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Protocols 1 Protocols 2 Calender

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	12	13	14	15	16	17
18	19	20	21	22	23	24
25	26	27	28	29	30	31

September
Mon	Tue	Wed	Thu	Fri	Sat	Sun
1	2	3	4	5	6	7
8	9	10	11	12	13	14
15	16	17	18	19	20	21
22	23	24	25	26	27	28
29	30

October
Mon	Tue	Wed	Thu	Fri	Sat	Sun
		1	2	3	4	5
6	7	8	9	10	11	12
13	14	15	16	17	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 Protein
This is BBa_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.

Amp

Ampicillin resistance gene

Cm

Chloramphenicol resistance gene.
This is pSB1C3.

RBS

Ribosome Binding Sites.
This is BBa_B0034.

DT

Double Terminator consisting of BBa_B0010 and BBa_B0012.
This is BBa_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.

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

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)

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-

August 16th

Liquid Culture

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

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

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-

August 21th

Reagent Preparation

Protein Extraction

SDS-PAGE

August 22th

Prorein Purification by use of Ni-NTA

SDS-PAGE

August 23th

Prorein Purification by use of Ni-NTA

SDS-PAGE

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

August 25th

Extraction of plasmid device

SDS-PAGE

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

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

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

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-

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

August 30th

Colony PCR

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

August 31th

Colony PCR

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

September 1st

Shaking Culture

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

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

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

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)

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

September 8th

SDS-PAGE

1. marker
2. supernatant
3. refined RFP

September 9th

HisTag refining

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

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

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-

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

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

September 15th

Ethanol Precipitation

Densitometry

reverse transcription PCR

PCR

September 16th

RNase treatment

reverse transcription PCR

cDNA synthesis

PCR for confirming existence of circular RNA

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)

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)-

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

September 21th

SDS-PAGE

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

September 23th

PCR

Ligation

Transformation

September 24th

Electrophoresis

September 25th

Colony PCR

Liquid culture

September 26th

Miniprep

SDS-PAGE

September 27th

Restriction Enzyme Digestion

Ligation

Transformation

September 28th

SDS-PAGE

Colony PCR

September 30th

SDS-PAGE

October 2nd

RNA extraction

PCR

October 3rd

PCR

October 4th

PCR

October 5th

PCR

October 6th

PCR

October 7th

PCR

October 8th

PCR

October 9th

PCR

October 10th

PCR

October 11th

PCR

October 12th

PCR

October 13th

PCR

October 14th

PCR

October 15th

PCR

October 16th

PCR

October 17th

PCR