EcoRI |
 5'-intron M | SpeI,EcoRI || Lac-3'intron-RBS | SpeI,EcoRI |
| His-RFP(without stop codon) | XbaI,EcoRI |
| RBS-Amp | SpeI,EcoRI |

### Ligation

1. EcoRI-His-RFP(with stop codon)-SpeI + XbaI-DT-Cm-EcoRI
2. EcoRI-5'intron M-SpeI + XbaI-DT-Cm-EcoRI
3. EcoRI-Lac-3'intron-RBS-SpeI + XbaI-His-RFP(without stop codon)-5'intron-DT-Cm-EcoRI
4. EcoRI-His-RFP(without stop codon)-SpeI + SpeI-Amp-EcoRI

### Transformation

1. -His-RFP(with stop codon)-DT-Cm-
2. -5'intron M-DT-Cm-
3. -Lac-3'intron-RBS-His-RFP(without stop codon)-5'intron-DT-Cm-
4. -His-RFP(without stop codon)-Amp-

### Densitometry

| sample | concentration(ng/µL) |
| --- | --- |
| His-RFP(with stop codon) EcoRI-SpeI | 162.6 |
| 5'intron M EcoRI-SpeI | 45.4 |
| Lac-3'intron-RBS EcoRI-SpeI | 120.9 |
| His-RFP(without stop codon) EcoRI-SpeI | 160.0 |
| His-RFP(without stop codon)-5'intron-DT-Cm XbaI-EcoRI | 174.0 |
| Amp EcoRI-SpeI | 101.8 |

[back to calender](#calender)

## August 12th

### Colony PCR

1. His-RFP(with stop codon)-DT-Cm
2. 5'intron M-DT-Cm
3. Lac-3'intron-RBS-His-RFP(without stop codon)-5'intron-DT-Cm:[1]turning red
4. Lac-3'intron-RBS-His-RFP(without stop codon)-5'intron-DT-Cm:[2]turning pink
5. Lac-3'intron-RBS-His-RFP(without stop codon)-5'intron-DT-Cm:[3]turning white
6. His-RFP(without stop codon)-Amp

### Electrophoresis

1. His-RFP(with stop codon)-DT
2. His-RFP
3. 5'intron M-DT
4. 5'intron M
5. Lac-3'intron-RBS-His-RFP(without stop codon)-5'intron-DT:[1]
6. Lac-3'intron-RBS-His-RFP(without stop codon)-5'intron-DT:[2]
7. Lac-3'intron-RBS-His-RFP(without stop codon)-5'intron-DT:[3]
8. His-RFP(without stop codon)-5'intron-DT
9. His-RFP(without stop codon)

### Liquid Culture

1. His-RFP(with stop codon)-DT
2. 5'intron M-DT
3. Lac-3'intron-RBS-His-RFP(without stop codon)-5'intron-DT:[1]
4. Lac-3'intron-RBS-His-RFP(without stop codon)-5'intron-DT:[3]
5. His-RFP(without stop codon)

[back to calender](#calender)

## August 13th

### Miniprep

### PCR

By use of KAPA HiFi DNA Polymerase

1. His-RFP(with stop codon)-DT-Cm
2. 5'intron M-DT-Cm

By use of Taq DNA Polymerase

1. Lac-3'intron-RBS-His-RFP(without stop codon)-5'intron-DT-Cm:[1]turning red
2. Lac-3'intron-RBS-His-RFP(without stop codon)-5'intron-DT-Cm:[3]turning white

### Electrophoresis

1. His-RFP(with stop codon)-DT-Cm
2. 5'intron M-DT-Cm
3. DNA Ladder Markers DNA Ladder One(Broad Range)
4. Lac-3'intron-RBS-His-RFP(without stop codon)-5'intron-DT-Cm:[1]turning red
5. Lac-3'intron-RBS-His-RFP(without stop codon)-5'intron-DT-Cm:[3]turning white

