Team:Kyoto/Notebook

From 2013.igem.org

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{{Kyoto/header}}
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<div id="kyoto-main">
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<div class="texts">
<div id="notebooks">
<div id="notebooks">
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{{Kyoto/Notebook/Calender}}
{{Kyoto/Notebook/Calender}}
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==template==
 
-
===Transformation===
 
-
<!-- こっから -->
 
-
<div class="experiment">
 
-
<span class="author">じっけんしゃ</span>
 
-
{| class="wikitable"
 
-
!Name||Well||Sample||Competent Cells||Total||Plate
 
-
|-
 
-
|いでんし||ぱーつのうぇる||なんちゃら&micro;L||ごにょごにょ&micro;L||ほにゃらら&micro;L||こーせーぶっしつ
 
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|-
 
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|いかどうよう|| || || || ||
 
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|-
 
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| || || || || ||
 
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|}
 
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</div>
 
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<!-- ここまでをコピーしてね -->
 
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===Restriction Enzyme Digestion===
 
-
<!-- こっから -->
 
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<div class="experiment">
 
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<span class="author">じっけんしゃ</span>
 
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{| class="wikitable"
 
-
!DNA||Buffer||restricion enzyme||restriction enzyme||MilliQ||total
 
-
|-
 
-
|（°°） &micro;L|| &micro;L|| &micro;L|| &micro;L|| &micro;L|| &micro;L
 
-
|}
 
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</div>
 
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<!-- ここまでをコピーしてね -->
 
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===Electrophoresis===
 
-
 
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<!-- こっから -->
 
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<div class="experiment">
 
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<span class="author">じっけんしゃ</span>
 
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{| class="wikitable"
 
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!Lane||Sample||Enzyme1||Enzyme2
 
-
|-
 
-
|（°Д°）|| || ||
 
-
|}
 
-
[[File:igku_xxxxxx.xxx]]<br>
 
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</div>
 
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<!-- ここまでをコピーしてね -->
 
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===Ligation===
 
-
 
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<!-- こっから -->
 
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<div class="experiment">
 
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<span class="author">じっけんしゃ</span>
 
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{| class="wikitable"
 
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!Name|| 目的（番号？）？||NCとか &micro;L
 
-
|-
 
-
|(＞o＜)|| ||
 
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|}
 
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</div>
 
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<!-- ここまでをコピーしてね -->
 
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-
===Gel Extraction===
 
-
<!-- こっから -->
 
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<div class="experiment">
 
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<span class="author">じっけんしゃ</span>
 
-
{| class="wikitable"
 
-
!Lane||DNA||Enzyme
 
-
|-
 
-
|（＞ω・）b || ||
 
-
|-
 
-
| || ||
 
-
|}
 
-
[[File:igku_xxbeforexx.xxx]]<br>
 
-
[[File:igku_xxafterxx.xxx]]<br>
 
-
{| class="wikitable"
 
-
!Name||quantity||concentration[&micro;g/mL]||260/280||260/230
 
-
|-
 
-
|（´ω｀）|| || || ||
 
-
|-
 
-
|（ΦωΦ^）|| || || ||
 
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|}
 
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</div>
 
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<!-- ここまでをコピーしてね -->
 
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===Colony PCR===
 
-
<!-- ここから -->
 
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<div class="experiment">
 
-
<span class="author">No name</span>
 
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{| class="wikitable"
 
-
!Sample||base pair
 
-
|-
 
-
|(｀・ω・´)||
 
-
|}
 
-
{| class="wikitable"
 
-
!PreDenature||Denature||Annealing||Extension||cycle
 
-
|-
 
-
|　&deg;C||　&deg;C||　&deg;C||　&deg;C||--
 
-
|-
 
-
|（◎Д◎）　||　||　||　||
 
-
|}
 
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</div>
 
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<!-- ここまでをコピーしてね -->
 
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===Liquid Culture===
 
-
<!-- ここから -->
 
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<div class="experiment">
 
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<span class="author">No name</span>
 
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{| class="wikitable"
 
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!Sample||medium
 
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|-
 
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|(・ω・) ||
 
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|-
 
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|（・Ｘ・） ||　
 
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|}
 
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</div>
 
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<!-- ここまでをコピーしてね -->
 
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===Miniprep===
 
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<div class="experiment">
 
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<span class="author">No name</span>
 
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{|class="wikitable"
 
-
!DNA||concentration[&micro;g/mL]||260/280||260/230
 
-
|-
 
-
|(・▽・)||　||　||
 
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|}
 
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</div>
 
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<!-- ここまでをコピーしてね -->
 
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===Master Plate===
 
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<!-- ここから -->
 
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<div class="experiment">
 
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<span class="author">No name</span>
 
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{| class="wikitable"
 
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!Number||Use LB plate (+抗生物質)
 
-
|-
 
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| ʅ（´◔౪◔）ʃ||
 
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|}
 
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</div>
 
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<!-- ここまでをコピーしてね -->
 
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{{Kyoto/Notebook/Aug_8}}
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{{Kyoto/Notebook/Aug_9}}
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{{Kyoto/Notebook/Aug_10}}
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{{Kyoto/Notebook/Aug_11}}
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{{Kyoto/Notebook/Aug_15}}
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{{Kyoto/Notebook/Aug_16}}
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{{Kyoto/Notebook/Aug_17}}
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{{Kyoto/Notebook/Aug_18}}
{{Kyoto/Notebook/Aug_19}}
{{Kyoto/Notebook/Aug_19}}
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{{Kyoto/Notebook/Sep_18}}
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{{Kyoto/Notebook/Sep_19}}
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{{Kyoto/footer}}

Latest revision as of 12:47, 10 October 2013

count down

Contents

Calender

Aug 8

Transformation

Inoue and Nakamoto

NameSampleCompetent CellsTotalPlate
BBa_J440002 µL20 µL (7/30)22 µLLB (+Amp)
BBa_J440002 µL20 µL (XL10-gold)22 µLLB (+Amp)
BBa_K7470282 µL20 µL (XL10-gold)22 µLLB (+CP)
BBa_K7470282 µL20 µL (7/30)22 µLLB (+CP)
BBa_J231002 µL20 µL (XL10-gold)22 µLLB (+Amp)
BBa_J231062 µL20 µL (XL10-gold)22 µLLB (+Amp)
BBa_E20302 µL20 µL (XL10-gold)22 µLLB (+Kan)

Aug 9

Transformation

Kojima and Nakamoto

NameSampleCompetent CellsTotalPlate
BBa_J440002 µL20 µL22 µLLB (+Amp)
BBa_K7470282 µL20 µL22 µLLB (+CP)
BBa_J231002 µL20 µL22 µLLB (+Amp)
BBa_J231062 µL20 µL22 µLLB (+Amp)
BBa_E20302 µL20 µL22 µLLB (+Kan)

Aug 10

Colony PCR

Nakamoto

Sample
8/9 BBa_J44000 -(1)
8/9 BBa_J44000 -(2)
NC
PreDenatureDenatureAnnealingExtensioncycle
94 °C94 °C55 °C68 °C--
5 min30 sec30 sec30 sec30 cycles
Sample
8/9 BBa_J23106
8/9 BBa_J23100 -(1)
8/9 BBa_J23100 -(2)
NC
PreDenatureDenatureAnnealingExtensioncycle
94 °C94 °C55 °C68 °C--
5 min30 sec30 sec1 min30 cycles

Electrophoresis

Tatsui and Nakamoto

LaneSample
100bp ladder
48/9 BBa_J44000 -1
58/9 BBa_J44000 -2
6NC

Igku Aug10 Electrophoresis BBa 1.jpg

LaneSample
1kbp ladder
18/9 BBa_J23106
28/9 BBa_J23100 -1
38/9 BBa_J23100 -2
4NC

Igku Aug10electrophoresis1.jpg

Liquid Culture

Nakamoto

Samplemedium
8/9 BBa-J23106LB(+Amp)
8/9 BBa-J23100-1LB(+Amp)
8/9 BBa-J23100-2LB(+Amp)
8/9 BBa-J44000-1LB(+Amp)
8/9 BBa-J44000-2LB(+Amp)

LB Medium Plate(+Ampicillin)

Nakamoto & Tatsui

volume200ml
Bacto T2ypton 2g
Bacto yeast extract 1g
NaCl1g
0.4M NaOHaq500µL
Agarose Pouder2g
Ampicillin40µL

Transformation

Hirano

NameSampleCompetent CellsTotalPlate
tetR3µL19µL22µLKan
RBS3µL19µL22µLAmp
lacZα3µL19µL22µLCP
lacI3µL19µL22µLCP
Plac3µL19µL22µLCP

Aug 11

Miniprep

Nakamoto

DNAconcentration[µg/mL]
8/9 BBa_J44000 -(2)100
8/9 BBa_J23106154
8/9 BBa_J23100144
8/10 T7-His-FT92

Colony PCR

Kojima and Yasuda

Sample
BBa_B0034 -(2)
BBa_B0034 -(1)
BBa_R0011 -(2)
BBa_R0011 -(1)
NC
PreDenatureDenatureAnnealingExtensioncycle
94 °C94 °C55 °C68 °C--
5 min30 s30 s20 s30 cycles
Sample
BBa_D0412 -(1)
BBa_D0412 -(2)
NC
PreDenatureDenatureAnnealingExtensioncycle
94 °C94 °C55 °C68 °C--
5 min30 s30 s1 min 30 s30 cycles

Restriction Enzyme Digestion

Kojima anda Yasuda

8/9 BBa_J44000 2EcoRIPstI10x buffer HMilliQtotal
20.20.216.610
20.2--16.810
2--0.216.810
2----17.010
8/9 BBa_J23106EcoRIPstI10x buffer HMilliQtotal
1.30.20.217.310
1.30.2--17.510
1.3--0.217.510
1.3----17.710
8/9 BBa_J23100 2EcoRIPstI10x buffer HMilliQtotal
1.30.20.217.310
1.30.2--17.510
1.3--0.217.510
1.3----17.710
  • incubated at 37 °C for 1 hour.

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
1100bp ladder----
28/9 BBa_J44000EcoRIPstI
38/9 BBa_J44000EcoRI--
48/9 BBa_J44000--PstI
58/9 BBa_J44000----
68/9 BBa_J23106EcoRIPstI
78/9 BBa_J23106EcoRI--
88/9 BBa_J23106--PstI
98/9 BBa_J23106----
108/9 BBa_J23100EcoRIPstI
118/9 BBa_J23100EcoRI--
128/9 BBa_J23100--PstI
138/9 BBa_J23100----

Igku Aug11electrophoresis1.jpg

LaneSample
1100bp ladder
2BBa_B0034 (1)
3BBa_B0034 (2)
4BBa_R0011 (1)
5BBa_R0011 (2)
6NC
7100bp ladder

11 electrophoresis2.jpg

LaneSample
11kb ladder
2BBa_P0412 (1)
3BBa_P0412 (2)
4NC

Igku Aug11electrophoresis3.jpg

Aug 15

Liquid Culture

Nakamoto

Samplemedium
8/10 BBa_B0034(1)LB (+Amp)
8/10 BBa_B0034(2)LB (+Amp)
8/10 BBa_R0011(1)LB (+CP)
8/10 BBa_R0011(2)LB (+CP)
8/10 BBa_P0412(1)LB (+CP)
8/10 BBa_P0412(2)LB (+CP)

Transformation

Nakamoto

NameSampleCompetent CellsTotalPlate
BBa_I0500(Pbad/araC)21719LB (+Kan)
BBa_F1610(RBS-luxI-DT)21719LB (+Amp)
BBa_R0061(Plux)21719LB (+CP)
BBa_I13504(RBS-GFP-DT)21719LB (+CP)
BBa_I0462(RBS-luxR-DT)21719LB (+Amp)
BBa_K117000(lysis1)21719LB (+CP)
BBa_K112806(lysis2)21719LB (+CP)
BBa_K748002(lysis3)21719LB (+CP)
BBa_B0015(DT)21719LB (+CP)
BBa_Q04400(RBS-tetR-DT-Ptet)21719LB (+Kan)
BBa_I732020(RBS-lacZα-DT)21719LB (+CP)
BBa_J2310021719LB (+Amp)

Miniprep

No name

DNAconcentration[µg/mL]260/280260/230
BBa_B0034(1)84.12.112.01
BBa_B0034(2)86.92.032.02
BBa_R0011(1)78.32.262.02
BBa_R0011(2)88.92.242.02
BBa_P0412(1)15.612.101.93
BBa_P0412(2)12.932.181.91

Aug 16

Colony PCR

Tatsui and Ashida

Samplebase pair
8/15 BBa_I13504(RBS-GFP-DT)(1)1174
8/15 BBa_I13504(RBS-GFP-DT)(2)1174
8/15 BBa_I0462(RBS-luxR-DT)(1)1174
8/15 BBa_I0462(RBS-luxR-DT)(2)1174
8/15 BBa_K748002(lysis3)1053
NC--
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s1min30cycles
Samplebase pair
8/15 BBa_R0061(Plux)344
8/15 BBa_K117000(lysis1)(1)456
8/15 BBa_K117000(lysis1)(2)456
8/15 BBa_B0015(DT)443
8/15 BBa_I732020(RBS-lacZα-DT)703
NC--
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s40s30cycles

Electrophoresis

No name

LaneSample
1100bp ladder
2BBa_I13504(RBS-GFP-DT) -(1)
3BBa_I13504(RBS-GFP-DT) -(2)
4BBa_I0462(RBS-luxR-DT) -(1)
5BBa_I0462(RBS-luxR-DT) -(2)
6BBa_K748002(lysis3)
7NC
8100bp ladder

Igku 8161.jpg

LaneSample
1100bp ladder
2BBa_R0061(Plux)
3BBa_K117000(lysis1) -(1)
4BBa_K117000(lysis1) -(1)
5BBa_B0015(DT)
6BBa_I732020(RBS-lacZα-DT)
7NC
8100bp ladder

Igku 8162.jpg

Master Plate

Tatsui

NumberUse LB plate (+CP)
1BBa_I13504(RBS-GFP-DT)(1)
2BBa_I13504(RBS-GFP-DT)(2)
3BBa_K748002(lysis3)
4BBa_R0061(Plux)
5BBa_K117000(lysis1)(1)
6BBa_K117000(lysis1)(2)
7BBa_B0015(DT)
8BBa_I732020(RBS-lacZα-DT)
NumberUse LB plate (+Amp)
1BBa_I0462(RBS-luxR-DT)(1)
2BBa_I0462(RBS-luxR-DT)(2)

Transformation

Hirano

NameSampleCompetent CellsTotalPlate
pBR3221µL100µL(XL-10gold)101µLAmp
pBR3221µL50µL(takara)51µLAmp
Pbad/araC2µL20µL22µLKan
RBS-luxI-DT2µL20µL22µLKan

Liquid Culture

Yoshdia

Samplemedium
8/15 BBa_I13504(RBS-GFP-DT))(1)Plusgrow medium (+CP)
8/15 BBa_I13504(RBS-GFP-DT)(2)Plusgrow medium (+CP)
8/15 BBa_I0462(RBS-luxR-DT)(2)Plusgrow medium (+Amp)
8/15 BBa_K748002(lysis3)Plusgrow medium (+CP)
8/15 BBa_R0061(Plux)Plusgrow medium (+CP)
8/15 BBa_K117000(lysis1)(1)Plusgrow medium (+CP)
8/15 BBa_K117000(lysis1)(2)Plusgrow medium (+CP)
8/15 BBa_B0015(DT)Plusgrow medium (+CP)
8/15 BBa_I732020(RBS-lacZα-DT)Plusgrow medium (+CP)

Aug 17

Colony PCR

Nakamoto

Samplebase pair
8/15 BBa_K112806(lysis2) -(1)828
NC--
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s1min30cycles
Samplebase pair
8/10 BBa_Q0400 -(1)3995
NC--
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s30s30cycles

Electrophoresis

No name

LaneSample
11kbp ladder
2K112806(lysis2) -1
3NC
4Q04400(tetR) -1
5NC
61kbp ladder

Igku Aug17colonyPCR.jpg

Liquid Culture

Nakamoto

Samplemedium
8/15 BBa_K112806(lysis2) -(1)Plusgrow medium(+CP)
8/15 BBa_Q04400(tetR) -(1)Plusgrow medium(+Amp)
8/16 pBR322 -(1)Plusgrow medium(+Amp)

Master Plate

Nakamoto

NumberUse LB plate (+CP)
1BBa_K112806(lysis2) -(1)
NumberUse LB plate (+Kana)
1BBa_Q04400(tetR) -(1)

Miniprep

Nakamoto and Murakami

DNAconcentration[µg/mL]260/280260/230
K748002(lysis3) -(1)3301.662.08
B0015(DT) -(1)1881.731.82
R0061(Plux) -(1)761.641.30
K117000(lysis1) -(1)2861.712.01
K117000(lysis1) -(2)2481.691.78
I732020(RBS-lacZα-DT) -(1)1981.711.93
I13504(RBS-GFP-DT) -(1)2481.692.03
I13504(RBS-GFP-DT) -(2)2461.551.73
I0462(RBS-luxR-DT) -(1)3261.651.74

Restriction Enzyme Digestion

Murata, Murakami, Nakamoto, Ashida, Matsumoto and Yoshida

8/11 J23100 -(2)SpeIPstIBufferMilliQtotal
2 cuts4.0µL1.0µL1.0µL3.0µL21µL30µL
1 cut1.3µL0.2µL--1.0µL7.5µL10µL
1 cut1.3µL--0.2µL1.0µL7.5µL10µL
NC1.3µL----1.0µL7.7µL10µL
8/15 B0034(RBS) -(2)SpeIPstIBufferMilliQtotal
2 cuts6.0µL1.0µL1.0µL3.0µL19µL30µL
1 cut2.0µL0.2µL--1.0µL6.8µL10µL
1 cut2.0µL--0.2µL1.0µL6.8µL10µL
NC2.0µL----1.0µL7.0µL10µL
8/17 K117000(lysis1) -(1)XbaIPstIBSABufferMilliQtotal
2 cuts2.0µL1.0µL1.0µL3.0µL3.0µL20µL30µL
1 cut1.0µL0.2µL--1.0µL1.0µL6.8µL10µL
1 cut1.0µL--0.2µL1.0µL1.0µL6.8µL10µL
NC1.0µL----1.0µL1.0µL7.0µL10µL
8/17 K748002(lysis3) -(1)XbaIPstIBSABufferMilliQtotal
2 cuts2.0µL1.0µL1.0µL3.0µL3.0µL20µL30µL
1 cut1.0µL0.2µL--1.0µL1.0µL6.8µL10µL
1 cut1.0µL--0.2µL1.0µL1.0µL6.8µL10µL
NC1.0µL----1.0µL1.0µL7.0µL10µL
8/17 I13504(RBS-GFP-DT) -(1)XbaIPstIBSABufferMilliQtotal
2 cuts2.0µL1.0µL1.0µL3.0µL3.0µL20µL30µL
1 cut1.0µL0.2µL--1.0µL1.0µL6.8µL10µL
1 cut1.0µL--0.2µL1.0µL1.0µL6.8µL10µL
NC1.0µL----1.0µL1.0µL7.0µL10µL
8/17 I0462(RBS-luxR-DT) -(1)XbaIPstIBSABufferMilliQtotal
2 cuts2.0µL1.0µL1.0µL3.0µL3.0µL20µL30µL
1 cut1.0µL0.2µL--1.0µL1.0µL6.8µL10µL
1 cut1.0µL--0.2µL1.0µL1.0µL6.8µL10µL
NC1.0µL----1.0µL1.0µL7.0µL10µL
8/17 I732020(RBS-lacZα-DT) -(1)XbaIPstIBSABufferMilliQtotal
2 cuts2.0µL1.0µL1.0µL3.0µL3.0µL20µL30µL
1 cut1.0µL0.2µL--1.0µL1.0µL6.8µL10µL
1 cut1.0µL--0.2µL1.0µL1.0µL6.8µL10µL
NC1.0µL----1.0µL1.0µL7.0µL10µL
8/11 R6061 -(1)SpeIPstIBufferMilliQtotal
2 cuts8.0µL1.0µL1.0µL3.0µL17µL30µL
1 cut3.0µL0.2µL--1.0µL5.8µL10µL
1 cut3.0µL--0.2µL1.0µL5.8µL10µL
NC3.0µL----1.0µL6.0µL10µL
  • incubate at 37 °C, for 1 h

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
1J23100 -②SpeIPstI
2J23100 -②SpeI--
3J23100 -②--PstI
4J23100 -②----
5B0034(RBS) -(2)SpeIPstI
6B0034(RBS) -(2)SpeI--
7B0034(RBS) -(2)--PstI
8B0034(RBS) -(2)----
91kb ladder----
10K11700(lysis1) -(1)XbaIPstI
11K11700(lysis1) -(1)XbaI--
12K11700(lysis1) -(1)--PstI
13K11700(lysis1) -(1)----
14K748002(lysis3) -(1)XbaIPstI
15K748002(lysis3) -(1)XbaI--
16K748002(lysis3) -(1)--PstI
17K748002(lysis3) -(1)----

