Team:Gifu/Notebook

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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

samplerestriction enzymes
His-RFP(with stop codon)SpeI,EcoRI
5'-intron MSpeI,EcoRI
Lac-3'intron-RBSSpeI,EcoRI
His-RFP(without stop codon)XbaI,EcoRI
RBS-AmpSpeI,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

sampleconcentration(ng/µL)
His-RFP(with stop codon) EcoRI-SpeI162.6
5'intron M EcoRI-SpeI45.4
Lac-3'intron-RBS EcoRI-SpeI120.9
His-RFP(without stop codon) EcoRI-SpeI160.0
His-RFP(without stop codon)-5'intron-DT-Cm XbaI-EcoRI 174.0
Amp EcoRI-SpeI101.8

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

samplerestriction enzymes
His-RFP(with stop codon)-DT-CmEcoRI,XbaI
5'intron M-DT-CmEcoRI,XbaI

Densitometry

sampleconcentration(ng/µL)
EcoRI-Lac-RBS-SpeI65.0
XbaI-His-RFP(with stop codon)-DT-Cm-EcoRI188.8
EcoRI-His-RFP(with stop codon)-SpeI160.0
EcoRI-His-RFP(without stop codon)-SpeI160.0
XbaI-5'intron M-DT-Cm-EcoRI167.8

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

samplerestriction 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

sampleconcentration(ng/µL)
EcoRI-Lac-3'intron-RBS-SpeI120.0
XbaI-His-RFP(with stop codon)-5'intron M-DT-Cm-EcoRI187.8
XbaI-His-RFP(without stop codon)-5'intron M-DT-Cm-EcoRI158.9

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

samplerestriction enzymes
His-RFP(with stop codon)EcoRI,PstI
His-RFP(without stop codon)EcoRI,PstI
5'intron MEcoRI,PstI
Lac-3'intron-RBSEcoRI,PstI
TTHA0175EcoRI,SpeI

Densitometry

sampleconcentration(ng/µL)
His-RFP(with stop codon)180.7
His-RFP(without stop codon)164.6
5'intron M37.7
Lac-3'intron-RBS107.5
TTHA0175112.7

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

sampleconcentration(ng/µL)
EcoRI-TTHA0175-5'intron M-DT-Cm-XbaI118.8

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



Retrieved from "http://2014.igem.org/Team:Gifu/Notebook"