Team:Kyoto/Notebook

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Contents

template

Transformation

じっけんしゃ

NameWellSampleCompetent CellsTotalPlate
いでんしぱーつのうぇるなんちゃらµLごにょごにょµLほにゃららµLこーせーぶっしつ
いかどうよう

Restriction Enzyme Digestion

じっけんしゃ

DNABufferrestricion enzymerestriction enzymeMilliQtotal
(°°) µL µL µL µL µL µL

Electrophoresis

じっけんしゃ

LaneSampleEnzyme1Enzyme2
(°Д°)

File:Igku xxxxxx.xxx

Ligation

じっけんしゃ

Name 目的(番号?)?NCとか µL
(>o<)

Gel Extraction

じっけんしゃ

LaneDNAEnzyme
(>ω・)b

File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx

Namequantityconcentration[µg/mL]260/280260/230
(´ω`)
(ΦωΦ^)

Colony PCR

No name

Samplebase pair
(`・ω・´)
PreDenatureDenatureAnnealingExtensioncycle
 °C °C °C °C--
(◎Д◎)    

Liquid Culture

No name

Samplemedium
(・ω・)
(・X・)  

Miniprep

DNAconcentration[µg/mL]260/280260/230
(・▽・)  

Master Plate

No name

NumberUse LB plate (+抗生物質)
ʅ(´◔౪◔)ʃ

Aug 19

Colony PCR

Kojima

Samplebase pair
8/18 J23100 control -(1)1142
8/18 J23100-RBS-GFP-DT -(1)1156
8/18 J23100-RBS-GFP-DT -(2)1156
8/18 J23100-RBS-luxR-DT -(1)1271
8/18 J23100-RBS-luxR-DT -(2)1271
8/18 J23100-RBS-lacZα-DT -(1)670
8/18 J23100-RBS-lacZα-DT -(2)670
8/18 RBS-lysis3 -(1)997
8/18 RBS-lysis3 -(2)997
8/18 P0440(RBS-tetR-DT) -(1)1154
8/18 P0440(RBS-tetR-DT) -(2)1154
negative control--
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s1min30cycles

File:Igku Aug19electrophoresis1.png

Samplebase pair
8/18 RBS control -(1)--
8/18 R0040(Ptet) -(1)686
8/18 R0040(Ptet) -(2)686
negative control--
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s30s30cycles

File:Igku Aug19electrophoresis2.png

Gel Extraction

Ashida

LaneDNAEnzyme
1100bp ladder--
38/18 K117000(lysis2) -1XbaI & PstI
58/18 K117000(lysis2) -1XbaI & PstI

File:Igku Aug19electrophoresis3
File:Igku Aug19electrophoresis4

Nameconcentration[µg/mL]260/280260/230
8/18 lysis2-1(XbaI & PstI)3.13.640.19
8/18 lysis2-1(XbaI & PstI)3.41.890.13

LB Medium Plate

Nakamoto

volume200ml
Bacto T2ypton 2g
Bacto yeast extract 1g
NaCl1g
Agar Pouder2g 

Restriction Enzyme Digestion

Tatsui

lysis2-1XbaIPstIBSA10xbufferMilliQtotal
10µL1µL1µL3µL3µL12µL30µL
2µL0.2µL--1µL1µL5.8µL10µL
2µL--0.2µL1µL1µL5.8µL10µL
2µLL----1µL6µL10µL
  • at 37°C, for 1h

Murata and Okazaki

8/19 lysis1-110xbufferXbaIPstIBSAMilliQtotal
2µL2µL0.5µL0.8µL2µL13µL20µL
0.5µL1µL0.2µL--1µL7.3µL10µL
0.5µL1µL--0.2µL1µL7.3µL10µL
0.5µL1µL----1µL7.5µL10µL
  • at 37°C, for 1h

Ligation

No name

Name NC
RBS0.30.3 lysis27.4
MilliQ7.4

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
1100bp ladder----
28/19 lysis1XbaIPstI
38/19 lysis1XbaI--
48/19 lysis1--PstI
58/19 lysis1----
68/19 lysis1(Gel Extraction Product)XbaIPstI

Transformation

Murata and Kojima

NameWellSampleCompetent CellsPlate
8/18 Plux+RBS-GFP-DT--2µL20µLCP
8/18 Plux(NC)--2µL20µLCP
8/19 RBS+lysis2 -- 2µL20µLAmp
8/15 Pbad-araCPlate5-14N2µL20µLKan
  • Incubate at 37°C