Igku Aug17 Electrophoresis(1).jpg

LaneSampleEnzyme1Enzyme2
18I13504(RBS-GFP-DT) -(1)XbaIPstI
19I13504(RBS-GFP-DT) -(1)XbaI--
20I13504(RBS-GFP-DT) -(1)--PstI
21I13504(RBS-GFP-DT) -(1)----
22I0462(RBS-luxR-DT) -(1)XbaIPstI
23I0462(RBS-luxR-DT) -(1)XbaI--
24I0462(RBS-luxR-DT) -(1)--PstI
25I0462(RBS-luxR-DT) -(1)----
261kb ladder----
27I732020(RBS-lacZα-DT) -(1)XbaIPstI
28I732020(RBS-lacZα-DT) -(1)XbaI--
29I732020(RBS-lacZα-DT) -(1)--PstI
30I732020(RBS-lacZα-DT) -(1)----
31R0061(Plux) -(1)SpeIPstI
32R0061(Plux) -(1)SpeI--
33R0061(Plux) -(1)--PstI
34R0061(Plux) -(1)----

Igku Aug17 Electrophoresis(2).jpg

Miniprep

Murata

DNAconcentration[µg/mL]260/280260/230
8/17 PBR322(TaKaRa) -(1)471.681.55
8/17 K112806(lysis2) -(1)641.671.70

Aug 18

Gel Extraction

Yoshida and Kojima

LaneDNAEnzyme
1100kb ladder--
28/11 J23100 -②--
38/11 J23100 -②--
4----
58/17 K117000(lysis1) -1--
68/17 K117000(lysis1) -1--
7----
88/17 K748002(lysis3) -1--
98/17 K748002(lysis3) -1--
10----
118/17 I13504(RBS-GFP-DT) -1--
128/17 I13504(RBS-GFP-DT) -1--

Igku Aug18 Gel Extraction before.jpg
Igku Aug18 Gel Extraction1-2.jpg

LaneDNAEnzyme
131kb ladder--
148/17 I0462(RBS-luxR-DT) -1--
158/17 I0462(RBS-luxR-DT) -1--
16----
178/17 I732020(RBS-lacZα-DT) -1--
188/17 I732020(RBS-lacZα-DT) -1--

Igku Aug18 Gel Extraction2 before.jpg
Igku Aug18 Gel Extraction2-2.jpg

Nameconcentration[µg/mL]260/280260/230
8/14 J2310001.71.610.04
8/17 K748002(lysis3) -15.42.390.45
8/17 I13504(RBS-GFP-DT) -13.43.330.19
8/17 I0462(RBS-luxR-DT) -14.42.070.37
8/17 I732020(RBS-lacZα-DT) -12.92.170.29

Ligation

Ashida

stateVectorInserterLigation High ver.2
experimentJ231009.0RBS-lacZα-DT8.63.5
experimentJ231009.0RBS-GFP-DT7.33.5
experimentRBS 1lysis34.62.8
experimentPlux 1GFP7.33.5
experimentJ23100 9.0----3.5
experimentRBS1.0----0.5
experimentPlux1.0----0.5

incubate 16°C 1hour

Restriction Enzyme Digestion

Murata and Okazaki

8/17K112806(lysis2)-1XbaIPstIBufferBSAMilliQtotal
2 cuts2.0µL1.0µL1.0µL3.0µL3.0µL 3.0µL30 µL
1 cuts1.0&mic6/8ro;L0.2µL--1.0µL1.0µL 6.8µL10 µL
1 cuts1.0µL--0.2µL1.0µL1.0µL 6.8µL10 µL

Electrophoresis

Okazaki and Honda

LaneSampleEnzyme1Enzyme2
1100bp ladder----
2lysis1 XbaIPstI
3lysis1XbaI--
4lysis1--PstI
5lysis1----
6lysis2XbaIPstI
7lysis2XbaI--
8lysis2--PstI
9lysis2----
10100bp ladder----

Igku Aug18electrophoresis.jpg

Colony PCR

No name

Samplebase pair
8/8 LBKana--
8/16 Pbad araC--
NC--
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s1min30

Liquid Culture

No name

Samplemedium
7/8 LB Kana Plusgrow(+Kana)
8/16 Pbad araCPlusgrow(+Kana)

LB Medium Plate

No name

volume200ml
Bacto Trypton2g
Bacto yeasy extract1g
NaCl1g
Agar Powder2g

Transformation

No name

NameSampleCompetent CellsTotalPlate
8/18 J23100+RBS-LacZα-DT2µL20µL22µLAmp
8/18 J23100+RBS-GFP-DT2µL20µL22µLAmp
8/18 J23100+RBS-luxR-DT 2µL20µL22µLAmp
RBS+lysis32µL20µL22µLAmp
Plux+GFP2µL20µL22µLCP
J231002µL20µL22µLAmp
RBS2µL20µL22µLAmp
Plux2µL20µL22µLCP

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
1100bp ladder-- --
28/16Pbad araC-1----
38/16Pbad araC-2----
4NC----

Igku Aug18electrophoresis2.jpg

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

Igku Aug19electrophoresis1(colony PCR).png

LaneDNA
1100bp ladder
28/18 NC J23100(1)
38/18 J23100-RBS-GFP-DT-(1)
48/18 J23100-RBS-GFP-DT-(2)
58/18 J23100-RBS-luxR-DT-(1)
68/18 J23100-RBS-luxR-DT-(2)
78/18 J23100-RBS-lacZα-DT-(1)
88/18 J23100-RBS-lacZα-DT-(2)
98/18 RBS-lysis3-(1)
108/18 RBS-lysis3-(2)
118/18 R0400(RBS-tetR-DT)-(1)
128/18 R0400(RBS-tetR-DT)-(2)
13NC
14100bp ladder

Igku Aug19electrophoresis1(Colony PCR).png

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


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

Gel Extraction

Ashida

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

Igku Aug19electrophoresis3(Gel Extraction).png
Igku Aug19electrophoresis4(Gel Extraction).png

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

lysis2-1XbaIPstI10xbufferBSAMilliQtotal
2 cuts10µL1µL1µL3µL3µL12µL30µL
1 cut2µL0.2µL--1µL1µL5.8µL10µL
1 cut2µL--0.2µL1µL1µL5.8µL10µL
NC2µL----1µL1µ6µL10µL
  • at 37°C, for 1h

Murakami and Okazaki

8/19 lysis1-1XbaIPstI10xbufferBSAMilliQtotal
2 cuts2µL0.5µL0.8µL2µL2µL13µL20µL
1 cut0.5µL0.2µL--1µL1µL7.3µL10µL
1 cut0.5µL--0.2µL1µL1µL7.3µL10µL
NC0.5µL----1µL1µL7.5µL10µL
  • at 37°C, for 1h

Igku Aug19 Restriction Enzyme Digestion(lysis2-1(1)).jpg

Ligation

No name

stateVectorInserterLigation High ver.2
experimentRBS0.3lysis27.44
NCRBS0.3MilliQ7.44

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

Igku Aug19 Electrophoresis lysis1.jpg

Transformation

Murata and Kojima

NameSampleCompetent CellsPlate
8/18 Plux+RBS-GFP-DT2µL20µLCP
8/18 Plux(NC)2µL20µLCP
8/19 RBS+lysis2 2µL20µLAmp
8/15 Pbad-araC2µL20µLKan
  • Incubate at 37°C

Gel Extraction

No name

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

Igku Aug19 Gel Extraction(lysis①-1)before.jpg
Igku Aug19 Gel Extraction(lysis①-1)after.jpg

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
2 cuts4.5113317.530
1 cut0.50.20117.310
1 cut0.500.2117.310
NC0.500117.510
8/20 RBS-tetR-DT -2(182µg/mL)EcoRISpeI10x buffer HMilliQtotal
2 cuts5.511319.530
1 cut0.60.2018.210
1 cut0.600.218.210
NC0.60018.410
8/20 J23100-RBS-GFP-DT(334µg/ml)EcoRISpeI10x buffer HMilliQtotal
2 cuts3.01132230
1 cut0.30.2018.510
1 cut0.300.218.510
NC0.30018.710
8/20 J23100-RBS-luxR-DT -2(344µg/mL)EcoRIXbaIBSA10x buffer MMilliQtotal
2 cuts2.9113319.130
1 cut0.30.20117.510
1 cut0.300.2117.510
NC0.300117.710
8/20 RBS-lysis3 -1(282µg/mL)EcoRISpeI10x buffer HMilliQtotal
2 cuts3.511321.530
1 cut0.40.2018.410
1 cut0.400.218.410
NC0.40018.610
8/17 DT -1(188µg/mL)EcoRIXbaI10x BSA10x buffer MMilliQtotal
2 cuts5.3113316.730
1 cut0.30.20117.510
1 cut0.300.2117.510
NC0.300117.710

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----

Igku Aug20Electrophoresis(Ptet E,X).jpg
Igku Aug20 Electrophoresis2(RBS‐lysis3-1).jpg
Igku Aug20Electrophoresis3(DT・E＋X).jpg

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

Igku Aug21 Gel Extraction(Ptet‐(1))before.jpg Igku Aug21 Gel Extraction 2-2.jpg

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
2 cuts4.5113317.530
1 cut0.50.20117.310
1 cut0.500.2117.310
NC0.500117.510
8/20 Pconst-RBS-luxR-DT-(2) EcoRIXbaI10x BSA10x buffer MMilliQtotal
2 cuts2.9113319.130
1 cut0.30.20117.510
1 cut0.300.2117.510
NC0.300117.710
8/17 DTEcoRIXbaI10x BSA10x buffer MMilliQtotal
2 cuts5.3113316.730
1 cut0.50.20117.310
1 cut0.500.2117.310
NC0.500117.510
  • incubate at 37°C for 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.

Igku Aug21 Electorophoresis Rtet E+X.jpg
Kojima

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

Igku Aug21 Electrophoresis(J23100‐RBS,DT-E+X)bykojima.jpg

Gel Extraction

Honda

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

Igku Aug21 Gel Extraction(J23100,Ptet①)before.jpg
Igku Aug21 Gel Extraction(J23100,Ptet)after.jpg

Electrophoresis

Honda

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

Igku Aug21electrophoresis.jpg

Ligation

No name

stateVectorInserterLigation High ver.2total
experiment8/21 Pcn-RBS-luxR-DT(+Amp)2.38/21 Pcn-RBS-GFP-DT1.726
NC8/21 Pcn-RBS-luxR-DT(+Amp)2.3MilliQ1.726
experiment8/18 Plux(+CP)1.18/19 RBS-GFP-DT53.19.2
NC8/18 Plux(+CP)1.1MilliQ53.19.2
experiment8/18 RBS(+Amp)2.28/19 lysis16.04.112.3
experiment8/18 RBS(+Amp)2.28/19 lysis23.62.98.7
NC8/18 RBS(+Amp)2.2MilliQ3.62.98.7
experiment8/21 Ptet1.58/20 RBS-tetR-DT (2)2.11.85.4
NC8/21 Ptet1.5MilliQ2.11.85.4


Transformation

Okazaki

NameSampleCompetent CellsTotalPlate
8/21 Pcon-RBS-GFP-DT+Pcon-RBS-GFP-DT1µL10µL11µLAmp
8/21 Pcon-RBS-GFP-DT NC1µL10µL11µLAmp
8/18 RBS+8/19 lysis11µL10µL11µLAmp
8/18 RBS+8/19 lysis21µL10µL11µLAmp
8/18 RBS NC1µL10µL11µLAmp
8/18 Plux+8/18 RBS-GFP-DT1µL10µL11µLCP
8/18 Plux+8/18 RBS-GFP-DT1µL10µL11µLCP
8/21 Ptet(pm)+8/20 RBS-tetR-DT(2)1µL10µL11µLCP
8/21 Ptet(pm)+8/20 RBS-tetR-DT(2) NC1µL10µL11µLCP
8/21 pSB1C31µL10µL11µLCP

LB Medium Plate

Gel Extraction

Okazaki

LaneDNAEnzyme
11kb ladder--
2----
38/21 DTEcoRI & XbaI
4

Miniprep

Tatsui, Kojima, and Nakamoto

DNAconcentration [µg/mL]260/280260/230
tRNA-spinach-(1)2341.642.14
tRNA-spinach-(2)4401.601.91
tetR-aptamer 12-p(1076)-(1)1501.612.03
tetR-aptamer 12-p(1076)-(2)3741.451.66
tetR-aptamer 12-1R(113)-(1)3041.682.21
tetR-aptamer 12-1R(113)-(2)3821.471.66
tetR-aptamer 12-1M(105)-(1)3741.682.20
tetR-aptamer 12-1M(105)-(2)2861.311.43
PT181 attenuator-(1)3301.662.11
PT181 attenuator-(2)2701.682.18
Fusion1 attenuator-(1)3061.672.20
Fusion1 attenuator-(2)2821.601.94
Fusion3m2 attenuator-(1)3321.682.25
Fusion3m2 attenuator-(2)3261.672.10
PT181 antisense-(1)1781.641.68
PT181 antisense-(2)1161.631.95
Fusion1 antisense-(1)3001.672.21
Fusion1 antisense-(2)1641.421.46
Fusion6 antisense-(1)3361.652.18
Fusion6 antisense-(2)1921.652.01

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

Restriction Enzyme Digestion

Tatsui, Kojima, Honda and Ashida

Fusion1 antisense (1) (300 µg/mL)EcoRISpeIbuffer HMilliQtotal
2 cuts3.311321.730
1 cut0.70.2018.110
1 cut0.700.218.110
NC0.70018.310
Fusion1 antisense (1) (300 µg/mL)XbaIPstIbuffer MBSAMilliQtotal
2 cuts3.3113318.730
1 cut0.70.20117.110
1 cut0.700.2117.110
NC0.700117.310
Fusion6 antisense (1) (336 µg/mL)EcoRISpeIbuffer HMilliQtotal
2 cuts31132230
1 cut0.60.2018.210
1 cut0.600.218.210
NC0.60018.410
Fusion6 antisense (1) (336 µg/mL)XbaIPstIbuffer MBSAMilliQtotal
2 cuts311331930
1 cut0.60.20117.210
1 cut0.600.2117.210
NC0.600117.410
pT181 antisense (1) (178 µg/mL)EcoRISpeIbuffer HMilliQtotal
2 cuts5.611319.430
1 cut1.10.2017.710
1 cut1.100.217.710
NC1.10017.910
pT181 antisense (1) (178 µg/mL)XbaIPstIbuffer MBSAMilliQtotal
2 cuts5.6113316.430
1 cut1.10.20116.710
1 cut1.100.2116.710
NC1.100116.910
pT181 attenuator (1)EcoRISpeIbuffer HMilliQtotal
2 cuts31132230
1 cut0.60.2018.210
1 cut0.600.218.210
NC0.60018.410
pT181 attenuator (1)XbaIPstIbuffer MBSAMilliQtotal
2 cuts311331930
1 cut0.60.20117.210
1 cut0.600.2117.210
NC0.600117.410
Fusion1 attenuator (1)EcoRISpeIbuffer HMilliQtotal
2 cuts3.311321.730
1 cut0.70.2018.110
1 cut0.700.218.110
NC0.70018.310
Fusion1 attenuator (1)XbaIPstIbuffer MBSAMilliQtotal
2 cuts3.3113318.730
1 cut0.70.20117.110
1 cut0.700.2117.110
NC0.700117.310
Fusion3m2 attenuator (1)EcoRISpeIbuffer HMilliQtotal
2 cuts31132230
1 cut0.60.2018.210
1 cut0.600.218.210
NC0.60018.410
Fusion3m2 attenuator (1)XbaIPstIbuffer MBSAMilliQtotal
2 cuts311331930
1 cut0.60.20117.210
1 cut0.600.2117.210
NC0.600117.410
Spinach (2)EcoRISpeIbuffer BBSAMilliQtotal
2 cuts2.30.50.530.323.430
1 cut0.50.1010.18.310
1 cut0.500.110.18.310
NC0.50010.18.410
tetR aptamer 12_P (2)EcoRISpeIbuffer BBSAMilliQtotal
2 cuts2.70.50.530.32330
1 cut0.50.1010.18.310
1 cut0.500.110.18.310
NC0.50010.18.410
tetR aptamer 12_1R (2)EcoRISpeIbuffer BBSAMilliQtotal
2 cuts2.60.50.530.323.130
1 cut0.50.1010.18.310
1 cut0.500.110.18.310
NC0.50010.18.410
tetR aptamer 12_1M (1)EcoRISpeIbuffer BBSAMilliQtotal
2 cuts2.70.50.530.32330
1 cut0.50.1010.18.310
1 cut0.500.110.18.310
NC0.50010.18.410
J23100SpeIPstIbuffer BBSAMilliQtotal
2 cuts70.50.530.318.730
1 cut1.40.1010.17.410
1 cut1.400.110.17.410
NC1.40010.17.510
Plac (2)SpeIPstIbuffer BBSAMilliQtotal
2 cuts11.20.50.530.314.530
1 cut2.20.1010.16.610
1 cut2.200.110.16.610
NC2.20010.16.710

Electrophoresis

No name

LaneSample
1100bp ladder
2RBS-lysis1 -(1)
3RBS-lysis1 -(2)
4RBS-lysis2 -(1)
5RBS-lysis2 -(2)
6RBS(NC) -(1)
7RBS(NC) -(2)
8NC

Igku Aug22 Electrophoresis(ColoP)-1.jpg

LaneSample
11kb ladder
2Ptet-RBS-tetR-DT -(1)
3Ptet-RBS-tetR-DT -(2)
4Ptet(NC) -(1)
5Ptet(NC) -(2)
6Plux-RBS-GFP-DT -(1)
7pSB1C3 -(1)
8pSB1C3 -(2)
9NC

Igku Aug22 Electrophoresis(ColoP)-2.jpg

LaneSample
11kb ladder
2Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT -(1)
3Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT -(2)
4Pcon-RBS-luxR -(1)
5Pcon-RBS-luxR -(2)
6NC
71kb ladder

Igku Aug22 Electrophoresis(ColoP)-3.jpg

Miniprep

Ashida

DNAconcentration[µg/mL]260/280260/230
8/22 RBS-lysis (1)1281.772.29
8/22 RBS-lysis (2)961.802.07
8/22 pSB1C3 (1)1081.551.45
8/22 pSB1C3 (2)1301.691.76

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
1100bp----
28/22 pT181 attenuator (1)EcoRISpeI
38/22 pT181 attenuator (1)EcoRI--
48/22 pT181 attenuator (1)--SpeI
58/22 pT181 attenuator (1)----
68/22 pT181 attenuator (1)XbaIPstI
78/22 pT181 attenuator (1)XbaI--
88/22 pT181 attenuator (1)--PstI
98/22 pT181 attenuator (1)----
108/22 Fusion6 antisense (1)EcoRISpeI
118/22 Fusion6 antisense (1)EcoRI--
128/22 Fusion6 antisense (1)--SpeI
138/22 Fusion6 antisense (1)----
14100bp----

Igku Aug22electrophoresis.jpg

LaneSampleEnzyme1Enzyme2
1100bp----
28/22 Fusion6 antisense (1)XbaIPstI
38/22 Fusion6 antisense (1)XbaI--
48/22 Fusion6 antisense (1)--PstI
58/22 Fusion6 antisense (1)----
68/22 Fusion1 antisense (1)EcoRISpeI
78/22 Fusion1 antisense (1)EcoRI--
88/22 Fusion1 antisense (1)--SpeI
98/22 Fusion1 antisense (1)----
108/22 Fusion1 antisense (1)XbaIPstI
118/22 Fusion1 antisense (1)XbaI--
128/22 Fusion1 antisense (1)--PstI
138/22 Fusion1 antisense (1)----
14100bp----

Igku Aug22electrophoresis2.jpg

Aug 23

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
1pT181 antisenseEcoRISpeI
2pT181 antisenseEcoRI--
3pT181 antisense--SpeI
4pT181 antisense----
5pT181 antisenseXbaIPstI
6pT181 antisenseXbaI--
7pT181 antisense--PstI
8pT181 antisense----
9100bp ladder----
10Fusion1 attenuatorEcoRISpeI
11Fusion1 attenuatorEcoRI--
12Fusion1 attenuator--SpeI
13Fusion1 attenuator----
14Fusion1 attenuatorXbaIPstI
15Fusion1 attenuatorXbaI--
16Fusion1 attenuator--PstI
17Fusion1 attenuator----

Igku Aug23electrophoresis.jpg

LaneSampleEnzyme1Enzyme2
1Fusion3m2 attenuatorEcoRISpeI
2Fusion3m2 attenuatorEcoRI--
3Fusion3m2 attenuator--SpeI
4Fusion3m2 attenuator----
5Fusion3m2 attenuatorXbaIPstI
6Fusion3m2 attenuatorXbaI--
7Fusion3m2 attenuator--PstI
8Fusion3m2 attenuator----
9100bp ladder----
10tetR aptamer 12_1MEcoRISpeI
11tetR aptamer 12_1MEcoRI--
12tetR aptamer 12_1M--SpeI
13tetR aptamer 12_1M----
14tetR aptamer 12_PEcoRISpeI
15tetR aptamer 12_PEcoRI--
16tetR aptamer 12_P--SpeI
17tetR aptamer 12_P----

Igku Aug23electrophoresis2.jpg
Kojima and Honda

LaneSampleEnzyme1Enzyme2
1PlacSpeIPstI
2PlacSpeI--
3Plac--PstI
4Plac----
5SpinachEcoRISpeI
6SpinachEcoRI--
7Spinach--SpeI
8Spinach----
9100bp ladder----
10J23100SpeIPstI
11J23100SpeI--
12J23100--PstI
13J23100----
14tetR aptamer 12_1REcoRISpeI
15tetR aptamer 12_1REcoRI--
16tetR aptamer 12_1R--SpeI
17tetR aptamer 12_1R----

Igku Aug23 Electrophoresis(N2).jpg

Restriction Enzyme Digestion

Nakamoto and Kojima

Fusion6 antisense (1)EcoRISpeIbuffer BBSAMilliQtotal
2 cuts30.50.530.322.730
1 cut0.60.1010.18.210
1 cut0.600.110.18.210
NC0.60010.18.310
Fusion6 antisense (1)XbaIPstIbuffer DBSAMilliQtotal
2 cuts30.50.530.322.730
1 cut0.60.1010.18.210
1 cut0.600.110.18.210
NC0.60010.18.310
Fusion1 antisense (1)EcoRISpeIbuffer BBSAMilliQtotal
2 cuts3.30.50.530.322.430
1 cut0.70.1010.18.110
1 cut0.700.110.18.110
NC0.70010.18.210
pT181 antisense (1)EcoRISpeIbuffer BBSAMilliQtotal
2 cuts5.60.50.530.320.130
1 cut1.10.1010.17.710
1 cut1.100.110.17.710
NC1.10010.17.810
pT181 antisense (1)XbaIPstIbuffer DBSAMilliQtotal
2 cuts5.60.50.530.320.130
1 cut1.10.1010.17.710
1 cut1.100.110.17.710
NC1.10010.17.810
  • incubate at 37°C from 17:29.