### Restriction Enzyme Digestion

| sample | restriction enzymes |
| --- | --- |
| His-RFP(with stop codon)-DT-Cm | EcoRI,XbaI |
| 5'intron M-DT-Cm | EcoRI,XbaI |

### Densitometry

| sample | concentration(ng/µL) |
| --- | --- |
| EcoRI-Lac-RBS-SpeI | 65.0 |
| XbaI-His-RFP(with stop codon)-DT-Cm-EcoRI | 188.8 |
| EcoRI-His-RFP(with stop codon)-SpeI | 160.0 |
| EcoRI-His-RFP(without stop codon)-SpeI | 160.0 |
| XbaI-5'intron M-DT-Cm-EcoRI | 167.8 |

### Ligation

1. EcoRI-Lac-RBS-SpeI + XbaI-His-RFP(with stop codon)-DT-Cm-EcoRI
2. EcoRI-His-RFP(with stop codon)-SpeI + XbaI-5'intron M-DT-Cm-EcoRI
3. EcoRI-His-RFP(without stop codon)-SpeI + XbaI-5'intron M-DT-Cm-EcoRI

### Transformation

1. -Lac-RBS-His-RFP(with stop codon)-DT-Cm-
2. -His-RFP(with stop codon)-5'intron M-DT-Cm-
3. -His-RFP(without stop codon)-5'intron M-DT-Cm-

[back to calender](#calender)

## August 16th

### Liquid Culture

[back to calender](#calender)

## August 18th

### Plasmid Extraction

1. -Lac-RBS-His-RFP(with stop codon)-DT-Cm-
2. -His-RFP(with stop codon)-5'intron M-DT-Cm-
3. -His-RFP(without stop codon)-5'intron M-DT-Cm-

### Plasmid Purification

1. -5'intron M-His-RFP(with stop codon)-
2. -5'intron M-His-RFP(without stop codon)-

### Miniprep

### Colony PCR

### Restriction Enzyme Digestion

| sample | restriction enzymes |
| --- | --- |
| -His-RFP(with stop codon)-5'intron M-DT-Cm- | EcoRI,XbaI |
| -His-RFP(without stop codon)-5'intron M-DT-Cm- | EcoRI,XbaI |

### Densitometry

| sample | concentration(ng/µL) |
| --- | --- |
| EcoRI-Lac-3'intron-RBS-SpeI | 120.0 |
| XbaI-His-RFP(with stop codon)-5'intron M-DT-Cm-EcoRI | 187.8 |
| XbaI-His-RFP(without stop codon)-5'intron M-DT-Cm-EcoRI | 158.9 |

### Ligation&Transformation

1. EcoRI-Lac-3'intron-RBS-SpeI + XbaI-His-RFP(with stop codon)-5'intron M-DT-Cm-EcoRI
2. EcoRI-Lac-3'intron-RBS-SpeI + XbaI-His-RFP(without stop codon)-5'intron M-DT-Cm-EcoRI

### Polyethylene glycol Precipitation

[back to calender](#calender)

## August 19th

### Colony PCR

1. -Lac-3'intron-RBS-His-RFP(with stop codon)-5'intron M-DT-Cm-
2. -Lac-3'intron-RBS-His-RFP(without stop codon)-5'intron M-DT-Cm-

### Electrophoresis

1. -Lac-3'intron-RBS-His-RFP(with stop codon)-5'intron M-DT-Cm-
2. -Lac-3'intron-RBS-His-RFP(without stop codon)-5'intron M-DT-Cm-
3. marker
4. -His-RFP(with stop codon)-5'intron M-DT-Cm-
5. -His-RFP(without stop codon)-5'intron M-DT-Cm-

### Shaking Culture

[back to calender](#calender)

## August 20th

### Colony PCR

1. -Lac-3'intron-RBS-His-RFP(without stop codon)-5'intron M-DT-Cm-

by use of another colony differs from 8/19

### Electrophoresis

1. -Lac-3'intron-RBS-His-RFP(without stop codon)-5'intron M-DT-Cm-
2. marker
3. -His-RFP(without stop codon)-5'intron M-DT-Cm-