Gel Extraction

No name

LaneDNAEnzyme
1100bp ladder--
28/19 lysis1 ①XbaI & PstI

[fig before] [fig after]

Nameconcentration[µg/mL]260/280260/230
8/19 lysis1 ①(XbaI & PstI)87.81.060.82

Liquid Culture

Ashida

Samplemedium
8/18 Ptet -1Plusgrow medium(+CP)
8/18 Ptet -2Plusgrow medium(+CP)
8/18 RBS-tetR-DT -1Plusgrow medium(+CP) 
8/18 RBS-tetR-DT -2Plusgrow medium(+CP) 
8/18 J23100-GFP -1Plusgrow medium(+Amp)
8/18 J23100-luxR -1Plusgrow medium(+Amp)
8/18 J23100-luxR -2Plusgrow medium(+Amp)
8/18 J23100-lacZα -1Plusgrow medium(+Amp)
8/18 RBS-lysis3 -1Plusgrow medium(+Amp)
8/18 RBS-lysis3 -2Plusgrow medium(+Amp)

Master Plate

Ashida

NumberUse LB plate(+Amp)
18/18 J23100-GFP -1
28/18 J23100-luxR -1
38/18 J23100-luxR -2
48/18 J23100-lacZα -1
58/18 RBS-lysis3 -1
68/18 RBS-lysis3 -2
NumberUse LB plate(+CP)
18/18 Ptet -1
18/18 Ptet -2
38/18 RBS-tetR-DT -1
48/18 RBS-tetR-DT -2

Aug 20

Miniprep

Kojima

DNAconcentration[µg/µL]260/280260/230
8/18 Ptet -12201.691.87
8/18 Ptet -22101.691.71
8/18 RBS-tetR-DT -12361.631.69
8/18 RBS-tetR-DT -21821.651.98
8/18 J23100-RBS-GFP-DT -13341.712.12
8/18 J23100-RBS-luxR-DT -14401.671.60
8/18 J23100-RBS-luxR-DT -23441.681.83
8/18 J23100-RBS-lacZα-DT -12801.701.81
8/18 RBS-lysis3 -12821.671.76
8/18 RBS-lysis3 -22121.301.35

LB Medium Plate

Okazaki

volume200mL
Bacto Trypton2g
Bacto yeast extract1g
Nacl1g
Agar Powder2g
Amp40µl

1. Measuring reagents and put they in the Erlenmeyer flask.
2. diluting in measuring cylinder to 200ml total.
3. autoclaving.
4. adding ampicirine 40ml.

Ristriction Enzyme Digestion

Kojima and Ashida

8/20 Ptet -1(220µg/mL)EcoRIXbaI10x BSA10x buffer MMilliQtotal
4.5113317.530
0.50.20117.310
0.500.2117.310
0.500117.510
8/20 RBS-tetR-DT -2(182µg/mL)EcoRISpeI10x buffer HMilliQtotal
5.511319.530
0.60.2018.210
0.600.218.210
0.60018.410
8/20 J23100-RBS-GFP-DT(334µg/ml)EcoRISpeI10x buffer HMilliQtotal
3.01132230
0.30.2018.510
0.300.218.510
0.30018.710
8/20 J23100-RBS-luxR-DT -2(344µg/mL)EcoRIXbaIBSA10x buffer MMilliQtotal
2.9113319.130
0.30.20117.510
0.300.2117.510
0.300117.710
8/20 RBS-lysis3 -1(282µg/mL)EcoRISpeI10x buffer HMilliQtotal
3.511321.530
0.40.2018.410
0.400.218.410
0.40018.610
8/17 DT -1(188µg/mL)EcoRIXbaI10x BSA10x buffer MMilliQtotal
5.3113316.730
0.30.20117.510
0.300.2117.510
0.300117.710