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
1100bp----
2pT181 attenuatorEcoRISpeI
3pT181 attenuatorXbaIPstI
4Fusion3m2 attenuatorEcoRISpeI
5Fusion1 attenuatorXbaIPstI
6Fusion1 attenuatorEcoRISpeI
7Fusion3m2 attenuatorXbaIPstI
8pT181 antisenseEcoRISpeI
9pT181 antisenseXbaIPstI
10Fusion1 antisenseEcoRISpeI
11Fusion1 antisenseXbaIPstI
12Fusion6 antisenseEcoRISpeI
13Fusion6 antisenseXbaIPstI
14tetR aptamer 12_1MEcoRISpeI
15tetR aptamer 12_PEcoRISpeI
16tetR aptamer 12_1REcoRISpeI
17100bp----

Igku Aug23 Electrophoresis (N3).jpg

LaneSampleEnzyme1Enzyme2
1100bp----
2SpinachEcoRISpeI
3J23100SpeIPstI
4PlacSpeIPstI
5100bp----
6J23100SpeIPstI

Igku Aug23 Electrophoresis(N3)-2.jpg

LaneSampleEnzyme1Enzyme2
1100bp----
2Fusion6 antisenseEcoRISpeI
3Fusion6 antisenseEcoRI--
4Fusion6 antisense--SpeI
5Fusion6 antisense----
6pT181 antisenseXbaIPstI
8100bp----
9100bp----
10Fusion6 antisenseXbaIPstI
11Fusion6 antisenseXbaI--
12Fusion6 antisense--PstI
13Fusion6 antisense----
14pT181 antisenseXbaI--
16100bp----

Igku Aug23electrophoresis4.jpg

LaneSampleEnzyme1Enzyme2
1100bp----
2Fusion1 antisenseEcoRISpeI
3Fusion1 antisenseEcoRI--
4Fusion1 antisense--SpeI
5Fusion1 antisense----
6pT181 antisense--PstI
8100bp----
9100bp----
10pT181 antisenseEcoRISpeI
11pT181 antisenseEcoRI--
12pT181 antisense--SpeI
13pT181 antisense----
14pT181 antisense----
16100bp----

Igku Aug23electrophoresis5.jpg

Gel Extraction

Nakamoto, Ashida, Kojima, Okazaki & Matsumoto

LaneDNAEnzyme
1100bp ladder--
2pT181 attenuatorXbaI & PstI
3
5Fusion3m2 attenuatorEcoRI & SpeI
6
8Fusion1 attenuatorXbaI & PstI
9
11Fusion1 attenuatorEcoRI & SpeI
12

Igku Aug23Gel Extraction.jpg
Igku Aug23 Gel Extraction(N6)-1.2.jpg

LaneDNAEnzyme
1100bp ladder--
2Fusion3m2 attenuatorXbaI & PstI
3
5pT181 antisenseEcoRI & SpeI
6
8pT181 antisenseXbaI & PstI
9
11Fusion1 antisenseXbaI & PstI
12

Igku Aug23 Gel Extraction(N7)-2.jpg

LaneDNAEnzyme
1100bp ladder--
2Fusion6 attenuatorXbaI & PstI
3
5aptamer 12_1MEcoRI & SpeI
6
LaneDNAEnzyme
1100bp ladder--
2aptamer 12_PEcoRI & SpeI
3
5aptamer 12_1REcoRI & SpeI
6

Igku Aug23 Gel Extraction(N6)-2.4.jpg
Igku Aug23 Gel Extraction(N6)-3-2.jpg
Ashida

LaneDNAEnzyme
1100bp ladder--
2Fusion6 antisenseEcoRI & SpeI
3
5Fusion6 attenuatorXbaI & PstI
6
8Fusion1 antisenseEcoRI & SpeI
9
11pT181 antisenseEcoRI & SpeI
12

Igku Aug23 Gel Extracton(N8)-1.jpg
Igku Aug23 Gel Extraction(N8)-1.2.jpg

LaneDNAEnzyme
1100bp ladder--
3pT181 antisenseXbaI & PstI
4

Igku Aug23 Gel Extraction(N7)-1.jpg
Igku Aug23 Gel Extraction(N7)1 2.jpg

LaneDNAEnzyme
1100bp ladder--
3PlacSpeI & PstI
4
LaneDNAEnzyme
1100bp ladder--
3J23100SpeI & PstI
5SpinachEcoRI & SpeI
6

Igku Aug23 Gel Extraction(N9)-1-1.jpg
Igku Aug23 Gel Extraction(N9)-1-2.jpg


Aug 24

Gel Extraction

Ashida

Nameconcentration[µg/mL]260/280260/230
8/22 aptamer 12_P (EcoRI & SpeI)8821.3--
8/22 aptamer 12_1R (EcoRI & SpeI)6323.1--
8/22 aptamer 12_1M (EcoRI & SpeI)4022.0--
8/23 Fusion6 antisense (EcoRI & SpeI)6.01.600.17
8/23 Fusion6 antisense (XbaI & PstI)6.01.710.25
8/23 pT181 antisense (EcoRI & SpeI)6.02.220.15
8/23 pT181 antisense (XbaI & PstI)6.02.100.04
8/22 Plac (SpeI & PstI)6.01.470.09
8/22 Fusion1 antisense (EcoRI & SpeI)102.160.01
8/22 J23100 (SpeI & PstI)6.01.520.35
Nameconcentration[µg/mL]260/280260/230
8/22 Spinach (EcoRI & SpeI)8.01.150.77
8/22 Fusion6 antisense (XbaI & PstI)441.070.96
8/22 Fusion1 antisense (XbaI & PstI)2--1.00
8/22 pT181 antisense (XbaI & PstI)3.2.010.40
8/22 pT181 antisense (EcoRI & SpeI)81.130.82
8/22 Fusion3m2 attenuator (XbaI & PstI)52.271.50
8/22 Fusion3m2 attenuator (EcoRI & SpeI)341.161.02
8/22 Fusion1 attenuator (XbaI & PstI)141.400.62
8/22 Fusion1 attenuator (EcoRI & SpeI)161.270.61
8/22 pT181 attenuator (XbaI & PstI)5--0.88
  • These samples were performed ethanol ethanl precipitation.

Restriction Enzyme Digestion

No name

tetR aptamer 12_P(2)EcoRISpeI10x Buffer B100x BSAMilliQtotal
2 cuts2.7µL0.5µL0.5µL3µL0.3µL23µL30µL
NC0.3µL0µL0µL1µL0.1µL8.6µL10µL
tetR aptamer 12_1R(2)EcoRISpeI10x Buffer B100x BSAMilliQtotal
2 cuts2.6µL0.5µL0.5µL3µL0.3µL23.1µL30µL
NC0.3µL0µL0µL1µL0.1µL8.6µL10µL
tetR aptamer 12_M(1)EcoRISpeI10x Buffer B100x BSAMilliQtotal
2 cuts2.7µL0.5µL0.5µL3µL0.3µL23µL30µL
NC0.3µL0µL0µL1µL0.1µL8.6µL10µL

Master Plate

No name

NumberUse LB plate(+Kan)
1entA- #1
2entA- #1
3entA- #1
4entA- #2
5entA- #2
6entA- #2

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
1100bp ladder--
2tetR aptamer 12_PEcoRISpeI
3tetR aptamer 12_P--
4tetR aptamer 12_1REcoRISpeI
5tetR aptamer 12_1R--
6tetR aptamer 12_1MEcoRISpeI
7tetR aptamer 12_1M--
8100bp ladder--

Igku Aug24 Electrophoresis(N1)-pic.jpg

LaneSampleEnzyme1Enzyme2
1100bp ladder--
2tetR aptamer 12_PEcoRISpeI
3tetR aptamer 12_1REcoRISpeI
4tetR aptamer 12_1MEcoRISpeI
5100bp ladder--


Igku Aug24 Electrophoresis(N2)-pic2.jpg


Aug 26

Gel Extraction

Matsumoto and Ashida

LaneDNAEnzyme
1100bp ladder-
28/24 tetR aptamer 12_PEcoRI & SpeI
48/24 tetR aptamer 12_1REcoRI & SpeI
68/24 tetR aptamer 12_MEcoRI & SpeI

Igku Aug26 Gel Extraction(N①)before.jpg
Igku Aug26 Gel Extraction(N②)after.jpg

Nameconcentration[µg/mL]260/280260/230
tetR aptamer 12_P (EcoRI & SpeI)2.30.380.13
tetR aptamer 12_1R (EcoRI & SpeI)12.00.631.19
tetR aptamer 12_M (EcoRI & SpeI)2.06.430.03

Ligation

Ashida

stateVectorInserterLigation High ver.2
experiment8/21 DT5.08/26 tetR aptamer 12_1R2.03.5
experiment8/21 DT5.08/22 pT181 atteniator5.05.0
experiment8/21 DT5.08/22 pT181 antisense3.04.0
experiment8/21 DT5.08/22 Spinach3.04.0
experiment8/22 Plac4.08/22 pT181 attenuator5.04.5
experiment8/22 J231004.08/22 pT181 attenuator5.04.5
experiment8/22 Plac4.08/22 pT181 antisense8.06.0
experiment8/22 J231004.08/22 pT181 antisense8.06.0

Transformation

No name

NameSampleCompetent CellsTotalPlate
8/26 tetR aptamer 12_1R-DT1µL10µL11µLCP
8/26 pT181 attenuator-DT1µL10µL11µLCP
8/26 pT181 antisense-DT1µL10µL11µLCP
8/26 Spinach-DT1µL10µL11µLCP
8/26 Plac-pT181 attenuator1µL10µL11µLCP
8/26 Pcon-pT181 attenuator1µL10µL11µLAmp
8/26 Plac-pT181 antisense1µL10µL11µLCP
8/26 Pcon-pT181 antisense1µL10µL11µLAmp
Pλ-RBS-luxI-DT1µL10µL11µAmp

Aug 27

Restriction Enzyme Digestion

No name

8/21 pT181 attenuator-1(330µg/mL)EcoRISpeI10x BufferB100x BSAMilliQtotal
2 cuts3.0µL0.5µL0.5µL3.0µL0.3µL22.7µL30µL
NC0.6µL0µL0µL1µL0.1µL8.3µL10µL
8/17 DT-1(188µg/mL)EcoRIXbaI10x BufferB100x BSAMilliQtotal
2 cuts3.0µL0.5µL0.5µL3.0µL0.3µL22.7µL30µL
NC1.0µl0µL0µL1µL0.1µL7.9µL10µL
8/20 Pcon-RBS-luxR-DT-2(344µg/mL)EcoRISpeI10x BufferB100x BSAMilliQtotal
2 cuts2.9µL0.5µL0.5µL3.0µL0.3µL22.8µL30µL
NC0.6µl0µL0µL1µL0.1µL8.3µL10µL

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
1100bp ladder----
28/21 pT181 attenuatorEcoISpeI
38/21 pT181 atteniator----
48/17 DT-1EcoRIXbaI
58/17 DT-1----
68/20 Pcon-RBS-luxR-DT-2EcoISpeI
78/20 Pcon-RBS-luxR-DT-2----
8100bp ladder----

Igku Aug27 Electrophoresis(N1)-1.jpg

Colony PCR

Kojima and Yoshida

Samplebase pair
8/26 Pλ-luxI(1)
8/26 Pλ-luxI(2)
NC
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s3min30cycles
LaneSample
1100bp ladder
2Pλ-luxI-(1)
3Pλ-luxI-(2)
4NC

Igku Aug27 Electrophoresis(ColoP)(N3)-2.jpg

Liquid Culture

No name

Samplemedium
8/26 Pλ-RBS-luxI-DT-1Plusgrow medium (+Amp)
8/26 Pλ-RBS-luxI-DT-2Plusgrow medium (+Amp)　

Restriction Enzyme Digestion

No name

8/21 pT181 attenuator-1(330µg/mL)EcoRISpeI10x BufferB100x BSAMilliQtotal
2 cuts3.0µL0.5µL0.5µL3.0µL0.3µL22.7µL30µL
NC0.6µL0µL0µL1µL0.1µL8.3µL10µL
8/17 DT-1(188µg/mL)EcoRIXbaI10x BufferB100x BSAMilliQtotal
2 cuts3.0µL0.5µL0.5µL3.0µL0.3µL22.7µL30µL
NC1.0µl0µL0µL1µL0.1µL7.9µL10µL
8/20 Pcon-RBS-luxR-DT-2(344µg/mL)EcoRISpeI10x BufferB100x BSAMilliQtotal
2 cuts2.9µL0.5µL0.5µL3.0µL0.3µL22.8µL30µL
NC0.6µl0µL0µL1µL0.1µL8.3µL10µL

Electrophoresis

Hirano

LaneSampleEnzyme1Enzyme2
100bp ladder----
1pT181 attenuatorEcoRISpeI
2pT181 attenuator----
3DT-(1)EcoRIXbaI
4DT-(1)----
5Pcon-RBS-luxR-DT-(2)EcoRISpeI
6Pcon-RBS-luxR-DT-(2)----
100bp ladder----

Igku Aug27 Electrophoresis(N2)-2.jpg

Gel Extraction

Inoue

LaneDNAEnzyme
1100bpladder--
2pT181 attenuator-1EcoRI&SpeI
3pT181 attenuator-1EcoRI&SpeI
4DT-1EcoRI&XbaI
5DT-1EcoRI&XbaI
6Pcon-RBS-luxR-DT-2EcoRI&SpeI
7Pcon-RBS-luxR-DT-2EcoRI&SpeI
8100bpladder--

Igku Aug27 Gel Extraction(N3)-1-3.jpg
Igku Aug27 Gel Extraction(N4)pic(1).jpg

Nameconcentration[µg/mL]260/280260/230
pT181 attenuator-1(EcoRI&SpeI)4.21.620.29
DT-1(EcoRI&XbaI)171.880.88

Transformation

Kojima and Hirano

NameSampleCompetent CellsTotalPlate
8/26 tetR aptamer 12_1R-DT1µL10µL11µLCP
8/26 pT181 attenuator-DT1µL10µL11µLCP
8/26 pT181 antisense-DT1µL10µL11µLCP
8/26 Spinach-DT1µL10µL11µLCP
8/26 Plac-pT181 attenuator1µL10µL11µLCP
8/26 Pcon-pT181 attenuator1µL10µL11µLAmp
8/26 Plac-pT181 antisense1µL10µL11µLCP
8/26 Pcon-pT181 antisense1µL10µL11µLAmp

Liquid culture

No name

Samplemedium
8/16 PluxPlusgrow medium(+CP)

Genome PCR

Yoshdia and Nakagawa

genome DNAKOD plus10x bufferdNTPMgSO4SasA_fwd primerSasA_rev primerMilliQtotal
0.50.52.52.51.50.750.751625
genome DNAKOD plus10x bufferdNTPMgSO4RpaA_fwd primerRpaA_rev primerMilliQtotal
0.50.52.52.51.50.750.751625
genome DNAKOD plus10x bufferdNTPMgSO4RpaB_fwd primerRpaB_rev primerMilliQtotal
0.50.52.52.51.50.750.751625
genome DNAKOD plus10x bufferdNTPMgSO4PkaiBC_fwd primerPkaiBC_rev primerMilliQtotal
0.50.52.52.51.50.750.751625
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C50°C68°C--
2 min10 sec30 sec38 sec30 cycles

Ligation

No name

stateVectorInserter
experiment8/27 DT (EcoRI & XbaI)2.88/21 RBS-lysis3 (EcoRI & SpeI)
experiment8/27 DT (EcoRI & XbaI)2.88/27 pT181 attenuator (EcoRI & SpeI)
  • Samples were evaporeted used evaporator into about 3 µL.
sampleMilliQLigation Hightotal
343.510.5
  • incubate overnight at 4 °C

Aug 28

Electrophoresis

No name

LaneSample
1100bp
2SasA
3RpaA
4RpaB
5PkaiBC
6PCR NC
7100bp

Igku Aug28 Electrophoresis(N①-1).jpg

Miniprep

No name

DNAconcentration[µg/mL]260/280260/230
8/27 Plux173.81.951.45

Ligation

No name

stateVectorInserter
experiment8/17 DT-13.08.0
  • Samples were evaporeted used evaporator into about 3 µL.
sampleMilliQLigation Hightotal
343.510.5
  • incubate one hour at 16 °C

Gel Extraction

No name

LaneDNA
1100bpladder
3SasA(8/27 Genome PCR production)
5RpaA(8/27 Genome PCR production)
7RpaB(8/27 Genome PCR production)
9PkaiBC(8/27 Genome PCR production)

Igku Aug28 Gel Extraction(N②pic)before.jpg
Igku Aug28 Gel Extraction(N①pic)after.jpg

Nameconcentration[µg/mL]260/280260/230
RpaA15.41.880.03
SasA21.61.660.79
PkaiBC16.31.790.68
RpaB26.71.770.82

Colony PCR

No name

Samplebase pair
8/21 RBS-lysis2-(3)772
8/21 RBS-lysis2-(4)772
8/21 RBS-lysis2-(5)772
8/21 RBS-lysis2-(6)772
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s48s30cycles

Igku Aug28 Colony PCR(N③pic).jpg

Restriction Enzyme Digestion

No name

8/22 RBS-lysis1-(1)EcoRISpeIBufferBBSAMilliQtotal
2 cuts9µL0.5µL0.5µL3µL0.3µL16.7µL30µL
NC1µL0µL0µL1µL0.1µL7.9µL10µL
8/17 RBS-GFP-DT-(2)XbaIPstIBufferDBSAMilliQtotal
2 cuts4µL0.5µL0.5µL3µL0.3µL21.7µL30µL
NC0.5µL0µL0µL1µL0.1µL8.4µL10µL
8/20 Pcon-RBS-GFP-DT-(1)EcoRISpeIBufferBBSAMilliQtotal
2 cuts3µL0.5µL0.5µL3µL0.3µL22.7µL30µL
NC0.3µL0µL0µL1µL0.1µL8.6µL10µL
8/20 Pcon-RBS-luxR-DT-(1)EcoRIXbaIBufferDBSAMilliQtotal
2 cuts2µL0.5µL0.5µL3µL0.3µL23.7µL30µL
NC0.3µL0µL0µL1µL0.1µL8.6µL10µL