[back to calender](#calender)

## August 21th

### Reagent Preparation

### Protein Extraction

### SDS-PAGE

[back to calender](#calender)

## August 22th

### Prorein Purification by use of Ni-NTA

### SDS-PAGE

[back to calender](#calender)

## August 23th

### Prorein Purification by use of Ni-NTA

### SDS-PAGE

[back to calender](#calender)

## August 24th

### Extraction of plasmid device

1. -Lac-RBS-His-RFP(with stop codon)-DT-Cm-
2. -Lac-RBS-3'intron-His-RFP(without stop codon)-5'intron-DT-Cm-
3. -Lac-RBS-3'intron-His-RFP(with stop codon)-5'intron M-DT-Cm-
4. -Lac-RBS-3'intron-His-RFP(without stop codon)-5'intron M-DT-Cm-

### PCR&Electrophoresis

1. EcoRI-His-RFP(with stop codon)-PstI
2. EcoRI-His-RFP(without stop codon)-PstI
3. EcoRI-5'intron M-PstI
4. EcoRI-Lac-3'intron-RBS-PstI

[back to calender](#calender)

## August 25th

### Extraction of plasmid device

### SDS-PAGE

1. Lac-3'intron-RBS-His-RFP-5'intron M-DT-Cm

[back to calender](#calender)

## August 26th

### PCR

### Restriction Enzyme Digestion

| sample | restriction enzymes |
| --- | --- |
| His-RFP(with stop codon) | EcoRI,PstI |
| His-RFP(without stop codon) | EcoRI,PstI |
| 5'intron M | EcoRI,PstI |
| Lac-3'intron-RBS | EcoRI,PstI |
| TTHA0175 | EcoRI,SpeI |

### Densitometry

| sample | concentration(ng/µL) |
| --- | --- |
| His-RFP(with stop codon) | 180.7 |
| His-RFP(without stop codon) | 164.6 |
| 5'intron M | 37.7 |
| Lac-3'intron-RBS | 107.5 |
| TTHA0175 | 112.7 |

### Ligation&Transformation

1. EcoRI-His-RFP(with stop codon)-PstI + EcoRI-Cm-PstI
2. EcoRI-His-RFP(without stop codon)-PstI + EcoRI-Cm-PstI
3. EcoRI-5'intron M-PstI + EcoRI-Cm-PstI
4. EcoRI-Lac-3'intron-RBS-PstI + EcoRI-Cm-PstI
5. EcoRI-TTHA0175-SpeI + EcoRI-5'intron M-DT-Cm-XbaI

[back to calender](#calender)

## August 27th

### Colony PCR

1. -His-RFP(with stop codon)-Cm-
2. -TTHA0175-Amp-
3. -TTHA0175-5'intron M-DT-Cm-

### Shaking Culture

1. -His-RFP(with stop codon)-Cm-
2. -TTHA0175-Amp-
3. -TTHA0175-5'intron M-DT-Cm-

### Enrichment Culture

[back to calender](#calender)

## August 28th

### Plasmid Purification

1. -His-RFP(with stop codon)-5'intron M-Cm-
2. -TTHA0175-5'intron M-DT-Cm-

### PCR

1. -His-RFP(with stop codon)-Cm-
2. -Lac-3'intron-RBS-Cm-
3. -His-RFP(without stop codon)-Cm-
4. -5'intron M-Cm-

[back to calender](#calender)

## August 29th

### Densitometry

| sample | concentration(ng/µL) |
| --- | --- |
| EcoRI-TTHA0175-5'intron M-DT-Cm-XbaI | 118.8 |

### Ligation

1. EcoRI-TTHA0175-5'intron M-DT-Cm-XbaI + EcoRI-Lac-3'intron-RBS-SpeI

### SDS-PAGE

### Electrophoresis

1. EcoRI-TTHA0175-5'intron M-DT-Cm-XbaI

[back to calender](#calender)

## August 30th

### Colony PCR

1. -Lac-3'intron-RBS-TTHA0715-5'intron M-DT-Cm-
2. Plate4 3G

[back to calender](#calender)

## August 31th

### Colony PCR

Because result of Colony PCR on 8/30 is not good, We chose 5 colonies and put them on PCR.