Transformation

Nakamoto

NameSampleCompetent Cells(XL10-gold)TotalPlate
tRNA-Spinach-tRNA1µL10µL11µLAmp
tetR-aptamer 12_PC(076)1µL10µL11µLAmp
tetR-aptamer 12_1R(113)1µL10µL11µLAmp
tetR-aptamer 12_1M(105)1µL10µL11µLAmp
pT181 attenuator1µL10µL11µLAmp
Fusin1 attenuator1µL10µL11µLAmp
Fusion3m2 attenuator1µL10µL11µLAmp
pT181 antisense1µL10µL11µLAmp
Fusion1 antisense1µL10µL11µLAmp
Fusion6 antisense1µL10µL11µLAmp

incubate 37°C overnight 20:34~

Electrophoresis

Kojima

LaneSampleEnzyme1Enzyme2
18/20 Ptet -1EcoRIXbaI
28/20 Ptet -1EcoRI--
38/20 Ptet -1--XbaI
48/20 Ptet -1----
58/20 RBS-tetR-DT -2EcoRISpeI
68/20 RBS-tetR-DT -2EcoRI--
78/20 RBS-tetR-DT -2--SpeI
88/20 RBS-tetR-DT -2----
91kbp ladder----
108/20 J23100-RBS-GFP-DT -1EcoRISpeI
118/20 J23100-RBS-GFP-DT -1EcoRI--
128/20 J23100-RBS-GFP-DT -1--SpeI
138/20 J23100-RBS-GFP-DT -1----
148/20 J23100-RBS-luxR-DT -1EcoRIXbaI
158/20 J23100-RBS-luxR-DT -1EcoRI--
168/20 J23100-RBS-luxR-DT -1--XbaI
178/20 J23100-RBS-luxR-DT -1----
181kbp ladder----
198/20 RBS-lysis3 -1EcoRISpeI
208/20 RBS-lysis3 -1EcoRI--
218/20 RBS-lysis3 -1--SpeI
228/20 RBS-lysis3 -1----
231kbp ladder----
248/17 DTEcoRIXbaI
258/17 DTEcoRI--
268/17 DT--XbaI
278/17 DT----

Aug 21

Plusgrow Medium

Tatsui

  • material
  • 8/17 Plusgrow medium 50ml
  • 7/22 5000x Ampicillin 10µl
  1. Measuring materials and putting them in a 50ml tube

Liquid culture

Tatsui

Samplemedium
8/20 tRNA-spinach -(1)8/21 Plusgrow medium(+Amp)
8/20 tRNA-spinach -(2)8/21 Plusgrow medium(+Amp)
8/20 TetR-aptamer 12_P(1076) -(1)8/21 Plusgrow medium(+Amp)
8/20 TetR-aptamer 12_P(1076) -(2)8/21 Plusgrow medium(+Amp)
8/20 TetR-aptamer 12_1R(113) -(1)8/21 Plusgrow medium(+Amp)
8/20 TetR-aptamer 12_1R(113) -(2)8/21 Plusgrow medium(+Amp)
8/20 TetR-aptamer 12_1M(105) -(1) 8/21 Plusgrow medium(+Amp)
8/20 TetR-aptamer 12_1M(105) -(2)8/21 Plusgrow medium(+Amp)
8/20 PT181 attenuator -(1)8/21 Plusgrow medium(+Amp)
8/20 PT181 attenuator -(2)8/21 Plusgrow medium(+Amp)
8/20 Fusion1 attenuator -(1) 8/21 Plusgrow medium(+Amp)
8/20 Fusion1 attenuator -(2) 8/21 Plusgrow medium(+Amp)
8/20 Fusion3m2 attenuator -(1) 8/21 Plusgrow medium(+Amp)
8/20 Fusion3m2 attenuator -(2) 8/21 Plusgrow medium(+Amp)
8/20 PT181 antisense -(1) 8/21 Plusgrow medium(+Amp)
8/20 PT181 antisense -(2) 8/21 Plusgrow medium(+Amp)
8/20 Fusion1 antisense -(1) 8/21 Plusgrow medium(+Amp)
8/20 Fusion1 antisense -(2) 8/21 Plusgrow medium(+Amp)
8/20 Fusion6 antisense -(1) 8/21 Plusgrow medium(+Amp)
8/20 Fusion6 antisense -(2) 8/21 Plusgrow medium(+Amp)
  • incubate 37°C 10hour

Gel Extraction

Nakamoto and TaTsui

LaneDNAEnzyme
11kbp ladder--
28/20 Ptet -(1)EcoRI & XbaI
3
58/20 RBS-tetR-DT -(2)EcoRI & SpeI
6
88/20 Pconst-RBS-GFP-DT -(1)EcoRI & SpeI
9
118/20 RBS-lysis3 -(1)EcoRI & SpeI
12

[fig bifore][fig after]

Nameconcentration[µg/mL]260/280260/230
Ptet (EcoRI & XbaI)211.550.78
RBS-tetR-DT (EcoRI & SpeI)121.450.62
Pconst-RBS-GFP-DT (EcoRI & SpeI)111.360.60
RBS-lysis3 (EcoRI & SpeI)101.260.50