No name

8/28 PluxSpeIPstIBufferDBSAMilliQtotal
2 cuts6µL0.5µL0.5µL3µL0.3µL19.7µL30µL
NC1µL0µL0µL1µL0.1µL7.9µL10µL
8/20 RBS-tetR-DT-(1)XbaIPstIBufferDBSAMilliQtotal
2 cuts5µL0.5µL0.5µL3µL0.3µL20.7µL30µL
NC0.5µL0µL0µL1µL0.1µL8.4µL10µL
  • incubated 37°C 1hour

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
18/22 RBS-lysis1-(1)EcoRISpeI
28/22 RBS-lysis1-(1)----
38/17 RBS-GFP-DT-(2)XbaIPstI
48/17 RBS-GFP-DT-(2)----
58/20 Pcon-RBS-GFP-DT-(1)EcoRISpeI
68/20 Pcon-RBS-GFP-DT-(1)----
7100bp ladder----
88/20 Pcon-RBS-luxR-DT-(1)EcoRIXbaI
98/20 Pcon-RBS-luxR-DT-(1)----
108/28 PluxSpeIPstI
118/28 Plux----
128/20 RBS-tetR-DTXbaIPstI
138/20 RBS-tetR-DT----

Igku Aug28 Electrophoresis(ColoP)(N4)pic.jpg

Gel Extraction

No name

LaneDNAEnzyme
1100bp ladder--
28/20 RBS-lysis1-(1)EcoRI & SpeI
3
4----
58/28 RBS-GFP-DT-(2)XbaI & PstI
6

Igku Aug28 Gel Extraction(N⑤-1,2)before.jpg
Igku Aug28 Gel Extraction4-2.jpg

LaneDNAEnzyme
1100bp ladder--
28/28 PluxSpeI & PstI
3
4----
58/20 RBS-tetR-DT-(1)XbaI & PstI
6

Igku Aug28 Gel Extraction(N⑤-3).jpg

Nameconcentration[µg/mL]260/280260/230
Pcon-RBS-GFP-DT (EcoRI&SpeI)7.11.880.07
Pcon-RBS-luxR-DT (EcoRI&XbaI)15.31.930.70
Plux (SpeI&PstI)4.51.790.31
RBS-GFP-DT (XbaI&PstI)6.02.160.03
RBS-tetR-DT (XbaI&PstI)7.61.910.38

Colony PCR

No name

Samplebase pair
8/27 pT181 attenuator-DT-1738
8/27 pT181 attenuator-DT-2738
8/27 pT181 attenuator-DT-3738
8/27 TetR-aptamer 12_1R-DT-1521
8/27 TetR-aptamer 12_1R-DT-2521
8/27 pT181 antisense-DT-1542
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s30s30cycles

Igku Aug28 c(N⑤-1,2)before.jpg

Samplebase pair
8/27 Pλ-luxI-1--
8/27 Pλ-luxI--
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s30min3cycles

Igku Aug28 ColonyPCR(N⑦-2).jpg

Colony PCR

No name

Samplebase pair
8/21 RBS-lysis2-9772
8/21 RBS-lysis2-10772
8/21 RBS-lysis2-11772
8/21 RBS-lysis2-12772
8/21 RBS-lysis2-13772
8/21 RBS-lysis2-14772
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s48s30cycles

Liquid Culture

No name

Samplemedium
8/16 Master Plate(CP)-6 DTPlusgrow medium(+CP)
J23100 Master Plate-1Plusgrow medium(+Amp)
Plac Master Plate-1Plusgrow medium(+CP)

Ligation

No name

stateVectorInserter
experiment8/27 DT (EcoRI & XbaI)2.98/28 RBS-lysis1 (EcoRI & SpeI)9.5
experiment8/28 Plux (SpeI & PstI)2.38/28 RBS-GFP-DT (XbaI & PstI)18
experiment8/28 Pcon-RBS-luxR-DT (EcoRI & XbaI)3.38/28 Pcon^RBS-GFP-DT-(1) (EcoRI & SpeI)28
experiment8/27 DT (EcoRI & XbaI)2.78/24 Spinach (EcoRI & SpeI)2.4
  • Samples were evaporeted used evaporator into about 1 µL.
sampleMilliQLigation Hightotal
1349
  • incubate overnight at 4 °C

Liquid Culture

No name

Samplemedium
8/26 Pλ-RBS-luxI-DT-1Plusgrow medium (+Amp)
8/26 Pλ-RBS-luxI-DT-2Plusgrow medium (+Amp)
  • incubate 37°C 10hour

Aug 29

Colony PCR

No name

Samplebase pair
8/27 Pcon-pT181 antisense-(1)542
8/26 pT181 attenuator-DT-(1)738
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s30s30cycles
LaneSample
1100bp ladder
28/27 Pcon-pT181antisense-1
38/26 pT181attenuator-DT-1
4100bp ladder

Igku Aug29 Electrophoresis(ColoP)(N1)pic.jpg

Electrophoresis

No name

LaneSample
1100bp ladder
28/21 RBS-lysis2-9(Colony PCR prodution)
38/21 RBS-lysis2-8(Colony PCR prodution)
48/21 RBS-lysis2-7(Colony PCR prodution)
58/21 RBS-lysis2-10(Colony PCR prodution)
68/21 RBS-lysis2-11(Colony PCR prodution)
78/21 RBS-lysis2-12(Colony PCR prodution)
88/21 RBS-lysis2-13(Colony PCR prodution)
98/21 RBS-lysis2-14(Colony PCR prodution)

Igku Aug29 Electrophoresis1(N②).jpg

Miniprep

No Name

DNAconcentration [µg/mL]260/280260/230
Plac153.72.032.10
Pλ-luxⅠ-(1)76.52.072.06
Pλ-luxⅠ-(2)86.42.082.10
DT166.21.652.12
J23100190.11.682.34

Liquid Culture

No name

Samplemedium
8/21 RBS-Lysis2-9Plusgrow medium(+Amp)
8/21 RBS-Lysis2-12Plusgrow medium(+Amp)
8/21 RBS-Lysis2-14Plusgrow medium(+Amp)

Restriction Enzyme Digestion

No name

8/22 psB1C3-(2)EcoRISpeIXbaIPstIBufferBBufferDBSAMilliQtotal
2 cuts8µL0.5µL0.5µL0µL0µL3µL0µL0.3µL17.7µL30µL
NC1µL0µL0µL0µL0µL1µL0µL0.1µL7.9µL10µL
2 cuts8µL0µL0µL0.5µL0.5µL0µL3µL0.3µL17.7µL30µL
NC1µL0µL0µL0µL0µL0µL1µL0.1µL7.9µL10µL
8/21 tRMA-spinach(1)XbaISpeIBufferBBSAMilliQtotal
2 cuts4µL0.5µL0.5µL3µL0.3µL21.7µL30µL
NC0.5µL0µL0µL1µL0.1µL8.4µL10µL
8/21 tetR aptamer12-1R(1)EcoRISpeIBufferBBSAMilliQtotal
2 cuts3µL0.5µL0.5µL3µL0.3µL22.7µL30µL
NC0.4µL0µL0µL1µL0.1µL8.5µL10µL
8/21 PT181 attenuator-(2)EcoRISpeIXbaIPstIBufferBBufferDBSAMilliQtotal
2 cuts4µL0.5µL0.5µL0µL0µL3µL0µL0.3µL21.7µL30µL
NC0.5µL0µL0µL0µL0µL1µL0µL0.1µL8.4µL10µL
2 cuts4µL0µL0µL0.5µL0.5µL0µL3µL0.3µL21.7µL30µL
NC0.5µL0µL0µL0µL0µL0µL1µL0.1µL8.4µL10µL
8/21 PT181 antisense-(2)EcoRISpeIXbaIPstIBufferBBufferDBSAMilliQtotal
2 cuts10µL0.5µL0.5µL0µL0µL3µL0µL0.3µL15.7µL30µL
NC1µL0µL0µL0µL0µL1µL0µL0.1µL7.9µL10µL
2 cuts10µL0µL0µL0.5µL0.5µL0µL3µL0.3µL15.7µL30µL
NC1µL0µL0µL0µL0µL0µL1µL0.1µL7.9µL10µL
  • incubated 37°C 1hour

Igku Aug29 Restriction Enzyme Digestion(N③).jpg

Transformation

No Name

NameSampleCompetent CellsTotalPlate
8/27 pT181 attenuator-DT2µL20µL22µLCP
8/27 RBS-lysis3-DT2µL20µL22µLCP
8/28 RBS-lysis3-DT2µL20µL22µLCP
8/28 Pcon-RBS-GFP-DT-Pcon-RBS-LuxR-DT2µL20µL22µLAmp
8/28 RBS-lysis1-DT2µL20µL22µLCP
8/28 Plux-RBS-GFP-DT2µL20µL22µLCP
8/28 Spinach-DT2µL20µL22µLCP

Gel Extraction

No name

LaneDNAEnzyme1Enzyme2
100bp ladder----
98/29 pT181attenuator(2)EcoRI SpeI
108/29 pT181attenuator(2)EcoRI SpeI
118/29 pT181attenuator(2)XbaIPstI
128/29 pT181attenuator(2)XbaIPstI
13pT181antisense(2)EcoRISpeI
14pT181antisense(2)EcoRISpeI
15pT181antisense(2)XbaIPstI
16pT181antisense(2)XbaIPstI
100bp ladder----

Igku Aug29 Transformation1(N④-1).jpg
Igku Aug29 Transformation(N④-2).jpg

LaneSampleEnzyme1Enzyme2
100bp ladder----
18/29 pSB1C3EcoRISpeI
28/29 pSB1C3EcoRISpeI
38/29 pSB1C3XbaIPstI
48/29 pSB1C3XbaIPstI
58/29 spinachEcoRISpeI
68/29 spinachEcoRISpeI
78/29 tetRaptarmer12-1R(1)EcoRISpeI
88/29 tetRaptarmer12-1R(1)EcoRISpeI
100bp ladder----


Igku Aug29 Transformation(N⑤).jpg
Igku Aug29 Gel Extraction4-2.jpg

Aug 30

Gel Extraction

No Name

Namequantityconcentration[µg/mL]260/280260/230
pT181 attenuator(2) (EcoRI & SpeI)305.3 2.2 1.66 0.36
pT181 attenuator(2) (XbaI & PstI)217.7 3.9 1.73 0.04
pT181 antisense(2) (EcoRI & SpeI)347.7 3.1 1.88 0.31
pT181 antisense(2) (XbaI & PstI)376.5 5.9 0.49 2.36
pSB1C3 (EcoRI & SpeI)436.6 30.7 1.03 1.28
pSB1C3 (XbaI & PstI)457.4 7.9 1.65 0.42
Spinach (EcoRI & SpeI)490.5 3.0 1.87 0.26
tetR aptamer 12_1R (EcoRI & SpeI)451.7 -12.8 5.56 -0.04

Miniprep

No name

DNAconcentration[µg/mL]260/280260/230
RBS-lysis2 9264.61.972.21
RBS-lysis2 12220.01.892.46
RBS-lysis2 14195.31.881.53

Colony PCR

No name

Samplebase pair
8/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT(8/20 LB Amp) (1)~(4)2143
8/29 Plux-RBS-GFP-DT(8/27 LB-CP) (1)~(2)1227
8/29 Pbad/araC-RBS-RFP{BBa_I13516} (8/21 LB-CP) (1)~(2)2256
8/29 RBS-lysis3-DT-(1)1210
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s2min30cycle

Igku Aug30 colonyPCR N1.jpg

Ethanol Precipitation

No name

DNA
pT181 attenuator-(2) (EcoRI & SpeI)
pT181 attenuator-(2) (XbaI & PstI)
pT181 antisense-(2) (EcoRI & SpeI)
pT181 antisense-(2) (XbaI & PstI)
pSB1C3 (EcoRI & SpeI)
pSB1C3 (XbaI & PstI)
Spinach (EcoRI & SpeI)
tetR aptamer 12_1R (EcoRI & SpeI)

Restriction Enzyme Digestion

No name

8/30 RBS-lysis2 (9)EcoRISpeI10x bufferB100x BSAMilliQtotal
2 cuts7.5 µL0.5 µL0.5 µL3 µL0.3 µL18.2 µL30 µL
NC0.5 µL0 µL0 µL1 µL0.1 µL8.4 µL10 µL
8/22 pSB1C3-(2)EcoRISpeI10x Buffer100x BSAMilliQtotal
2 cuts8 µL0.5 µL0.5 µL3 µL0.3 µL17.7 µL30 µL
NC1 µL0 µL0 µL1 µL0.1 µL7.9 µL10 µL
8/22 pSB1C3-(2)XbaIPstIBufferDBSAMilliQtotal
2 cuts8 µL0.5 µL0.5 µL3 µL0.3 µL17.7 µL30 µL
NC1 µL0 µL0 µL1 µL0.1 µL7.9 µL10 µL
8/21 Spinach (1)EcoRISpeI10x BufferB100x BSAMilliQtotal
2 cuts4 µL0.5 µL0.5 µL3 µL0.3 µL21.9 µL30 µL
NC0.5 µL0 µL0 µL1 µL0.1 µL8.4 µL10 µL
8/21 tetR aptamer 12_1R (1)EcoRISpeI10xBufferB100xBSAMilliQtotal
2 cuts3 µL0.5 µL0.5 µL3 µL0.3 µL22.7 µL30 µL
NC0.4 µL0 µL0 µL1 µL0.1 µL8.4 µL10 µL
8/21 pT181 attenuator-(2)EcoRISpeIXbaIPstI10x BufferB10x BufferD100x BSAMilliQtotal
2 cuts4 µL0.5 µL0.5 µL0 µL0 µL3 µL0 µL0.3 µL21.7 µL30 µL
NC0.5 µL0 µL0 µL0 µL0 µL1 µL0 µL0.1 µL8.4 µL10 µL
2 cuts4 µL0 µL0 µL0.5 µL0.5 µL0 µL3 µL0.3 µL21.7 µL30 µL
NC0.5 µL0 µL0 µL0 µL0 µL0 µL1 µL0.1 µL8.4 µL10 µL
8/21 pT181 antisense-(2)EcoRISpeIXbaIPstI10x BufferB10x BufferD100x BSAMilliQtotal
2 cuts9 µL0.5 µL0.5 µL0 µL0 µL3 µL0 µL0.3 µL16.7 µL30 µL
NC1 µL0 µL0 µL0 µL0 µL1 µL0 µL0.1 µL7.9 µL10 µL
2 cuts9 µL0 µL0 µL0.5 µL0.5 µL0 µL3 µL0.3 µL16.7 µL30 µL
NC1 µL0 µL0 µL0 µL0 µL0 µL1 µL0.1 µL7.9 µL10 µL

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
1100bp ladder----
2RBS-lysis2-(9)EcoRISpeI
3RBS-lysis2-(9)----
4pSB1C3-(2)EcoRISpeI
5pSB1C3-(2)----
6pSB1C3-(2)XbaIPstI
7pSB1C3-(2)----
8tRNA-Spinach-(1)EcoRISpeI
9tRNA-Spinach-(1)----
10tetR aptamer12_1REcoRISpeI
11tetR aptamer12_1R----
12pT181 attenuatorEcoRISpeI
13pT181 attenuatorEcoRISpeI
14100bp ladder----

Igku Aug30 Electrophoresis(N2-1).jpg


LaneSampleEnzyme1Enzyme2
1100bp ladder----
2pT181 antisenseEcoRISpeI
3pT181 antisense----
4pT181 antisenseXbaIPstI
5pT181 antisense----
6pT181 attenuatorXbaIPstI
7pT181 attenuator----
8100bp ladder----

Igku Aug30 Electrophoresis(N2-2).jpg

LaneSample
11kb ladder
28/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(1)(Colony PCR product)
38/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(2)(Colony PCR product)
48/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(3)(Colony PCR product)
58/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(4)(Colony PCR product)
6Pbad/araC-RBS-RFP-DT-(1)(Colony PCR product)
7Pbad-araC-RBS-RFP-DT-(2)(Colony PCR product)

Igku Aug30 Electrophoresis(N2-3).jpg

Liquid Culture

No name

Samplemedium
8/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(1)Plusgrow medium(+Amp)
8/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(2)Plusgrow medium(+Amp)
8/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(3)Plusgrow medium(+Amp)
8/29 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(4)Plusgrow medium(+Amp)
8/29 Plux-RBS-GFP-DT-(1)Plusgrow medium(+CP)
8/29 Plux-RBS-GFP-DT-(2)Plusgrow medium(+CP)
8/29 Pbad/araC-RBS-RFP-DT-(1)Plusgrow medium(+CP)
8/29 Pbad-araC-RBS-RFP-DT-(2)Plusgrow medium(+CP)
RBS-lysis3-DT-(1)Plusgrow medium(+CP)

Aug 31

Gel Extraction

No name

LaneDNAEnzyme
1100bp ladder--
2----
3RBS-lysis2-(9)EcoRI & SpeI
4
5----
6pSB1C3(1)EcoRI & SpeI
7
8----
9pSB1C3(1)XbaI & PstI
10

Igku Aug30 Gel Extraction(N1).jpg
Igku Aug30 Gel Extraction(N2-1).jpg

LaneDNAEnzyme
1100bp ladder--
2tRNA-SpinachEcoRI & SpeI
3
4----
5pT181 attenuator-(2)EcoRI & SpeI
6
7pT181 attenuator-(2)XbaI & PstI
8
9----
10pT181 antisense-(2)EcoRI & SpeI
11

Igku Aug31 Gel Extraction(N2-2).jpg
Igku Aug31 Gel Extraction(N2-3).jpg

LaneDNAEnzyme
1100bp ladder--
2pT181 antisenseXbaRI & PstI
3
4----
5tetR aptamer 12_1R-(1)EcoRI & SpeI
6

Igku Aug31 Gel Extraction(N3-1).jpg
Igku Aug31 Gel Extraction(N3-2).jpg

Nameconcentration[µg/mL]260/280260/230
RBS-lysis2(EcoRI&SpeI)4.02.690.24
pSB1C3(EcoRI&SpeI)6.02.480.05
pSB1C3(XbaI&PstI)------
tRNA-Spinach------
pT181 attenuator-(2)(EcoRI&SpeI)------
pT181 anttenuator-(2)(SbaI&PstI)------
pT181 antisense-(2)(EcoRI&SpeI)------
pT181 antisense-(2)(XbaI&PstI)------
tetR aptamer 12_1R(EcoRI&SpeI)------

Ligation

Nakamoto and Hirano

stateVectorInserterLigation High ver.2
experiment8/30 pSB1C3 (EcoRI & SpeI)6.68/30 tRNA-Spinach (EcoRI & SpeI)12.53.5
experiment8/30 pSB1C3 (EcoRI & SpeI)6.68/30 pT181 antisense (EcoRI & SpeI)12.53.5
experiment8/30 pSB1C3 (XbaI & Pst)12.58/30 pT181 attenuator (XbaI & PstI)253.5
experiment8/30 pSB1C3 (XbaI & Pst)12.58/30 pT181 antisense (XbaI & PstI)253.5
experiment8/20 DT (EcoRI & XbaI)6.68/30 pT181 attenuator(2) (EcoRI & SpeI)12.53.5
experiment8/20 DT (EcoRI & XbaI)6.68/30 pT181 antisense (EcoRI & SpeI)12.53.5
experiment8/27 DT (EcoRI & XbaI)3.08/30 tRNA-Spinach (EcoRI & SpeI)12.53.5
experiment8/20 DT (EcoRI & XbaI)6.68/30 RBS-lysis2 (EcoRI & SpeI)12.53.5
experiment8/22 J23100 (SpeI & PstI)--RBS-tetR-DT (XbaI & PstI)--3.5
experiment8/22 Plac (SpeI & PstI)8.08/18 RBS-lacZα-DT (XbaI & PstI)103.5

Samples were evaporeted used evaporator into about 7 µL

Miniprep

Kojima and Nakamoto

DNAconcentration[µg/mL]260/280260/230
8/30 Plux-RBS-GFP-DT-(1)1241.771.87
8/30 Plux-RBS-GFP-DT-(2)2131.751.52
8/30 Pbad/araC-RBS-RFP4271.701.56
8/30 Pbad/araC-RBS-RFP6701.300.83
8/30 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(1)3321.732.04
8/30 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(2)4781.742.09
8/30 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(3)1461.772.11
8/30 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT-(4)4541.752.19

Sep 1

Transformation

No name

NameSampleCompetent CellsTotalPlate
8/31 tRNA-Spinach(pSB1C3)2µL20µL22µLCP
8/31 pT181 antisense(pSB1C3)2µL20µL22µLCP
8/31 pT181 attenuator(pSB1C3)2µL20µL22µLCP
8/31 pT181 antisense(pSB1C3)2µL20µL22µLCP
8/31 pT181 attenuator(2)-DT2µL20µL22µLCP
8/31 pT181 antisense-DT2µL20µL22µLCP
8/31 tRNA-Spniach-DT2µL20µL22µLCP
8/31 RBS-lysis2-DT2µL20µL22µLCP
8/31 Pconst-RBS-tetR-DT2µL20µL22µLAmp
8/31 Plac-RBS-lacZα-DT2µL20µL22µLCP
8/31 Ptet-RBS-lacZα-DT2µL20µL22µLCP
8/31 2µL20µL22µLCP

ligation production PCR

No name

Samplebase pair
8/31 tRNA Spinach(pSB1C3)459
8/31 pT181 antisense(2)(pSB1C3)(EcoRI+SpeI)415
8/31 pT181 attenuator(2)(pSB1C3)(XbaI+PstI)601
8/31 pT181 antisense(2)(pSB1C3)(XbaI+PstI)415
8/31 pT181 attenuator(2)-DT754
8/31 pT181 antisense(2)-DT577
8/31 tRNA Spinach-DT596
8/22 pSB1C3(2)(controll)1383
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s30min30cycle

Igku Sep1 ligation production PCR.jpg

Ligation

Nakamoto

stateVectorInserter
experimentDT(EcoRI+SpeI)5.58/28 RBS-lysis1(EcoRI+SpeI)2.4ng/µL2.4

1. Samples were evaporeted used evaporator into about 7 µL.
2. Add Ligation High 3.5&micro:L and incubate 16°C 1hour.

Liquid Culture

Tatsui,Stephane,Nakamoto

Samplemedium
8/21 pSB1C3(BBa_J04450)-(1)Plusgrow medium(+CP)
8/21 pSB1C3(BBa_J04450)-(2)Plusgrow medium(+CP)
RBS-lysis3-DTPlusgrow medium(+CP)


M9 Medium

No name 5xM9 liquid medium

volume100mL
NaHPO430g
KH2PO3g
Nacl0.25g
NH4Cl0.5g
3.75% agar solution400mL
1M MgSO40.5mL
2M Glucose2.8mL
1% Vitamin B10.5mL
1M CaCl20.05mL

1. mix 5xM9 medium and 3.75%Agar solution 400mL
2. Add 1M MgSO4, 2M Glucose and 1 VitaminB1 0.5mL
3. Add CaCl2 while shaking Erlenmeyer flask.