[back to calender](#calender)

## September 1st

### Shaking Culture

1. -Lac-3'intron-RBS-TTHA0715-5'intron M-DT-Cm-

### Cell body Crush&SDS-PAGE(gel 10%)

[back to calender](#calender)

## September 3rd

### Culture

1. -Lac-3'intron-RBS-RFP(without stop codon)-5'intron M-DT-Cm- 20 mL
2. -Lac-3'intron-RBS-RFP(with stop codon)-5'intron M-DT-Cm- 5 mL

[back to calender](#calender)

## September 4th

### Make circular RNA

1. TTHA0715
2. RFP(without stop codon)
3. RFP(with stop codon)

### Cell body Crush

1. TTHA0715
2. RFP(without stop codon)
3. RFP(with stop codon)

[back to calender](#calender)

## September 5th

### SDS-PAGE

1. GFP
2. TTHA0715 supernatant1(no wash)
3. TTHA0715 supernatant2(first wash)
4. TTHA0715 supernatant3(second wash)
5. TTHA0715 deposition
6. TTHA0715 deposition two fold dilution
7. marker
8. RFP(without stop codon) supernatant1
9. RFP(without stop codon) deposition
10. RFP(with stop codon) supernatant1
11. RFP(with stop codon) deposition
12. RFP(without stop codon) supernatant2
13. RFP(without stop codon) supernatant3
14. RFP(with stop codon) supernatant2
15. RFP(with stop codon) supernatant3

[back to calender](#calender)

## September 8th

### SDS-PAGE

1. marker
2. supernatant
3. refined RFP

###

###

[back to calender](#calender)

## September 9th

### HisTag refining

###

###

[back to calender](#calender)

## September 10th

### SDS-PAGE with 10% gel

1. TTHA0715 deposition
2. TTHA0715 supernatant
3. marker
4. RFP and fungus bodies
5. dialyzed RFP
6. guanidine deposition

[back to calender](#calender)

## September 11th

### SDS-PAGE

1. culture supernatant on September 1st
2. TTHA0715 supernatant on September 1st
3. TTHA0715 deposition on September 1st
4. marker
5. TTHA0715 supernatant on September 4th
6. TTHA0715 deposition on September 4th
7. TTHA0715 guanidine deposition

[back to calender](#calender)

## September 12th

### Ligation

1. promoter + RBS-GFP-DT-Cm
2. TTHA0715 + 5'intron-DT-Cm
3. Lac + RBS-GFP-DT-Cm

### Transformation

1. -promoter-RBS-GFP-DT-Cm-
2. -Lac-3'intron-RBS-TTHA0715-5'intron-DT-Cm-
3. -BBa\_K519010-pSB1C3-
4. -Lac-RBS-GFP-DT-Cm-

[back to calender](#calender)

## September 13th

### RNA Extraction

1. circular RNA (RFP without stop codon)

### Densitometry

1. circular RNA (RFP without stop codon)

### DNase treatment

### RNase treatment

### reverse transcription PCR

### cDNA synthesis

### PCR for confirming existence of circular RNA

[back to calender](#calender)

## September 14th

### Miniprep

1. -BBa\_K519010-pSB1C3-
2. -TTHA-5'intron-DT-Cm-

### PCR

1. -BBa\_K519010-pSB1C3-
2. -TTHA-5'intron-DT-Cm-

### Ethanol Precipitation

[back to calender](#calender)

## September 15th

### Ethanol Precipitation

### Densitometry

### reverse transcription PCR

### PCR

[back to calender](#calender)

## September 16th

### RNase treatment

### reverse transcription PCR

### cDNA synthesis

### PCR for confirming existence of circular RNA

###

[back to calender](#calender)