Ristriction Enzyme Digestion

Kojima and Nakamoto

8/20 Ptet-(1) EcoRIXbaI10x BSA10x buffer MMilliQtotal
4.5113317.530
0.50.20117.310
0.500.2117.310
0.500117.510
8/20 Pconst-RBS-luxR-DT-(2) EcoRIXbaI10x BSA10x buffer MMilliQtotal
2.9113319.130
0.30.20117.510
0.300.2117.510
0.300117.710
8/17 DTEcoRIXbaI10x BSA10x buffer MMilliQtotal
5.3113316.730
0.50.20117.310
0.500.2117.310
0.500117.510
  • incubate 37°C 1h

Electrophoresis

Kojima and Nakamoto

LaneSampleEnzyme1Enzyme2
11kbp ladder----
28/20 Ptet -(1)EcoRIXbaI
38/20 Ptet -(1)EcoRI--
48/20 Ptet -(1)--XbaI
58/20 Ptet -(1)----
68/20 J23100-RBS-luxR-DT -(2)EcoRIXbaI
78/20 J23100-RBS-luxR-DT -(2)EcoRI--
88/20 J23100-RBS-luxR-DT -(2)--XbaI
98/20 J23100-RBS-luxR-DT -(2)----
108/20 DTEcoRIXbaI
118/20 DTEcoRI--
128/20 DT--XbaI
138/20 DT----
141kbp ladder----
158/20 Ptet -(1)EcoRIXbaI
  • The reason why lane 2 and 15 were same is that the well of lane 2 might be broken.

[fig]
Kojima

LaneSampleEnzyme1Enzyme2
11kbp ladder----
28/20 J23100-RBS-luxR-DT -(2)EcoRIXbaI
38/20 DTEcoRIXbaI

[fig]

Gel Extraction

Honda

LaneDNAEnzyme
11kb ladder--
28/21 J23100-RBS-luxR-DT 25µLE+X
38/21 J23100-RBS-luxR-DT 25µLE+X
4----
58/21 Ptet 25µLE+X
68/21 Ptet 25µLE+X

File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx

Electrophoresis

Honda

LaneSampleEcoRIXbaI
11kb ladder----
28/21 DT++
38/21 DT+--
48/21 DT--+
58/21 DT----

File:Igku xxxxxx.xxx

Ligation

No name

Name-?-NC
8/21 Pcn-RBS-luxR-DT(+Amp)2.32.3
8/21 Pcn-RBS-GFP-DT1.70
Ligation High buffer22
MilliQ01.7
total66
Name-?-NC
8/18 Plux(+CP)1.11.1
8/19 RBS-GFP-DT50
Ligation High buffer3.13.1
MilliQ05
total9.29.2
Name-?-NC
8/18 RBS(+Amp)2.2--
8/19 lysis16.0--
Ligation High buffer4.1--
MilliQ0--
total12.3--
Name-?-NC
8/18 RBS(+Amp)2.22.2
8/19 lysis23.60
Ligation High buffer2.92.9
MilliQ03.6
total8.78.7

Transformation

LB Medium Plate

Gel Extraction

Okazaki

LaneDNAEnzyme
11kb ladder3µL
2----
38/21 DT E+X12.5µL
48/21 DT E+X12.5µL

File:Igku xxbeforexx.xxx
File:Igku xxafterxx.xxx

Miniprep

Tatsui, Kojima, and Nakamoto

DNAconcentration[µg/mL]260/280260/230実濃度
tRNA-spinach-(1)11.71.642.14234
tRNA-spinach-(2)22.01.601.91440
tetR-aptamer 12-p(1076)-(1)7.51.612.03150
tetR-aptamer 12-p(1076)-(2)18.71.451.66374
tetR-aptamer 12-1R(113)-(1)15.21.682.21304
tetR-aptamer 12-1R(113)-(2)19.11.471.66382
tetR-aptamer 12-1M(105)-(1)18.71.682.20374
tetR-aptamer 12-1M(105)-(2)14.31.311.43286
PT181 attenuator-(1)10.51.662.11330
PT181 attenuator-(2)18.51.682.18270
Fusion1 attenuator-(1)15.31.672.20306
Fusion1 attenuator-(2)14.11.601.94282
Fusion3m2 attenuator-(1)16.61.682.25332
Fusion3m2 attenuator-(2)16.31.672.10326
PT181 antisense-(1)8.91.641.68178
PT181 antisense-(2)5.81.631.95116
Fusion1 antisense-(1)15.01.672.21300
Fusion1 antisense-(2)8.21.421.46164
Fusion6 antisense-(1)16.81.652.18336
Fusion6 antisense-(2)9.61.652.01192