Miniprep

DNAconcentration[µg/mL]260/280260/230
8/21 pSB1C3(1)309.21.691.72
8/21 pSB1C3(2)201.61.651.76

Kanamycin applicaton

Hirano

Add 20µL Kanamycin into 80µL MilliQ and apply it on a 9/1 M9 plate

Plating

Hirano

Apply entA-colony liquid culture (100µL) on M9(Km)plate and incubate 37°C

2 fold diluted OD 600 2.107

Liquid Culture

Hirano

Samplemedium
9/1 entA- Master Plate-(4)SOB Medium(+Km) 　

Sep 2

Colony PCR

Tatsui

Samplebase pair
9/1 RBS-lysis2-DT-(1)985
Ptet-RBS-lacZα-DT-(1)765
Plac-RBS-lacZα-DT-(1)765
Plac-RBS-lacZα-DT-(2)765
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s1min30cycles

Electrophoresis

No name

LaneSample
1100bp ladder
29/1 RBS-lysis2+DT -(1)
39/1 Ptet+RBS-lacZα-DT -(1)
49/1 Plac+RBS-lacZα-DT -(1)
59/1 Plac+RBS-lacZα-DT -(2)
6100bp ladder

Igku Sep2 ColonyPCR (N1-2).jpg

Restriction Enzyme Digestion

Nakamoto

pSB1C3-(1)EcoRISpeIXbaIPstIBufferBBufferDBSAMilliQtotal
2 cuts(EcoRI+SpeI)7µL1µL1µL0µL0µL3µL0µL0.3µL17.7µL30µL
NC(EcoRI+SpeI)0.3µL0µL0µL0µL0µL1µL0µL0.1µL8.6µL10µL
2 cuts(XbaI+PstI)7µL0µL0µL1µL1µL0µL3µL0.3µL17.7µL30µL
NC(XbaI+PstI)0.3µL0µL0µL0µL0µL0µL1µL0.1µL8.6µL10µL
8/21 tRMA-spinach(1)EcoRISpeIBufferBBSAMilliQtotal
2 cuts8µL1.0µL1.0µL3µL0.3µL17.6µL30µL
NC0.5µL0µL0µL1µL0.1µL8.4µL10µL
8/21 tetR aptamer12_1R-(1)EcoRISpeIBufferBBSAMilliQtotal
2 cuts6µL1.0µL1.0µL3µL0.3µL18.7µL30µL
NC0.4µL0µL0µL1µL0.1µL8.5µL10µL
8/21 pT181 attenuator-(2)EcoRISpeIXbaIPstIBufferBBufferDBSAMilliQtotal
2 cuts(EcoRI+SpeI)8µL1µL1µL0µL0µL3µL0µL0.3µL16.3µL30µL
NC(EcoRI+SpeI)0.5µL0µL0µL0µL0µL1µL0µL0.1µL8.4µL10µL
2 cuts(XbaI+PstI)8µL0µL0µL1µL1µL0µL3µL0.3µL16.3µL30µL
NC(XbaI+PstI)0.5µL0µL0µL0µL0µL0µL1µL0.1µL8.4µL10µL

Igku Sep2 Restriction Enzyme Digestion (N1-1).jpg

Liquid Culture

Kojima

Samplemedium
tRNA-Spinach-1Plusgrow medium(+CP)
tetR aptamaer12_1R-1Plusgrow medium(+CP)
pT181 attenuator-1Plusgrow medium(+CP)　
pT181 antisense-1Plusgrow medium(+CP)

Master Plate

Kojima

NumberUse LB plate(+CP)
1tRNA Spinach-1
2tetR aptamaer12_1R
3tetR aptamaer12_P
4tetR aptamaer12_1M
5pT181 attenuator-2
6Fusion1 attenuator-1
7Fusion3m2 attenuator-1
8pT181 antisense-1
9Fusion1 antisense-1
10Fuaion6 antisense-1

Electrophoresis

Nakamoto & Tatsui

LaneSampleEnzyme1Enzyme2
1100bp ladder----
2pSB1C3 -(1)EcoRISpeI
3pSB1C3 -(1)----
5pSB1C3 -(1)XbaIPstI
6pSB1C3 -(1)----
7pT181 attenuator (2)EcoRISpeI
8pT181 attenuator (2)----
9pT181 attenuator (2)XbaISpeI
10pT181 attenuator (2)----
11tetR-aptamer 12_1REcoRISpeI
12tetR-aptamer 12_1R----
13tRNA Spinach (1)EcoRISpeI
14100bp ladder----
15tRNA Spinach (1)----

Igku Sep2 Restriction Enzyme Digestion (N1-1).jpg

Gel Extraction

No name

LaneDNAEnzyme
1100bp ladder-
2pSB1C3(EcoRI+SpeI)EcoRI&SpeI
3
4----
58/24 tetR aptamer 12_1REcoRI&SpeI
6

Igku Sep1 Gel Extraction(N2-1).jpg
Igku Sep1 Gel Extraction(N2-2).jpg

LaneDNAEnzyme
1100bp ladder-
2pT181 attenuator(2)(EcoRI+SpeI)EcoRI&SpeI
3
4----
5pT181 attenuator(2)(XbaI+PstI)EcoRI&SpeI
6
7----
8pT181 attenuator(2)(XbaI+PstI)EcoRI&SpeI
9
10----
11SpinachEcoRI&SpaI
12

Igku Sep1 Gel Extraction(N3-1).jpg
Igku Sep1 Gel Extraction(N3-2).jpg

Nameconcentration[µg/mL]260/280260/230
pSB1C3 (EcoRI&SpeI)3.02.180.31
pSB1C3 (XbaI&PstI)4.72.250.36
pT181 attenuator-(2)(EcoR&SpeI)8.62.740.01
pT181 attenuator-(2) (XbaI&PstI)16.52.460.03
Spinach (EcoRI&SpeI)2.82.980.27
tetR aptamer12_1R (EcoRI&SpeI)50.428.071.97

Colony PCR

No name

Samplebase pair
9/1 Spinach(pSB1C3)about 450
9/1 Spinach-DT596
9/1 RBS-lysis1-DT613
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s40s30cycles
Samplebase pair
9/1 Ptet-RBS-lacZα-DT-2738
9/1 Ptet-RBS-lacZα-DT-3738
9/1 Ptet-RBS-lacZα-DT-4738
9/1 Ptet-RBS-lacZα-DT-5738
9/1 Ptet-RBS-lacZα-DT-6738
9/1 RBS-lysis2-DT-2985
9/1 RBS-lysis2-DT-3985
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s1min30cycles

Igku Sep3 ColonyPCR (N1).jpg

Electrophoresis

No name

LaneSample
1100bp ladder
2Spinach(pSB1C3)
3Spinach-DT
4RBS-lysis1-DT
5100bp ladder

Igku Sep2 ColonyPCR (N4).jpg

Master Plate

No name

NumberUse LB plate(+CP)
19/1 Spinach(pSB1C3)
29/1 Spinach-DT-(1)

Liquid Culture

No name

Samplemedium
pT181 attenuatorPlusgrow medium (+CP)
pT181 antisensePlusgrow medium (+CP)
SpinachPlusgrow medium (+CP)
  • incubated at 37 °C for 1 hour

Transformation

No name

NameSampleCompetent CellsTotalPlate
8/28 RBS-lysis3-DT3µL30µL33µLCP

Sep 3

Electrophoresis

Kojima

LaneSample
1100bp ladder
2pT181 attenuator(2)-DT2(738)
3Ptet-RBS-lacZα-DT-2(756)
4Ptet-RBS-lacZα-DT-3
5Ptet-RBS-lacZα-DT-4
6Ptet-RBS-lacZα-DT-5
7Ptet-RBS-lacZα-DT-6
8RBS-lysis2-DT-2(985)
9RBS-lysis2-DT-3
10100bp ladder

Igku Sep3 Electrophoresis(N1).jpg

Miniprep

Tatsui

DNAconcentration[µg/mL]260/280260/230
9/2 14:00 pT181 attenuator418.41.821.80
9/2 14:00 pT181 antisense362.21.921.96
9/2 14:00 Spinach438.31.861.99
9/2 14:00 tetR-apt 12_1R567.51.901.99
9/2 21:00 pT181 attenuator320.91.902.15
9/2 21:00 Spinach457.01.882.24

Liquid Culture

Kojima

Samplemedium
Spinach-DT(9/2 master plate)plusgrow(CP)4ml

Restriction Enzyme Digestion

Kojima

DNAEcoRISpeIXbaIPstI10xBuffer B(EcoRI&SpeI)10xBuffer D(XbaI&PstI)100xBSAMilliQtotal
9/3 pT181attenuator 14:00 418.4ng/µL 4.8µL 1.0µL 1.0µL 0µL 0µL 3µL 0µL 0.3µL 19.9µL 30µL
9/3 pT181attenuator 14:00 418.4ng/µL 0.2µL 0µL 0µL 0µL 0µL 1µL 0µL 0.1µL 8.7µL 10µL
9/3 pT181attenuator 14:00 418.4ng/µL 4.8µL 0µL 0µL 1.0µL 1.0µL 0µL 3µL 0.3µL 19.9µL 30µL
9/3 pT181attenuator 14:00 418.4ng/µL 0.2µL 0µL 0µL 0µL 0µL 0µL 1µL 0.1µL 8.7µL 10µL
DNAEcoRISpeIXbaIPstI10xBuffer B(EcoRI&SpeI)10xBuffer D(XbaI&PstI)100xBSAMilliQtotal
9/3 pT181antisense 14:00 362.2ng/µL 5.5µL 1.0µL 1.0µL 0µL 0µL 3µL 0µL 0.3µL 19.2µL 30µL
9/3 pT181attenuator 14:00 418.4ng/µL 0.3µL 0µL 0µL 0µL 0µL 1µL 0µL 0.1µL 8.6µL 10µL
9/3 pT181attenuator 14:00 418.4ng/µL 5.5µL 0µL 0µL 1.0µL 1.0µL 0µL 3µL 0.3µL 19.2µL 30µL
9/3 pT181attenuator 14:00 418.4ng/µL 0.3µL 0µL 0µL 0µL 0µL 0µL 1µL 0.1µL 8.6µL 10µL
DNAEcoRISpeI10xBuffer B100xBSAMilliQtotal
9/3 apt 12_1R 14:00 507.5ng/µL 3.9µL 1.0µL 1.0µL 3µL 0.3µL 20.8µL 30µL
9/3 apt 12_1R 14:00 507.5ng/µL 0.2µL 0µL 0µL 1µL 0.1µL 8.7µL 10µL
DNAEcoRIXbaI10xBuffer D100xBSAMilliQtotal
8/29 DT 166.2ng/µL 12µL 1.0µL 1.0µL 3µL 0.3µL 12.7µL 30µL
8/29 DT 166.2ng/µL 0.6µL 0µL 0µL 1µL 0.1µL 8.3µL 10µL
DNAEcoRISpeI10xBuffer B100xBSAMilliQtotal
8/22 RBS-lysis1(2) 96ng/µL 21µL 1.0µL 1.0µL 3µL 0.3µL 3.7µL 30µL
8/22 RBS-lysis1(2) 96ng/µL 1.0µL 0µL 0µL 1µL 0.1µL 7.9µL 10µL
DNAEcoRISpeI10xBuffer B100xBSAMilliQtotal
8/30 RBS-lysis2 220ng/µL 9.1µL 1.0µL 1.0µL 3µL 0.3µL 15.6µL 30µL
8/30 RBS-lysis2 220ng/µL 0.5µL 0µL 0µL 1µL 0.1µL 8.4µL 10µL
DNAEcoRISpeI10xBuffer B100xBSAMilliQtotal
8/20 RBS-lysis3(1) 282ng/µL 7.1µL 1.0µL 1.0µL 3µL 0.3µL 17.6µL 30µL
8/20 RBS-lysis3(1) 282ng/µL 0.4µL 0µL 0µL 1µL 0.1µL 8.5µL 10µL

PCR

Hirano

Plac(1) plasmid 153.7ng/µL10x buffer KOD plus-ver.22mM dNTPs25mM MgSO4primer (KaiA_BsaI_Mut_fwd)primer (KaiA_BsaI_Mut_rer)KOD plus-ver.2MilliQtotal
0.13 2.52.51.50.750.750.516.3725
KaiB (plasmid) 20.2ng/µL10x buffer KOD plus-ver.22mM dNTPs25mM MgSO4primer (GG2_RBS_KaiB_fwd)primer (KaiB_GG3_rer)KOD plus-ver.2MilliQtotal
12.52.51.50.750.750.515.525
KaiC (plasmid) 4.4ng/µL10x buffer KOD plus-ver.22mM dNTPs25mM MgSO4primer primer KOD plus-ver.2MilliQtotal
12.52.51.50.750.750.515.525
DT-1 (plasmid) 153.7ng/µL10x buffer KOD plus-ver.22mM dNTPs25mM MgSO4primer primer KOD plus-ver.2MilliQtotal
0.12.52.51.50.750.750.516.425
PKaiBC(PCR product) 16.3ng/µL10x buffer KOD plus-ver.22mM dNTPs25mM MgSO4primer primer KOD plus-ver.2MilliQtotal
0.72.52.51.50.750.750.515.825
SasA(PCR product) 21.6ng/µL10x buffer KOD plus-ver.22mM dNTPs25mM MgSO4primer primer KOD plus-ver.2MilliQtotal
0.52.52.51.50.750.750.51625
RpaA(PCR product) 15.4ng/µL10x buffer KOD plus-ver.22mM dNTPs25mM MgSO4primer primer KOD plus-ver.2MilliQtotal
0.62.52.51.50.750.750.515.925
RpaB(PCR product) 26.7ng/µL10x buffer KOD plus-ver.22mM dNTPs25mM MgSO4primer primer KOD plus-ver.2MilliQtotal
0.42.52.51.50.750.750.516.125
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C68°C--
5min10sec30sec30
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C68°C--
5min10sec30sec30


Mutatation PCR

No name

KaiABC(12.6pg/µL)Primer(Kai_BsaIMut_fwd)Primer(Kai_BsaIMut_rer)Prime STAR Max PremixMilliQtotal
1 11252250
Primer(Kai_BsaIMut_fwd)Primer(Kai_BsaIMut_rer)Prime STAR Max PremixMilliQtotal
11252350
PreDenatureDenatureAnnealingExtensioncycle
　94°C　98°C　65°C　72°C--
5min10sec　15sec　40sec　30

Colony PCR

Samplebase pair
9/1 pT181antisense(pSB1C3)400bp
9/1 pT181antisense-DT547bp
PreDenatureDenatureAnnealingExtensioncycle
　94°C　94°C　55°C　68°C--
5m　30s　30s　30s　30

Electrophoresis

Kojima

LaneSample
1100bp ladder
29/1 pT181antisense(pSB1C3)
39/1 pT181antisense

Igku Sep3 Electrophoresis (N2).jpg

Electrophoresis

LaneSample
1 100bp ladder
2 attenuator EcoRI&SpeI
3 attenuator negative EcoRI&SpeI
4 attenuator XbaI&PstI
5 attenuator negative XbaI&PstI
6 antisense EcoRI&SpeI
7 antisense negative EcoRI&SpeI
8 100bp ladder
LaneSample
1 100bp ladder
2 antisense XbaI&PstI
3 antisense negative XbaI&PstI
4 apt EcoRI&SpeI
5 apt negative EcoRI&SpeI
6 DT EcoRI&XbaI
7 DT negative EcoRI&XbaI
8 100bp ladder

Igku Sep3 Electrophoresis(N3 2).jpg

LaneSample
1 100bp ladder
2 RBS-lysis1 EcoRI&SpeI
3 RBS-lysis1 negative
4 RBS-lysis2 EcoRI&SpeI
5 RBS-lysis2 negative
6 RBS-lysis3 EcoRI&SpeI
7 RBS-lysis3 negative
8 100bp ladder

Igku Sep3 Electrophoresis(N3 3).jpg

Gel Extraction

LaneDNAEnzyme
1 100bp ladder
3 pT181 attenuatorEcoRI&SpeI
4 pT181 attenuatorEcoRI&SpeI
6 pT181 attenuatorXbaI&PstI
7 pT181 attenuatorXbaI&PstI
9 pT181 antisenseEcoRI&SpeI
10 pT181 antisenseEcoRI&SpeI

Igku Sep3 Electrophoresis(N4 1).jpg

LaneDNAEnzyme
1 100bp ladder
2 pT181 antisenseXbaI&PstI
3 pT181 antisenseXbaI&PstI
5 apt12_1REcoRI&SpeI
6 apt12_1REcoRI&SpeI

Igku Sep3 Electrophoresis(N4 2).jpg

LaneDNAEnzyme
1 100bp ladder
2 DTEcoRI&XbaI
3 DTEcoRI&XbaI
5 RBS-lysis1EcoRI&SpeI
6 RBS-lysis1EcoRI&SpeI

Igku Sep3 Electrophoresis(N5 3).jpg

LaneDNAEnzyme
1 100bp ladder
2 RBS-lysis2EcoRI&SpeI
3 RBS-lysis2EcoRI&SpeI
5 RBS-lysis3EcoRI&SpeI
6 RBS-lysis3EcoRI&SpeI

Igku Sep3 Electrophoresis(N5 4).jpg

Nameconcentration[µg/mL]260/280260/230
pT181 attenuator(EcoRI&SpeI)10.0 1.86 0.39
pT181 attenuator(XbaI&PstI) 11.7 1.65 0.36
pT181 antisense(EcoRI&SpeI) 4.2 1.95 0.25
pT181 antisense(XbaI&PstI)6.6 1.78 0.33
tRNA 12_1R apt(EcoRI&SpeI)5.7 1.73 0.29
DT(EcoRI&XbaI) 16.6 1.99 0.76
RBS-lysis1(EcoRI&SpeI)8.8 1.65 0.32
RBS-lysis2(EcoRI&SpeI)13.4 1.61 0.48
RBS-lysis3(EcoRI&SpeI)10.2 2.19 0.51