## September 17th

### PCR

1. EcoRI-SmtA-DT-Cm-XbaI
2. EcoRI-circularSmtA-SpeI
3. EcoRI-TTHA0715-5'intron-DT-Cm-XbaI

### Gel Extraction

### Ligation

1. Lac-RBS + SmtA-DT-Cm
2. circularSmtA + 5'intronM-DT-Cm
3. Lac-3'intron-RBS + TTHA0715-5'intron-DT-Cm
4. BBa\_K823012(promoter) + BBa\_E0240(GFP generator)

[back to calender](#calender)

## September 19th

### Colony PCR

1. -Lac-RBS-SmtA-DT-
2. -circularSmtA-5'intronM-DT-
3. -Lac-3'intron-RBS-TTHA0715-5'intron-DT-
4. -BBa\_K823012(promoter)-BBa\_E0240(GFP generator)-

[back to calender](#calender)

## September 20th

### Plasmid Extraction

1. -BBa\_K823012(promoter)-GFP-Cm-
2. -Lac-RBS-SmtA-DT-Cm-
3. -Lac-3'intron-RBS-TTHA0715-5'intron-DT-Cm-
4. -SmtA-5'intronM-DT-Cm-

### PCR

1. -BBa\_K823012(promoter)-GFP-Cm-
2. -Lac-RBS-SmtA-DT-Cm-
3. -Lac-3'intron-RBS-TTHA0715-5'intron-DT-Cm-
4. -SmtA-5'intronM-DT-Cm-
5. EcoRI-SmtA-5'intronM-DT-Cm-XbaI

### purification

### Restriction Enzyme Digestion

### purification

### Ligation

[back to calender](#calender)

## September 21th

### SDS-PAGE

[back to calender](#calender)

## September 22th

### SDS-PAGE

1. long-chain RFP precipitation
2. long-chain RFP supernatant
3. monomeric TTHA0715 precipitation
4. monomeric TTHA0715 supernatant
5. long-chain TTHA0715 precipitation
6. long-chain TTHA0715 supernatant
7. monomeric SmtA precipitation
8. monomeric SmtA supernatant
9. long-chain SmtA precipitation
10. long-chain SmtA supernatant

### Miniprep

1. -TTHA0715-5'intronM-DT-Cm-
2. -Lac-3'intron-RBS-SmtA-5'intronM-DT-Cm-
3. -BBa\_K823012(promoter)-GFP-

### PCR

[back to calender](#calender)

## September 23th

### PCR

### Ligation

### Transformation

[back to calender](#calender)

## September 24th

### Electrophoresis

[back to calender](#calender)

## September 25th

### Colony PCR

### Liquid culture

[back to calender](#calender)

## September 26th

### Miniprep

### SDS-PAGE

[back to calender](#calender)

## September 27th

### Restriction Enzyme Digestion

### Ligation

### Transformation

[back to calender](#calender)

## September 28th

### SDS-PAGE

### Colony PCR

[back to calender](#calender)

## September 30th

### SDS-PAGE

[back to calender](#calender)

## October 2nd

### RNA extraction

### PCR

[back to calender](#calender)

## October 3rd

### PCR

[back to calender](#calender)

## October 4th

### PCR

[back to calender](#calender)

## October 5th

### PCR

[back to calender](#calender)

## October 6th

### PCR

[back to calender](#calender)

## October 7th

### PCR

[back to calender](#calender)

## October 8th

### PCR

[back to calender](#calender)

## October 9th

### PCR

[back to calender](#calender)

## October 10th

### PCR

[back to calender](#calender)

## October 11th

### PCR

[back to calender](#calender)

## October 12th

### PCR

[back to calender](#calender)

## October 13th

### PCR

[back to calender](#calender)

## October 14th

### PCR

[back to calender](#calender)

## October 15th

### PCR

[back to calender](#calender)

## October 16th

### PCR

[back to calender](#calender)

## October 17th

### PCR

[back to calender](#calender)