Aug 22

Liquid culture

Nakamoto

SampleMedium
8/21 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT (1)8/21 Plusgrow medium (+Amp)
8/21 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT (2)8/21 Plusgrow medium (+Amp)
8/21 Pcon-RBS-GFP-DT control (1)8/21 Plusgrow medium (+Amp)
8/21 Pcon-RBS-GFP-DT control (2)8/21 Plusgrow medium (+Amp)
8/21 RBS-lysis1 (1)8/21 Plusgrow medium (+Amp)
8/21 RBS-lysis1 (2)8/21 Plusgrow medium (+Amp)
8/21 RBS control (1)8/21 Plusgrow medium (+Amp)
8/21 RBS control (2)8/21 Plusgrow medium (+Amp)
8/21 Ptet(pm)-RBS-tetR-DT (1)8/21 Plusgrow medium (+Amp)
8/21 Ptet(pm)-RBS-tetR-DT (2)8/21 Plusgrow medium (+Amp)
8/21 Ptet(pm) control (1)8/21 Plusgrow medium (+Amp)
8/21 Ptet(pm) control (2)8/21 Plusgrow medium (+Amp)
8/21 Plux-RBS-GFP-DT (1)8/21 Plusgrow medium (+Amp)
8/21 pSB1C3(BBa_J04450) (1)8/21 Plusgrow medium (+Amp)
8/21 pSB1C3(BBa_J04450) (2)8/21 Plusgrow medium (+Amp)

incubate 37°C 10hour

Colony PCR

Kojima

Samplebase pair
8/21 RBS-lysis1(1)400
8/21 RBS-lysis1(2)400
8/21 RBS control(1)--
8/21 RBS control(2)--
negative control--
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s30s30cycles
Samplebase pair
8/21 Pcon-GFP-DT-Pcon-RBS-luxR-DT (1)2143
8/21 Pcon-GFP-DT-Pcon-RBS-luxR-DT (2)2143
8/21 Pcon-RBS-luxR control(1)--
8/21 Pcon-RBS-luxR control(2)--
negative control--
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s2min30cycles
Samplebase pair
8/21 Ptet-RBS-tetR-DT (1)1216
8/21 Ptet-RBS-tetR-DT (2)1216
8/21 Ptet control (1)--
8/21 Ptet control (2)--
8/21 Plux-RBS-GFP-DT (1)1227
8/21 pSB1C3(BBa_J04450) (1)1353
8/21 pSB1C3(BBa_J04450) (2)1353
negative control--
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s1min30cycles

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
1pT181antisenseEcoRISpeI
2pT181antisenseEcoRI--
3pT181antisense--SpeI
4pT181antisense----
5pT181antisenseXbaIPstI
6pT181antisenseXbaI--
7pT181antisense--PstI
8pT181antisense----
9100bp ladder----
10Fusion1 attenuatorEcoRISpeI
11Fusion1 attenuatorEcoRI--
12Fusion1 attenuator--SpeI
13Fusion1 attenuator----
14Fusion1 attenuatorXbaIPstI
15Fusion1 attenuatorXbaI--
16fusion1 attenuator--PstI
17fusion1 attenuator----

File:Igku xxxxxx.xxx

LaneSampleEnzyme1Enzyme2
1Fusion3 3m2 attenuatorEcoRISpeI
2Fusion3 3m2 attenuatorEcoRI--
3Fusion3 3m2 attenuator--SpeI
4Fusion3 3m2 attenuator----
5Fusion3 3m2 attenuatorXbaIPstI
6Fusion3 3m2 attenuatorXbaI--
7Fusion3 3m2 attenuator--PstI
8Fusion3 3m2 attenuator----
9100bp ladder----
10aptamer 12-1MEcoRISpeI
11aptamer 12-1MEcoRI--
12aptamer 12-1M--SpeI
13aptamer 12-1M----
14aptamer 12-PEcoRISpeI
15aptamer 12-PEcoRI--
16aptamer 12-1P--SpeI
17aptamer 12-1P----

File:Igku xxxxxx.xxx

Restriction Enzyme Digestion

Miniprep

Electrophoresis

Aug 23

Electrophoresis

Restriction Enzyme Digestion

Electrophoresis

===Gel Extraction===