Liquid Culture

No name

Samplemedium
8/16master plate7(DT)
8/21RBS-lysis1 　

Sep 4

Ligation

No name

stateVectorInserterLigation High ver.2
experiment9/3 DT(EcoRI&XbaI)3.2µL9/3 RBS-lysis12.4µL2.7µL
experiment9/3 DT(EcoRI&XbaI) 3.0µL 9/3 RBS-lysis24.7µL 4.0µL
experiment9/3 DT(EcoRI&XbaI) 3.0µL 9/3 RBS-lysis38.8µL 4.0µL
experiment9/3 DT(EcoRI&XbaI) 3.0µL 9/3 aptamer12_1R(EcoRI&SpeI) 1.9µL 2.45µL
experiment9/2 pSB1C3(XbaI&PstI)10.6µL 9/3 pT181attenuator (XbaI& PstI)3.2µL 4.0µL
experiment9/2 pSB1C3(XbaI&PstI)9.6µL 9/3 pT181antisense (XbaI& PstI)2.1µL 4.0µL
experiment9/2 pSB1C3(EcoRI&SpeI)16.7µL 9/3 aptamer12_1R(EcoRI&SpeI) 2.0µL 4.0µL

incubate 16 °C 1 hour

Transformation

Tatsui

NameSampleCompetent CellsPlate
9/4 RBS-lysis1-DT 2µL20µLAmp
9/4 RBS-lysis2-DT 2µL20µLAmp
9/4 RBS-lysis3-DT 2µL20µLAmp
9/4 aptamer12_1R-DT2µL20µLAmp
9/4 pT181attenuator –pSB1C32µL20µLAmp
9/4 pT181antisense-pSB1C3 2µL20µLAmp
9/4 aptamer12_1R –pSB1C32µL20µLAmp
9/4 pSB4K5(2013plate5 56) 2µL20µLAmp
9/3 Mutagenesis product 1(KaiABC) 2µL20µLAmp
9/3 Mutagenesis product 2(NC) 2µL20µLAmp

Miniprep

No name

DNAconcentration[µg/mL]260/280260/230
DT193.11.931.81
Spinach-DT373.31.921.69
RBS-lysis1332.01.951.62

Master Plate

Hirano

NumberUse LB plate (+CP)
18/10 Plac(CP)-2
2 8/10 Plac(CP)-2
38/10 Plac(CP)-2
NumberUse LB plate (+Amp)
18/9 J23100(Amp)-1
28/9 J23100(Amp)-1
38/9 J23100(Amp)-1

Liquid Culture

Hirano

Samplemedium
8/10 Plac(CP)-2Plusgrow medium(+CP)
8/9 J23100(Amp)-1Plusgrow medium(+Amp)

PCR

Hirano

8/29 Pλ-RBS-luxI-DTKOD plus10x bufferdNTPMgSO4F primer (RBS-luxI-DT-cloning-fwd) R primer(RBS-luxI-DT-cloning-rev)MilliQtotal
0.3µL0.5µL 2.5µL 2.5µL 1.5µL 0.75µL 0.75µL 16.2µL 25µL
8/31 Pbad/araC-RBS-RFP(1)KOD plus10x bufferdNTPMgSO4F primer (Pbad/araC-pSB1C3-cloning-fond) R primer(Pbad/araC-cloning-rex)MilliQtotal
0.3µL0.5µL 2.5µL 2.5µL 1.5µL 0.75µL 0.75µL 16.2µL 25µL
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C65°C68°C--
5min10s30s3min10s30cycles

Restriction Enzyme Digestion

No name

8/21 PconstSpeIPstIBufferBSAMilliQtotal
2 cuts10.5µL1.0µL1.0µL3.0µL0.3µL14.2µL30 µL
NC0.5µL0µL0µ1.0µL0.1µL8.4µL10 µL
8/29 PlacSpeIPstIBufferBSAMilliQtotal
2 cuts13.0µL1.0µL1.0µL3.0µL0.3µL11.7µL30 µL
NC0.7µL0µL0µ1.0µL0.1µL 8.2µL10 µL
8/20 PtetSpeIPstIBufferBSAMilliQtotal
2 cuts9.0µL1.0µL1.0µL3.0µL0.3µL15.7µL30 µL
NC0.5µL0µL0µ1.0µL0.1µL 8.4µL10 µL
8/20 RBS-tetR-DTXbaIPstIBufferBSAMilliQtotal
2 cuts8.5µL1.0µL1.0µL3.0µL0.3µL16.2µL30 µL
NC0.5µL0µL0µ1.0µL0.1µL 8.4µL10 µL
spinach -DTXbaIPstIBufferBSAMilliQtotal
2 cuts5.4µL1.0µL1.0µL3.0µL3.0µL16.6µL30 µL
NC0.3µL0µL0µ1.0µL1.0µL 7.7µL10 µL
8/17 RBS-lacZα-DT XbaIPstIBufferBSAMilliQtotal
2 cuts11µL1.0µL1.0µL3.0µL3.0µL11µL30 µL
NC0.5µL0µL0µ1.0µL1.0µL7.5µL10 µL

Transformation

No name

NameSampleCompetent CellsTotalPlate
Ptrc KaiC1µL10µL11µLAmp
Ptrc KaiB1µL10µL11µLAmp
pSΩ 1µL10µL11µLAmp

Electrophoresis

No name
1

LaneSampleEnzyme1Enzyme2
1100bp ladder----
2PconstSpeIPstI
3Pconst NC----
4PlacSpeIPstI
5Plac NC----
6100bp ladder----

2

LaneSampleEnzyme1Enzyme2
1100bp ladder----
2PtetSpeIPstI
3Ptet NC----
4RBS-tetR-DTXbaIPstI
5RBS-tetR-DT NC----
6100bp ladder----

3

LaneSampleEnzyme1Enzyme2
1100bp ladder----
2Spinach-DTXbaIPstI
3Spinach-DT NC----
4RBS-lacZα-DTXbaIPstI
5RBS-lacα-DT NC----
6100bp ladder----

Igku Sep4 Electrophoresis(N1).jpg
Igku Sep4 Electrophoresis N2 3.jpg

Gel Extraction

No name

1

LaneDNAEnzyme
1100bp ladder--
2PconSpeI&PstI
3PconSpeI&PstI
4----
5PlacSpeI&PstI
6PlacSpeI&PstI

2

LaneDNAEnzyme
1100bp ladder--
2PtetSpeI&PstI
3PtetSpeI&PstI
4----
5RBS-tetR-DTXbaI&PstI
6RBS-tetR-DTXbaI&PstI

Igku Sep4 Electrophoresis(N2).jpg
Igku Sep4 Gel Extraction(N2) 2.jpg

Namequantityconcentration[µg/mL]260/280260/230
Pcon--13.2 1.99 -1.64
Plac-- 15.3 2.07 -0.39
Ptet-- 30.11.97 -0.97
RBS-tetR-DT--3.02.44 0.88

Gel Extraction

No name
LaneDNAEnzyme
1100bp ladder--
2spinach-DTXbaI&PstI
3spinach-DT XbaI&PstI
4----
5RBS-lacZα-DTXbaI&PstI
6RBS-lacZα-DTXbaI&PstI

Igku Sep4 Gel Extraction(N3) 1.jpg
Igku Sep4 Gel Extraction(N3) 2.jpg

Namequantityconcentration[µg/mL]260/280260/230
spinach-DT--2.12.10-0.05
RBS-lacZα-DT--3.32.59-0.07

Master Plate

No name

NumberUse LB plate (+Amp)
48/9 BBa_J23100-3
58/9 BBa_J23100-4
68/9 BBa_J23100-5
NumberUse LB plate
1JM109-1
2JM109-2
3JM109-3
4JM109-4

Liquid Culture

Hirano

Samplemedium
8/9 BBa_J23100-3Plusgrow medium(+Amp)
JM109-1(Master plate)LB medium

Colony PCR

Hirano

Samplebase pair
8/9 BBa_J23100 3--
8/9 BBa_J23100 4--
8/9 BBa_J23100 5--
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s30s30cycles

Sep 5

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
1100bp ladder----
2J23100-3----
3J23100-4----
4J23100-5----
5100bp ladder----

Igku Sep5 electrophoresis N1.jpg

Miniprep

Nakamoto

DNAconcentration[µg/mL]260/280260/230
8/3 J23100-3349.11.691.851
8/10 Plac-2331.31.841.42

Ligation

Kojima

stateVectorInserterLigation High ver.2
experimentPconst13.2spinach-DT2.13.5
experimentPlac15.3spinach-DT2.13.5
experimentPconst13.2pT181 antisense (XbaI & PstI)6.63.5
experimentPlac15.3pT181 antisense (XbaI & PstI)6.63.5
experimentPtet30.1pT181 antisense (XbaI & PstI)6.63.5
experimentPconst13.2pT181 attenuator11.73.5
experimentPlac15.3pT181 attenuator11.73.5
experimentPtet30.1RBS-lacZα-DT3.03.5

incubate 16 °C 1 hour

Electrophoresis

Tatsui

LaneSample
1100bp ladder
2RBS-luxI-DT
3Pbad/araC
4NC
5100bp ladder

Igku Sep5 electrophorsis N2 1.jpg
Igku Sep5 electrophorsis N2 2.jpg

Gel Extraction

No name

LaneDNAEnzyme
11kbp ladder
2RBS-luxI-DT
3RBS-luxI-DT
4
5Pbad/araC
6Pbad/araC

Igku Sep5 Gel Extraction(N3) 1.jpg
Igku Sep5 Gel Extraction(N3) 2.jpg

Nameconcentration[µg/mL]260/280260/230
RBS-luxI-DT23.20.837.89
Pbad/araC51.81.200.11

Colony PCR

No name

Samplebase pair
9/4 aptamer 12_1R(pSB1C3)-1384
9/4 aptamer 12_1R(pSB1C3)-2384
9/4 pT181 antisense(pSB1C3)-1415
9/4 pT181 antisense(pSB1C3)-2415
9/4 pT181 antisense(pSB1C3)-3415
9/4 pT181 antisense(pSB1C3)-4415
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s30s30cycles
Samplebase pair
9/4 aptamer 12_1R-DT-1521
9/4 aptamer 12_1R-DT-2521
9/4 aptamer 12_1R-DT-3521
9/4 aptamer 12_1R-DT-4521
9/4 pT181 attenuator(pSB1C3)601
9/4 pT181 attenuator(pSB1C3)601
9/4 RBS-lysis1-DT613
9/4 RBS-lysis1-DT613
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s36s30cycles
Samplebase pair
9/4 RBS-lysis2-DT-1985
9/4 RBS-lysis2-DT-2985
9/4 RBS-lysis2-DT-3985
9/4 RBS-lysis2-DT-4985
9/4 RBS-lysis3-DT-11210
9/4 RBS-lysis3-DT-21210
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s72s30cycles

Liquid Culture

Nakamoto

Samplemedium
8/9 J23100-5Plusgrow medium(+Amp)

37°C

Transformation

No name

NameSample(µL)Competent Cells(µL)Total(µL)Plate
9/5 Pconst+spinach-DT22022--
9/5 Plac+spinach-DT22022--
9/5 Pconst+pT181 antisense22022--
9/5 Plac+pT181 antisense22022--
9/5 Ptet+pT181 antisense22022--
9/5 Plux+pT181 attenuator22022--
9/5 Ptet+pT181 attenuator22022--
9/5 Ptet+pT181 attenuator22022--
9/5 Pconst+RBS-tetR+DT22022--
9/16(2012) T7-His-FT22022--
9/16(2012) pBr32222022--

Restriction Enzyme Digestion

No name

9/5 Pbad/araCEcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts4µL 0µL 1µL 0µL 1µL 3µL 3µL 18µL 30µL
9/5 RBS-luxI-DTEcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts4µL 0µL 0µL 1µL 1µL 3µL 3µL18µL 30µL
8/28 PluxEcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts 12µL 0µL 1µL 0µL 1µL 3µL 3µL10µL30µL
NC 0.6µL0µL0µL 0µL 0µL1µL1µL7.4µL10µL
9/5 Pconst(J23100)EcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts5.7µL0µL1µL0µL1µL3µL3µL16.3µL30µL
NC 0.3µL0µL0µL 0µL 0µL1µL1µL7.7µL10µL
9/5 PlacEcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts6.0µL0µL1µL0µL1µL3µL3µL16µL30µL
NC 0.3µL0µL0µL 0µL 0µL1µL1µL7.7µL10µL
8/31 Plux-RBS-GFP-DT-1EcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts9.4µL1µL1µL0µL0µL3µL3µL12.6µL30µL
NC 0.5µL0µL0µL 0µL 0µL1µL1µL7.5µL10µL
8/20 Pconst-RBS-luxR-DTEcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts4.5µL1µL0µL1µL0µL3µL3µL13.5µL30µL
NC 0.2µL0µL0µL 0µL 0µL1µL1µL7.8µL10µL
DNAconcentration[µg/mL]260/280260/230
9/5 RBS-luxI-DT7.61.56-1.42
9/5 Pbad/araC7.51.543.84

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
1100bp ladder----
2aptamer 12_1R (pSB1C3)-1----
3aptamer 12_1R (pSB1C3)-2----
4pT181 antisense(pSB1C3)-1----
5pT181 antisense(pSB1C3)-2----
6pT181 antisense(pSB1C3)-3----
7pT181 antisense(pSB1C3)-4----
8100bp ladder----

Igku Sep5 Electrophoresis(N4).jpg

LaneSampleEnzyme1Enzyme2
1100bp ladder----
2aptamer 12_1R-DT-1----
3aptamer 12_1R-DT-2----
4aptamer 12_1R-DT-3----
5aptamer 12_1R-DT-4----
6pT181 attenuator(pSB1C3)-1----
7pT181 attenuator(pSB1C3)-2----
8RBS-lysis1-DT-1----
9RBS-lysis1-DT-2----
10100bp ladder----

Igku Sep5 Electrophoresis(N6) 2.jpg

LaneSampleEnzyme1Enzyme2
1100bp ladder----
2RBS-lysis2-DT-1----
3RBS-lysis2-DT-2----
4RBS-lysis2-DT-3----
5RBS-lysis2-DT-4----
6RBS-lysis3-DT-1----
7RBS-lysis3-DT-2----
8100bp ladder----

Igku Sep5 Electrophoresis(N5).jpg

LaneSampleEnzyme1Enzyme2
11kbp ladder----
2PconstSpeIPstI
3Pconst NC----
4PlacSpeIPstI
5Plac NC----
6PluxSpeIPstI
7Plux NC----
8Pconst-RBS-luxR-DTEcoRIXbaI
9Pconst-RBS-luxR-DT NC----
10Plux-RBS-GFP-DTEcoRISpeI
11Plux-RBS-GFP-DT NC----
121kbp ladder----

Igku Sep5 Electrophoresis(N6) 3.jpg

5x M9 Medium (+EDTA)

Hirano

volume10ml
Na2HPO4 60mg
KH2PO430mg
NaCl5mg
NH4Cl10mg
Fe(III)-EDTA1263.27mg　
MilliQup to 10 mL
  • autoclave at 121 °C for 20 min

Gel Extraction

No name

LaneDNAEnzyme
11kbp ladder
2PconstSpeI & PstI
3PconstSpeI & PstI

Igku Sep5 gel extraction N7 1.jpg
Igku Sep5 gel extraction N7 2.jpg

Nameconcentration[µg/mL]260/280260/230
Pconst5.31.980.06
LaneDNAEnzyme
1100bp ladder
2PlacSpeI & PstI
3PlacSpeI & PstI
5PluxSpeI & PstI
6PluxSpeI & PstI

Igku Sep5 gel extraction N7 3.jpg
Igku Sep5 gel extraction N7 4.jpg

Nameconcentration[µg/mL]260/280260/230
Plac (SpeI & PstI)5.21.820.36
Plux(SpeI & PstI)8.01.870.25
LaneDNAEnzyme
11kbp ladder
2Pconst-RBS-luxR-DTEcoRI & XbaI
3Pconst-RBS-luxR-DTEcoRI & XbaI
5Plux-RBS-GFP-DTEcoRI & SpeI
6Plux-RBS-GFP-DTEcoRI & SpeI

Igku Sep5 gel extraction N7 5.jpg
Igku Sep5 gel extraction N7 6.jpg

Nameconcentration[µg/mL]260/280260/230
Pconst-RBS-luxR-DT(EcoRI & XbaI)30.61.841.16
Plux-RBS-GFP-DT (EcoRI & SpeI)21.81.870.98

DNA Purification

No name

DNAconcentration[µg/mL]260/280260/230
9/5 RBS-luxI-DT7.61.56-1.42
9/5 Pbad/araC7.51.543.84

Miniprep

No name

DNAconcentration[µg/mL]260/280260/230
8/9 J23100-5243.01.901.81

Colony PCR

No name

Samplebase pair
9/4 RBS-lysis3-DT-31210
9/4 RBS-lysis3-DT-41210
9/4 RBS-lysis3-DT-51210
9/4 RBS-lysis1-DT-3613
9/4 Ptrc KaiC-1--
9/4 pSB4K51370
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s1min 25s30cycles

Liquid Culture

Hirano

Samplemedium
9/4 aptamer 12_1R-DT-1Plusgrow medium (+CP)
9/4 pT181 attenuator(pSB1C3)-1Plusgrow medium (+CP)
9/4 RBS-lysis2-DT-1Plusgrow medium (+CP)
9/4 Ptrc-KaiC -1Plusgrow medium (+Amp)
9/4 pSB4K5 -1Plusgrow medium (+Kan)

Sep 6

Electrophoresis

Hirano

LaneSample
1100bp ladder
29/4 RBS-lysis3-DT -3
39/4 RBS-lysis3-DT -4
49/4 RBS-lysis3-DT -5
59/4 RBS-lysis1-DT -3
69/4 Ptrc-KaiC -1
79/4 pSB4K5 -1
8100bp ladder

Igku Sep6 ColonyPCRN1.jpg

Colony PCR

No name

Samplebase pair
Pconst-spinach-DT-(1~4)563
Plac-spinach-DT-(1~4)659
Pcon-pT181 antisense-(1~4)468
Ptet-pT181 antisense-(1~4)604
Pconst-pT181 attenuator-(1~4)568
Pconst+RBS-tetR-DT-(1~4)1121
Ptet+RBS-lacZα-DT-(1~4)1162
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s40s30cycles

Liquid Culture

No name

Samplemedium
9/4 RBS-Lysis3+DT3Plusgrow medium(+CP)
9/4 RBS-Lysis1+DT3Plusgrow medium(+CP)
9/4 pSB4K5Plusgrow medium(+Kan)

Electrophoresis

No name

LaneSample
1100bp ladder
2Pconst-spinach-DT -(1)
3Pconst-spinach-DT -(2)
4Pconst-spinach-DT -(3)
5Pconst-spinach-DT -(4)
6Plac-spinach-DT -(1)
7Plac-spinach-DT -(2)
8Plac-spinach-DT -(3)
9Plac-spinach-DT -(4)
10Pcon-pT181 antisense -(1)
11Pcon-pT181 antisense -(2)
12Pcon-pT181 antisense -(3)
13Pcon-pT181 antisense -(4)
LaneSample
1100bp ladder
2Ptet-pT181 antisense -(1)
3Ptet-pT181 antisense -(2)
4Ptet-pT181 antisense -(3)
5Ptet-pT181 antisense -(4)
6Plac-pT181 attenuator -(1)
7Plac-pT181 attenuator -(2)
8Plac-pT181 attenuator -(3)
9Plac-pT181 attenuator -(4)

Igku Sep6 COlonyPCRN1 1a.jpg
Igku Sep6 colonyPCR N2 1b.jpg

Miniprep

Honda and Kojima

DNAconcentration [µg/mL]260/280260/230
pT181 antisense-(1)238.71.681.44
pT181 antisense-(2)248.81.831.77
pT181 antisense-(3)223.21.781.61
pT181 antisense-(4)220.81.891.92
tetR aptamer-(1)252.51.891.90
tetR aptamer-(2)211.21.901.99
DT181 attenuator102.21.982.17
121R aptamer-DT251.71.922.01
pSB4K5212.91.801.90
RBS-lacZα-DT185.71.781.79
RBS-lysis2-DT-(1)84.81.852.24

Restriction Enzyme Digestion

Tatsui

9/6 RBS-lysis2-DT (78.3 µg/mL)XbaIPstIBSABufferMilliQtotal
2 cuts22 µL1 µL1 µL3 µL3 µL0 µL30 µL
NC1.3 µL0 µL0 µL1 µL1 µL6.7 µL10 µL
aptamer-DT (251.7 µg/mL)XbaIPstIBSABufferMilliQtotal
2 cuts0.4 µL1 µL1 µL3 µL3 µL14.1 µL30 µL
NC0.4 µL0 µL0 µL1 µL1 µL7.6 µL10 µL
Pλ-luxI (86.4 µg/mL)EcoRISpeIBSABufferMilliQtotal
2 cuts23.1 µL1 µL1 µL3 µL3 µL1.9 µL30 µL
NC1.2 µL0 µL0 µL1 µL1 µL6.8 µL10 µL
  • at 37 °C, for 1h

Electrophoresis

Hirano

LaneSample
1100bp ladder
2Pconst+RBS-tetR-DT -(1)
3Pconst+RBS-tetR-DT -(2)
4Pconst+RBS-tetR-DT -(3)
5Pconst+RBS-tetR-DT -(4)
6Ptet+RBS-lacZα-DT -(1)
7Ptet+RBS-lacZα-DT -(2)
8100bp ladder
9Ptet+RBS-lacZα-DT -(3)
10Ptet+RBS-lacZα-DT -(4)
11100bp ladder

Igku Sep6 electrophoresis N3.jpg

Gel Extraction

No name

LaneDNAEnzyme
1100bp ladder--
2RBS-lysis2-DTXbaI & PstI
3
4----
5aptamer 12_1R-DTXbaI & PstI
6
LaneDNAEnzyme
1100bp ladder--
2Pλ-luxIEcoRI & SpeI
3

Igku Sep6 gel extraction N4 2.jpg
Igku Sep6 gel extraction N4 3.jpg

Nameconcentration[µg/mL]260/280260/230
RBS-lysis2-DT (XbaI & PstI)7.11.880.07
aptamer 12_1R-DT (XbaI & PstI)15.31.930.70
Pλ-luxI (EcoRI & SpeI)4.51.790.31

Igku Sep6 gel extraction N5 1.jpg
Igku Sep6 gel extraction N5 2.jpg

Liquid Culture

No name

Samplemedium
9/5 Pconst-spinach-DT-(1)Plusgrow medium(+Amp&CP)
9/5 Ptet-pT181 antisense-(1)Plusgrow medium(+CP)
9/5 Pconst+RBS-tetR-DT-(1)Plusgrow medium(+Amp&CP)
9/5 Ptet+RBS-lacZα-DT-(1)Plusgrow medium(+CP)
9/5 Pcon-pT181 attenuator-(1)Plusgrow medium(+CP)
9/4 pT181 antisense-(1)Plusgrow medium(+CP)
9/4 aptamer 12_1R-(1)Plusgrow medium(+CP)

Colony PCR

No name

Samplebase pair
9/5 Plac-spinach-DT-(5~12)563
9/5 Pcon-antisense-(5~12)468
9/5 Plac-attenuator-(5~12)664
9/5 pT181 antisense+Plac-(1)-
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s42s30cycles

Sep 7

Miniprep

No name

DNAconcentration[µg/mL]260/280260/230
RBS-lysis1-DT195.3
RBS-lysis3-DT255.5 1.91 2.04
Ptet-pT18183.6 1.85 1.48
Pcon-RBS-tetR-DT134.3 1.94 2.20
Ptet-RBS-lacZα-DT67.7 1.92 1.79
Pcon-pT181 attenuator232.3 1.95 1.90
pSB4K5237.0 1.61 1.50
pT181 antisense(pSB1C3)256.4 1.63 1.78
apt12_1R(pSB1C3)230.0 1.98 2.28

Electrophoresis

No name

LaneSample
1100bp ladder
2Plac-Spinach-DT -5
3Plac-Spinach-DT -6
4Plac-Spinach-DT -7
5Plac-Spinach-DT -8
6Plac-Spinach-DT -9
7Plac-Spinach-DT -10
8100bp ladder
1100bp ladder
2Plac-Spinach-DT -11
3Plac-Spinach-DT -12
4Pcon-antisense -5
5Pcon-antisense -6
6Pcon-antisense -7
7Pcon-antisense -8
8100bp ladder

Igku Sep7electrophoresis1.jpg

LaneSample
1100bp ladder
2Pcon-antisense -9
3Pcon-antisense -10
4Pcon-antisense -11
5Pcon-antisense -12
6Plac-attenuator -5
7Plac-attenuator -6
8100bp ladder
1100bp ladder
2Plac-attenuator -7
3Plac-attenuator -8
4Plac-attenuator -9
5Plac-attenuator -10
6Plac-attenuator -11
7Plac-attenuator -12
8100bp ladder

Igku Sep7electrophoresis2.jpg

LaneSample
1100bp ladder
2Plac-pT181 antisense

Igku Sep7electrophoresis3.jpg

Colony PCR

No name

Sample
Plac-spinach-DT -13~20
Plac-pT181 attenuator -13~20
PreDenatureDenatureAnnealingExtensioncycle
94 °C94 °C55 °C68°C--
5 min30 s30 s42 sec30 cycles

Ligation

No Name

stateVectorInserterLigation High ver.2
experiment9/6 Plux (SpeI&PstI)2 µL9/7 RBS-lysis2-DT (EcoRI&XbaI) µL3.5 µL
experiment9/6 Plac (SpeI&PstI) µL9/7 RBS-lysis2-DT (EcoRI&XbaI) µL3.5 µL
experiment9/6 Pcon-RBS-luxR-DT (EcoRI&XbaI)1.9 µL9/6 Plux-RBS-GFP-DT(SpeI&PstI)2.2 µL3.5 µL
experiment9/6 Pbed/araC (SpeI&PstI)1.6 µL9/5 RBS-luxI-DT (EcoRI&XbaI)16 µL3.5 µL
experiment9/6 Plac (SpeI&PstI)1.5 µL9/5 pT181 antisense (EcoRI&XbaI)2.0 µL3.5 µL
experiment9/6 Pcon (SpeI&PstI)3.0 µL9/5 RBS-luxI-DT (EcoRI&XbaI)10 µL3.5 µL

Electrophoresis

Nakamoto

LaneSample
1100bp ladder
2Plac-Spinach-DT -13
3Plac-Spinach-DT -14
4Plac-Spinach-DT -15
5Plac-Spinach-DT -16
6Plac-Spinach-DT -17
7Plac-Spinach-DT -18
8Plac-Spinach-DT -19
9Plac-Spinach-DT -20
10Plac-pT181 attenuator -13
11Plac-pT181 attenuator -14
12100bp ladder

Igku 974.jpg

LaneSample
1100bp ladder
2Plac-pT181 attenuator -15
3Plac-pT181 attenuator -16
4Plac-pT181 attenuator -17
5Plac-pT181 attenuator -18
6Plac-pT181 attenuator -19
7Plac-pT181 attenuator -20
8100bp ladder

Igku 975.jpg

Transformation

Tatsui

NameSampleCompetent CellsTotalPlate
Pcon-RBS-luxR-DT+Plux-RBS-GFP-DT2µL20µL22µL
Pbed/araC+RBS-luxI-DT2µL 20µL 22µL
Plac+pT181 antisense 2µL 20µL 22µL
Pcon+RBS-luxI-DT 2µL 20µL 22µL

Restriction Enzyme Digestion

No Name

DNAXbaIPstIBufferBSAMilliQtotal
9/7 RBS-lysis1-DT(XbaI & PstI)10 µL1 µL1 µL3 µL3 µL12 µL30 µL
NC0.5 µL0 µL0 µL1 µL1 µL7.5 µL10 µL
DNAXbaIPstIBufferBSAMilliQtotal
9/7 RBS-lysis3-DT (XbaI & PstI)7.8 µL1 µL1 µL3 µL3 µL14.2 µL30 µL
NC0.4 µL0 µL0 µL1 µL1 µL7.6 µL10 µL
DNASpeIPstIBufferBSAMilliQtotal
9/5 Plac (SpeI & PstI)6.0 µL1 µL1 µL3 µL3 µL16.0 µL30 µL
NC0.3 µL0 µL0 µL1 µL1 µL7.7 µL10 µL
DNAEcorISpeIBufferBSAMilliQtotal
9/7 pSB4K5 (EcoRI & SpeI)8.4 µL1 µL1 µL3 µL3 µL16.0 µL30 µL
NC0.4 µL0 µL0 µL1 µL1 µL7.7 µL10 µL
DNArestricion enzymerestriction enzymeBufferBSAMilliQtotal
Pcon-pT181 attenuator (SpeI & PstI)8.6 µL1 µL1 µL3 µL3 µL13.4 µL30 µL
Pcon-pT181 attenuator (EcorI & SpeI)8.6 µL1 µL1 µL3 µL3 µL13.4 µL30 µL
NC0.4 µL0 µL0 µL1 µL1 µL7.6 µL10 µL
DNAXbaIPstIBufferBSAMilliQtotal
9/5 Spinach-DT (XbaI & PstI)5.4 µL1 µL1 µL3 µL3 µL16.6 µL30 µL
NC0.3 µL0 µL0 µL1 µL1 µL7.7 µL10 µL
DNASpeIPstIBufferBSAMilliQtotal
Ptet-anticodone (SpeI & PstI)11 µL1 µL1 µL3 µL3 µL11 µL30 µL
NC0.5 µL0 µL0 µL1 µL1 µL7.5 µL10 µL
DNAXbaIPstIBufferBSAMilliQtotal
9/6 RBS-lacZα-DT (XbaI & PstI)11 µL1 µL1 µL3 µL3 µL11 µL30 µL
NC0.5 µL0 µL0 µL1 µL1 µL7.5 µL10 µL
DNAEcoRISpeIBufferBSAMilliQtotal
pSB1C3 (1) (EcoRI & SpeI)6.6 µL1 µL1 µL3 µL3 µL15.4 µL30 µL
NC0.3 µL0 µL0 µL1 µL1 µL7.7 µL10 µL
DNAXbaIPstIBufferBSAMilliQtotal
pSB1C3 (3) (XbaI & PstI)6.6 µL1 µL1 µL3 µL3 µL15.4 µL30 µL
NC0.3 µL0 µL0 µL1 µL1 µL7.7 µL10 µL

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
1100bp ladder----
2RBS-lysis1-DT (9/7)XbaIPstI
3RBS-lysis1-DT (9/7)----
4RBS-lysis3-DT (9/7)XbaIPstI
5RBS-lysis3-DT (9/7)----
6Pcon-pT181 attenuatorEcoRISpeI
7Pcon-pT181 attenuator----
89/5 Spinach-DTXbaIPstI
99/5 Spinach-DT----
10100bp ladder----
11100bp ladder----
12RBS-lacZα-DTXbaIPstI
13RBS-lacZα-DT----

Igku 971.jpg

LaneSampleEnzyme1Enzyme2
1100bp ladder----
2Ptet-antisenseSpeIPstI
3Plac (NC)----
4pSB4K5EcoRISpeI
5pSB4K5----
6Pcon-pT181 attenuatorSpeIPstI
71kbp ladder----

Igku 972.jpg

LaneSampleEnzyme1Enzyme2
1100bp ladder----
2PlacSpeIPstI
3Ptet-antisense----
4pSB1C3EcoRISpeI
5pSB1C3----
6pSB1C3XbaIPstI
7pSB1C3----
81kbp ladder----

Igku 973.jpg

Liquid Culture

No name

Samplemedium
entA- strainSOB medium (+Kan)
9/15 Pcon-pT181 antisensePlusgrow (+Amp)

Sep 8

Gel Extraction

No name

LaneDNAEnzyme
1100bp ladder
2RBS-lysis1-DTXbaI & PstI
3RBS-lysis1-DTXbaI & PstI
5RBS-lysis3-DTXbaI & PstI
6RBS-lysis3-DTXbaI & PstI

Igku 981b.png
Igku 981a.png

Nameconcentration[µg/mL]260/280260/230
RBS-lysis1-DT (XbaI & PstI)5.31.980.06
RBS-lysis3-DT (XbaI & PstI)5.31.980.06
LaneDNAEnzyme
1100bp ladder
3Pcon-pT181 attenuatorEcoRI & SpeI
4Pcon-pT181 attenuatorEcoRI & SpeI
6Spinach-DTXbaI & PstI
7Spinach-DTXbaI & PstI
9RBS-lacZα-DTXbaI & PstI
10RBS-lacZα-DTXbaI & PstI

Igku 982b.png
Igku 982a.png

Nameconcentration[µg/mL]260/280260/230
Pcon-pT181 attenuator (EcoRI & SpeI)5.21.820.36
Spinach-DT (XbaI & PstI)8.01.870.25
RBS-lacZα-DT (XbaI & PstI)10.31.710.54
LaneDNAEnzyme
11kbp ladder
2Ptet-pT181 antisenseEcoRI & PstI
3Ptet-pT181 antisenseEcoRI & PstI
5pSB4K5EcoRI & SpeI
6pSB4K5EcoRI & SpeI

Igku 983b.png
Igku 983a.png

Nameconcentration[µg/mL]260/280260/230
Ptet-pT181 antisense(EcoRI & PstI)30.61.841.16
pSB4K5 (EcoRI & SpeI)21.81.870.98
LaneDNAEnzyme
11kbp ladder
2Pcon-pT181 attenuatorSpeI & PstI
3Pcon-pT181 attenuatorSpeI & PstI
5PlacSpeI & PstI
6PlacSpeI & PstI
8pSB1C3EcoRI & SpeI
9pSB1C3EcoRI & SpeI
11pSB1C3XbaI & PstI
12pSB1C3XbaI & PstI

Igku 984b.png
Igku 984a.png

Nameconcentration[µg/mL]260/280260/230
Pcon-pT181 attenuator (SpeI & PstI)14.51.911.09
Plac (SpeI & PstI)11.61.741.19
pSB1C3 (EcoRI & SpeI)5.71.740.34
pSB1C3 (XbaI & PstI)5.62.000.37

Miniprep

No name

DNAconcentration[µg/mL]260/280260/230
Pcon-pT181 anti243.01.901.81
Pcon-Spinach-DT253.71.571.51

Ligation

Kojima

stateVector(µL)Inserter(µL)Ligation High ver.2
experiment9/8 Plac SpeI+PstI4.39/3 pT181 attenuator3.13.5
experiment9/8 Plac SpeI+PstI4.39/7 aptamer12-1R-DT XbaI+PstI2.13.2
experiment9/8 Plac SpeI+PstI4.39/8 spinach-DT XbaI+PstI4.63.5
experiment9/6 Plac SpeI+PstI29/7 RBS-lysis2-DT XbaI+PstI103.5
experiment9/8 Plac SpeI+PstI4.39/7 RBS-lysis2-DT XbaI+PstI103.5
experiment9/8 Ptet-pT181 anti1.69/8 spinach-DT XbaI+PstI4.33.0
experiment9/6 Pcon SpeI+PstI1.49/8 RBS-laczα-DT XbaI+PstI4.83.1
experiment9/8 Pcon-pT181 attenuator SpeI+PstI3.49/7 aptamer12-1R-DT XbaI+PstI1.92.7
experiment9/6 Pcon SpeI+PstI1.49/7 aptamer12-1R-DT XbaI+PstI2.11.3
experiment9/8 DT3.09/8 Pcon-pT181 attenuator EcoRI+SpeI7.93.5
experiment9/6 Plux SpeI+PstI29/7 RBS-lysis1-DT XbaI+PstI7.23.5
experiment9/8 Plac SpeI+PstI4.39/7 RBS-lysis1-DT XbaI+PstI7.23.5
experiment9/4 Plac SpeI+PstI1.79/8 RBS-laczα-DT XbaI+PstI4.83.3

Restriction Enzyme Digestion

Kojima

9/8 Pcon-pT181 antiEcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts8.2µL 0µL 1µL 0µL 1µL 3µL 3µL 13.8µL 30µL
NC 0.4µL 0µL 0µL 0µL 0µL 1µL 1µL 7.6µL 10µL
9/8 Pcon-spinach-DTEcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts7.9µL 1µL 1µL 0µL 0µL 3µL 3µL14.1µL 30µL
NC 0.4µL 0µL 0µL 0µL 0µL 1µL 1µL7.6µL 10µL
8/21 F1 attenuatorEcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts 6.5µL 0µL 0µL 1µL 1µL 3µL 3µL15.5µL30µL
NC 0.3µL0µL0µL 0µL 0µL1µL1µL7.7µL10µL
8/21 F3m2 attenuatorEcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts6.0µL0µL0µL1µL1µL3µL3µL16µL30µL
NC 0.3µL0µL0µL 0µL 0µL1µL1µL7.7µL10µL
8/21 F1 antiEcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts6.7µL0µL0µL1µL1µL3µL3µL15.3µL30µL
NC 0.3µL0µL0µL 0µL 0µL1µL1µL7.7µL10µL
8/21 F6 antiEcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts6.0µL0µL0µL1µL1µL3µL3µL16µL30µL
NC 0.3µL0µL0µL 0µL 0µL1µL1µL7.7µL10µL
8/21 tetR aptamer 12_1MEcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts5.3µL1µL1µL0µL0µL3µL3µL16.7µL30µL
NC 0.3µL0µL0µL 0µL 0µL1µL1µL7.7µL10µL
8/21 tetR aptamer 12_PEcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts13.3µL1µL1µL0µL0µL3µL3µL8.7µL30µL
NC0.7µL0µL0µL0µL0µL1µL1µL7.3µL10µL
9/4 spinach-DTEcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts5.4µL0µL0µL1µL1µL3µL3µL16.6µL30µL
NC0.3µL0µL0µL0µL0µL1µL1µL7.7µL10µL
9/7 RBS-lysis1-DTEcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts10µL0µL0µL1µL1µL3µL3µL12µL30µL
NC0.5µL0µL0µL0µL0µL1µL1µL7.5µL10µL
9/6 RBS-lysis2-DTEcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts13µL0µL0µL1µL1µL3µL3µL9µL30µL
NC0.7µL0µL0µL0µL0µL1µL1µL7.3µL10µL
9/7 RBS-lysis3-DTEcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts10µL0µL0µL1µL1µL3µL3µL12µL30µL
NC0.5µL0µL0µL0µL0µL1µL1µL7.5µL10µL
8/20 J23100 RBS-lacZα-DT-(1)EcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts7µL0.5µL0.5µL0µL0µL3µL3µL16µL30µL
NC0.5µL0µL0µL0µL0µL1µL1µL7.5µL10µL
8/20 RBS-lacZα-DTEcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts10µL0.5µL0.5µL0µL0µL3µL3µL13µL30µL
NC0.5µL0µL0µL0µL0µL1µL1µL7.5µL10µL

Transformation

Nakamoto & Ashida

NameSample(µL)Competent Cells(µL)Total(µL)Plate
9/8 Pcont+aptamer 12-1R-DT11011Amp
9/8 Pcon+RBS-lacZα-DT11011Amp
9/8 Pcon-pT181 attenuator+aptamer 12-1R-DT11011Amp
9/8 Plac+RBS-lysis1-DT22022CP
9/8 Plac+RBS-lysis2-DT22022CP
9/8 Plac+aptamer 12-1R-DT22022CP
9/8 Plac+spinach22022CP
9/8 Plac+pT181 attenuator22022CP
9/8 Plux+RBS-lysis1-DT11011CP
9/8 Plux+RBS-lysis2-DT11011CP
9/8 Ptet+RBS-lacZα-DT11011CP
9/8 Ptet-pT181 anti+spinach-DT11011CP
9/8 Pcon-pT181 attenuator+DT11011CP

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
1100bp ladder
2Pcon-spinach-DTEcoRISpeI
3Pcon-spinach-DT
4Fusion1 attenuatorXbaIPstI
5Fusion1 attenuator
6Fusion3m2 attenuatorXbaIPstI
7Fusion3m2 attenuator
8Fusion1 antiXbaIPstI
9Fusion1 anti
10Fusion6 antiXbaIPstI
11Fusion6 anti
12aptamer 12-1MXbaIPstI
13aptamer 12-1M
14aptamer 12-PXbaIPstI
15aptamer 12-P
16spinach-DTXbaIPstI

Igku 985.png

LaneSampleEnzyme1Enzyme2
1spinach-DT
2J23100-RBS-lacZα-DTEcoRISpeI
3J23100-RBS-lacZα-DT
4100bp ladder
5RBS-lacZα-DTEcoRISpeI
6RBS-lacZα-DT
7RBS-lysis1-DTXbaIPstI
8RBS-lysis1-DT
LaneSampleEnzyme1Enzyme2
1100bp ladder
2RBS-lysis2-DTXbaIPstI
3RBS-lysis2-DT
4RBS-lysis3-DTXbaIPstI
5RBS-lysis3-DT
6Pcon pT181 antiSpeIPstI
7Pcon pT181 anti

Igku 986.png

Gel Extraction

Ashida and Nakamoto

LaneDNAEnzyme
1100bp ladder
2Pcon-spinach-DTEcoRI+SpeI
3Pcon-Spinach-DTEcoRI+SpeI
5Fusion 1 attenuatorXbaI+PstI
6Fusion 1 attenuatorXbaI+PstI
8Fusion3m2 attenuatorXbaI+PstI
9Fusion3m2 attenuatorXbaI+PstI
11Fusion 1 antisenseXbaI+PstI
12Fusion 1 antisenseXbaI+PstI

Igku 987.png

Namequantityconcentration[µg/mL]260/280260/230
Pcon-spinach-DT(EcoRI+SpeI) 10.51.860.39
Fusion 1 attenuator(XbaI+PstI) 7.01.710.27
Fusion3m2 attenuator(XbaI+PstI) 8.61.750.47
Fusion 1 antisense(XbaI+PstI) 7.61.870.43
LaneDNAEnzyme
1100bp ladder
2Fusion6 antisenseXbaI+PstI
3Fusion6 antisenseXbaI+PstI
5aptamer12_1MXbaI+PstI
6aptamer12_1MXbaI+PstI

Igku 988b.png
Igku 988a.png

Namequantityconcentration[µg/mL]260/280260/230
Fusion6 antisense(XbaI+PstI) 5.01.760.20
aptamer12_1M(XbaI+PstI) 3.32.310.04
LaneDNAEnzyme
1100bp ladder
2aptamer12_PXbaI+PstI
3aptamer12_PXbaI+PstI
5spinach-DTEcoRI+SpeI
6spinach-DTEcoRI+SpeI

Igku 989b.png
Igku 989a.png

Namequantityconcentration[µg/mL]260/280260/230
aptamer12_P(XbaI+PstI) 3.41.720.26
spinach-DT(EcoRI+SpeI) 10.11.570.47
LaneDNAEnzyme
1100bp ladder
2J23100-RBS-laczα-DTEcoRI+SpeI
3J23100-RBS-laczα-DTEcoRI+SpeI
5RBS-laczα-DTEcoRI+SpeI
6RBS-laczα-DTEcoRI+SpeI
8RBS-lysis1-DTXbaI+PstI
9RBS-lysis1-DTXbaI+PstI
11RBS-lysis2-DTXbaI+PstI
12RBS-lysis2-DTXbaI+PstI

Igku 9810b.png
Igku 9810a.png

Namequantityconcentration[µg/mL]260/280260/230
Pcon-RBS-laczα-DT(EcoRI+SpeI) 5.62.030.25
RBS-laczα-DT(EcoRI+SpeI) 4.42.040.33
RBS-lysis1-DT(XbaI+PstI) 5.92.220.53
RBS-lysis2-DT(XbaI+PstI) 5.11.670.48
LaneDNAEnzyme
1100bp ladder
2RBS-lysis3-DTXbaI+PstI
3RBS-lysis3-DTXbaI+PstI
5Pcon-pT181 antisenseSpeI+PstI
6Pcon-pT181 antisenseSpeI+PstI

Igku 9811b.png
Igku 9811a.png

Namequantityconcentration[µg/mL]260/280260/230
RBS-lysis3-DT(XbaI+PstI) 23.41.720.88
Pcon-pT181 antisense(SpeI+PstI) 31.31.860.92

Ligation

No name

stateVectorInserter
experimentPcon-spinach-DT(EcoRI&SpeI)10.5µg/mLpSB6A1(EcoRI&SpeI)8.9µg/mL
experimentPcon-spinach-DT(EcoRI&SpeI)10.5µg/mLpSB4K5(EcoRI&SpeI)21.8µg/mL
experimentPcon-pT181 anti(SpeI+PstI)31.3µg/mLspinach-DT(XbaI+PstI)10.1µg/mL
experimentPlac(SpeI+PstI)11.6µg/mLRBS-lysis3-DT23.4µg/mL
experimentPlux(SpeI+PstI)25µg/mLRBS-lysis3-DT23.4µg/mL
experimentPtet-pT181 anti(SpeI+PstI)30.6µg/mLspinach-DT(XbaI+PstI)10.1µg/mL
experimentPcon-pT181 attenuator(SpeI+PstI)14.5µg/mLRBS-laczα-DT(XbaI+PstI)4.4µg/mL

PCR

No name

KaiA(plasmid)KOD plus10x bufferdNTPMgSO4Primer(661-RBS-KaiA-fwd)Primer(kaiA-662-rev-ver2)MilliQtotal
10.52.52.51.50.750.7515.525
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C57°C68°C--
2min10s30s28s30cycles
KaiB(plasmid)KOD plus10x bufferdNTPMgSO4Primer(662-RBS-KaiB-fwd-ver2)Primer(kaiB-663-rev-ver2)MilliQtotal
10.52.52.51.50.750.7515.525
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C57°C68°C--
2min10s30s28s30cycles
DT(plasmid)KOD plus10x bufferdNTPMgSO4Primer(664-DT-fwd-ver2)Primer(DT-suffix-660-rev-ver2)MilliQtotal
0.10.52.52.51.50.750.7516.425
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C57°C68°C--
2min10s30s28s30cycles
P-KaiB(PCR prpduct)KOD plus10x bufferdNTPMgSO4Primer(prefix-PkaiBC-fwd-ver2)Primer(PkaiBC-suffix--662-rev-ver2)MilliQtotal
0.70.52.52.51.50.750.7515.825
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C57°C68°C--
2min10s30s28s30cycles
Plac(1)(plasmid)KOD plus10x bufferdNTPMgSO4Primer(GG0-pSBIC3-fwd-ver2)Primer(Plac-GG1-rev-ver2)MilliQtotal
0.130.52.52.51.50.750.7516.3725
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C57°C68°C--
2min10s30s60s30cycles
KaiC(plasmid)KOD plus10x bufferdNTPMgSO4Primer(GG3-RBS-KaiC-fwd-ver2)Primer(KaiC-GG4-rev-ver2)MilliQtotal
10.52.52.51.50.750.7515.525
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C57°C68°C--
2min10s30s60s30cycles

Gel Extraction

Yoshida

LaneDNA
1100bp ladder
2KaiA
3KaiA
4KaiB
5KaiB
6DT
7DT
8P-KaiBC
9P-KaiBC
10nega
11100bp ladder

9898.jpg
9897.jpg

LaneDNA
1Plac
2Plac
3KaiC
4KaiC
5nega
61kb ladder

9899.jpg
991.jpg

Sep 9

PCR

Nakagawa

KaiA(plasmid)10x bufferdNTPsMgSO4Primer(661-RBS-KaiA-fwd)Primer(kaiA-662-rev-ver2)Kod-plus ver2MilliQtotal
1.02.52.51.50.750.750.515.525
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C58.5°C68°C--
2min10s30s30s30cycles
KaiB(plasmid)10x bufferdNTPsMgSO4Primer(661-RBS-KaiA-fwd-ver2)Primer(kaiB-663-rev-ver2)Kod-plus ver2MilliQtotal
1.02.52.51.50.750.750.515.525
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C58.5°C68°C--
2min10s30s30s30cycles
P-KaiBC(PCR products)10x bufferdNTPsMgSO4Primer(prefix-P-kaiBC-fwd-ver2)Primer(P-KaiBC-suffix-rev-verw)Kod-plus ver2MilliQtotal
0.72.52.51.50.750.750.515.825
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C58.5°C68°C--
2min10s30s30s30cycles
DT(plasmid)10x bufferdNTPsMgSO4Primer(664-DT-fwd-ver2)Primer(DT-suffix-660-rev-ver2)Kod-plus ver2MilliQtotal
1.02.52.51.50.750.750.515.525
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C58.5°C68°C--
2min10s30s30s30cycles

Gel Extraction

Yoshida

LaneDNAEnzyme
1100bp ladder--
2KaiA--
3
4KaiB--
5
6P-KaiBC--
7
8DT--
9
10NC--
11100bp ladder--

Igku 992.jpg
Igku 993.jpg

Nameconcentration[µg/mL]260/280260/230
KaiC28.51.2611.92
Plac53.91.821.61
KaiB59.61.612.20
KaiB76.51.861.44
DT98.71.471.43

Miniprep

No name

DNAconcentration[µg/mL]260/280260/230
9/8 pSB1C383.21.450.76
9/8 RBS-lysis2-DT72.81.670.97
9/8 spinach-DT152.61.881.97
Nuclease-Free water-0.4-14.300.25

Restriction Enzyme Digestion

No name

9/9 pSB1C3XbaIPstIBufferBSAMilliQtotal
2 cuts24µL1µL1µL3µL3µL22µL30µL*
NC1.2µL0µL0µL1µL1µL6.8 µL10µL
9/9 pSB1C3XbaIPstIBufferBSAMilliQtotal
2 cuts27.4µL1µL1µL3µL3µL22µL30µL*
NC1.3µL0µL0µL1µL1µL6.7µL10µL
9/9 spinach-DTXbaIPstIBufferBSAMilliQtotal
2 cuts10.8µL1µL1µL3µL3µL22µL30µL*
NC0.5µL0µL0µL1µL1µL7.5µL10µL
  • "*"=be evaporated

Transformation

No name

NameSampleCompetent CellsTotalPlate
Pcon+RBS-luxI-DT2µL20µL22µLAmp
Pcon-RBS-luxR-DT+Plux-GFP2µL20µL22µL
Ptet-PT181 antisense+spinach-DT-Pbad/araC+RBS-lux-DT2µL20µL22µL

Ligation

No Name

stateVectorInserterLigation High ver.2
experiment9/8 F1-attenuator(XbaI&PstI)5.3µg9/8 pSB1C3(XbaI&PstI)8.9µg3.5µg
experiment9/8 F3m2-attenuator(XbaI&PstI)4.5µg9/8 pSB1C3(XbaI&PstI)8.9µg3.5µg
experiment9/8 F1-antisense(XbaI&PstI)1.7µg9/8 pSB1C3(XbaI&PstI)8.9µg3.5µg
experiment9/8 F6-antisense(XbaI&PstI)2.9µg9/8 pSB1C3(XbaI&PstI)8.9µg3.5µg
experiment9/8 aptamer12-P(EcoRI&SpeI)3.1µg9/8 pSB1C3(EcoRI&SpeI)8.8µg3.5µg
experiment9/8 aptamer12-1M(EcoRI&SpeI)3.1µg9/8 pSB1C(EcoRI&SpeI)38.8µg3.5µg

M9 medium

Shimizu & Hirano

EDTA consenlationNa2HPO4KH2PO4NaClNH4ClFe(Ⅲ)-EDTAdistilled watertotal
1mMol300mg150mg25mg50mg21mgup to 10mL10mL
10mMol300mg150mg25mg50mg210.5mgup to 10mL10mL
50mMol300mg150mg25mg50mg1052.7mgup to 10mL10mL

Colony PCR

No name

Samplebase pair
9/8 Plac-spinach-DT-(1)659
9/8 Plac-spinach-DT-(2)659
9/8 Pcon-pt181attenuator-DT-(1)584
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s40s30cycles

Gel Extraction

No name

LaneDNAEnzyme
1100bp ladder--
2Spinach-DTXbaI+PstI
3
5RBS-lacZα-DTXbaI+PstI
6

Igku 995.jpg

LaneDNAEnzyme
1100bp ladder--
3pSB1C3XbaI+PstI
4

Igku 996.jpg

Ligation

No name

stateVectorInserterLigation High ver.2
experimentpSB1C3(XbaI+PstI)xx µL9/8 F1-attenuator(XbaI+PstI)xx µLxx µL
experimentpSB1C3(XbaI+PstI)xx µL9/8 F3m2-attenuator(XbaI+PstI)xx µLxx µL

PCR

Tatsui and Yoshida

KaiC10x buffer KOD plus ver22M dNTPMgSO4Primer Mix(F&R)Kod-plus ver2MilliQtotal
0.52.52.51.51.50.516.025.0
1.02.52.51.51.50.515.525.0
2.02.52.51.51.50.514.525.0
4.02.52.51.51.50.512.525.0
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C57°C68°C--
2min10s30s2min30cycles
Plac(100pg)10x buffer KOD plus ver22M dNTPMgSO4Primer Mix(F&R)Kod-plus ver2MilliQtotal
0.32.52.51.51.50.516.225.0
0.62.52.51.51.50.515.925.0
1.22.52.51.51.50.515.325.0
2.42.52.51.51.50.514.125.0
0.02.52.51.51.50.516.525.0
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C57°C68°C--
2min10s30s2min30cycles

Liquid Culture

No name

Samplemedium
pSB2C3(master)-2Plusgrow medium(+CP)

Restriction Enzyme Digestion

No name

8/17 lysis1-2XbaIPstIBSABufferMilliQtotal
2 cuts8.1µL1µL1µL3µL3µL13.9µL30µL
NC0.4µL0µL0µL1µL1µL7.6µL10µL
8/17 lysis3-1XbaIPstIBSABufferMilliQtotal
2 cuts6.1µL1µL1µL3µL3µL15.9µL30µL
NC0.3µL0µL0µL1µL1µL7.7µL10µL
9/15 PlacXbaIPstIBSABufferMilliQtotal
1 cuts12.1µL0µL1µL3µL3µL10.9µL30µL
NC0.3µL0µL0µL1µL1µL7.7µL10µL

PCR

Yoshida

KaiA(KaiABC) 100pg/µL10x buffer KOD plus ver2dNTPsMgSO4Primer Mix(Fwd&Rev)KOD plus ver2MilliQtotal
2.02.52.51.51.50.514.525
4.02.52.51.51.50.512.525
8.02.52.51.51.50.58.525
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C60°C68°C--
2min10s30s1min30cycles
PKaiBC 400pg/µL10x buffer KOD plus ver2dNTPsMgSO4Primer Mix(Fwd&Rev)KOD plus ver2MilliQtotal
0.52.52.51.51.50.516.025
1.02.52.51.51.50.515.525
2.02.52.51.51.50.514.525
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C60°C68°C--
2m10s30s1m30cycles

Liquid Culture

No name

Samplemedium
9/2 Spinach-DTPlusgrow medium(+CP)
8/29 Plux-RBS-GFP-DTPlusgrow medium
9/6 master plate Plusgrow medium(+Amp)
3 Pcon-PT181 attenuatorPlusgrow medium

Gel Extraction

No name

LaneDNAEnzyme
11kbp ladder--
3lysis1XbaI+PstI
4
5
6

Igku 9910.jpg
Igku 9911.jpg

LaneDNAEnzyme
11kbp ladder--
3lysis3XbaI+PstI
4
5
6

Igku 9912.jpg
Igku 9913.jpg

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

Electrophoresis

Yoshida

LaneSampleEnzyme1Enzyme2
11kb ladder----
2Plac 100pg----
3Plac 200pg----
4Plac 400pg----
5Plac 800pg----
6NC----
71kbp ladder----

Igku 9914.jpg

Sep 10

Ligation

Nakamoto

stateVectorInserterLigation High ver.2
experiment9/6 Pcon-RBS-luxR-DT1.9µL9/6Plux-RBS-GFP-DT22 µL3.5µL
experiment9/6 Plux2µL9/8 RBS-lysis1-DT6.6µL3.5µL
experiment9/6 Plux2µL9/8 RBS-lysis2-DT6.3µL3.5µL
experiment9/8 Ptet-PT181 antisense1.6µL9/8 spinach-DT4.4µL3.0µL
experiment9/8 Pcon-PT181 attenuator3.4µL9/8 apz 12-1R-PT2.1µL2.8µL
experiment9/8 Pcon3.4µL9/8 apz 12-1R-PT2.3µL2,9µL
experiment9/8 DT3.0µL9/8 Pcon-PT181 attenuator7.9µL3.5µL
experiment9/10 pSB1C31.8µL9/8 F3m2 attenuator4.6µL3.2µL
experiment9/10 pSB1C31.8µL9/8 F1 attenuator3.4µL2.6µL

Miniprep

Nakamoto

DNAconcentration[µg/mL]260/280260/230
spinach-DT202.41.971.93
Plux-RBS-GFP-DT164.22.802.06
Pcon-DT attenuator205.41.812.39
RSBJC3-2240.81.731.43
Plaz-1240.81.731.43

Restriction Enzyme Digestion

Nakamoto

9/10　Plux-RBS-GFP-DTEcoRISpeIBufferBSAMilliQtotal
2cuts12.1µL1µL1µL3µL3µL9.9µL30µL
NC0.6µL0µL0µL1µL1µL7.4µL10µL
9/10　Spinach-DTXbaIPstIBufferBSAMilliQtotal
2cuts9.9µL1µL1µL3µL3µL12.1µL30µL
NC0.5µL0µL0µL1µL1µL7.5µL10µL
9/6 apt12-1R -DTXbaIPstIBufferBSAMilliQtotal
2cuts8.0µL1µL1µL3µL3µL14.0µL30µL
NC0.4µL0µL0µL1µL1µL7.6µL10µL
9/10　Pcon-pt81 attenuator EcoRISpeIBufferBSAMilliQtotal
2cuts9.8µL1µL1µL3µL3µL12.2µL30µL
NC0.5µL0µL0µL1µL1µL7.5µL10µL

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
1100bp ladder----
2 Plux-RBS-GEP-DTEcoRISpeI
3 Plux-RBS-GEP-DT----
4Spinach-DTXbaIPstI
5Spinach-DT----
6aptamer 121R-DTXbaIPstI
7aptamer 121R-DT----
8Pcon-pt181 attenuatorEcoRISpeI
9 Pcon-pt181 attenuator----
10100bp ladder----

Igku 9104.jpg

Colony PCR

Tatsui

Samplebase pair
9/9 Pcon-pT181 antisense--
9/8 J223100(entA)--
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s1min10sec30

Electrophoresis

Tatsui

LaneSample
1100bp ladder
2Pcon Pt181 antisense1+spinach-DT
3Pcon Pt181 antisense2+spinach-DT
4Pcon Pt181 antisense3+spinach-DT
5Pcon Pt181 antisense4+spinach-DT
6Pcon Pt181 antisense5+spinach-DT
7Pcon Pt181 antisense6+spinach-DT
8Pcon Pt181 antisense7+spinach-DT
9Pcon Pt181 antisense8+spinach-DT
10Pcon Pt181 antisense9+spinach-DT
11Pcon Pt181 antisense10+spinach-DT
12Pcon Pt181 antisense11+spinach-DT
13Pcon Pt181 antisense12+spinach-DT
14100bp ladder

Igku 0910 E2.jpg

LaneSample
11kbp ladder
2Pcon entA1
3Pcon entA2
4Pcon entA3
51kbp ladder

Igku 0910 E3.jpg

Gel Extraction

Tatsui

LaneDNAEnzyme
11kbp ladder--
2KaiA(200pg)--
3KaiA(200pg)--
4----
5KaiA(400pg)--
6KaiA(400pg)--
7----
8KaiA(800pg--
9KaiA(800pg)--
101kbp ladder--

Igku 9101.jpg
Igku 9102.jpg

LaneDNAEnzyme
11kbp ladder--
2PKaiBC(200pg)--
3PKaiBC(200pg)--
4----
5PKaiBC(400pg)--
6PKaiBC(400pg)--
7----
8PKaiBC(800pg--
9PKaiBC(800pg)--
101kbp ladder--

Igku 0910 G3.jpg

Liquid Culture

Hirano

Samplemedium
9/4 J23100①(JM109)LB(Amp)
9/8 J23100① (entA)LB(Amp)　

Gel Extraction

Tatsui

LaneDNAEnzyme
11kbp ladder--
2KaiC--
3KaiC--
4KaiC--
5----
61kbp ladder--
71kbp ladder--
8KaiC--
9KaiC--
10KaiC--
11----
121kbp ladder--

Igku 9105.jpg
Igku 9106.jpg

Liquid Culture

Tatsui

Samplemedium
9/10 Pcon-pt181 antisense-Spinach-DTPlusgrow 　

Colony PCR

No name

Samplebase pair
9/9 Phad larac-1 RBS-lux1-DT2331
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s2min30
Samplebase pair
9/9 Pcon-lacZα (psB4KS)712
9/9 Pcon-Spinach-DT(psB4KS)605
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s42s30
Samplebase pair
9/9 Plac+RBS-lysis3}-DT1273
9/9 Pcon+RBS-lux1-DT1079
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s1min12s30

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
1100bp ladder----
29/9 Pcou-RBS-lacZα-DT(p3B4KS9)-1----
39/9 Pcou-RBS-lacZα-DT(p3B4KS9)-2----
49/9 Pcou-Spinach-DT(pB4KS)-1----
59/9 Pcou-Spinach-DT(pB4KS)-2----
6100bp ladder----

Igku 91014.jpg

Transformation

Nakamoto

NamePlate
F1 attenuator +p3B1C3CP
F3n2 attenuator +p3B1C3CP
F1 antisense +p3B1C3CP
F6 antisense+p3B1C3CP
apt12-P +p3B1C3CP
apt12-1M +p3B1C3CP
Pcon-luxR+Plux-GFPAmp
Plux+RBS-lysis1-DTCP
Plux+RBS-lysis2-DTCP
Ptet-pT181antisense+Spinach-DTCP
Pcon-pT181 attenuator+apt121R-DTAmp
Pcon-1 apt12-1R-DTAmp
DT+Pcon-pT181 attenuatorCP

Electrophoresis

Okazaki

LaneSampleEnzyme1Enzyme2
1100bp ladder----
2Spinach-DTXbaIPstI
3Spinach-DTXbaIPstI
4Spinach-DTXbaIPstI
5Spinach-DTXbaIPstI
6100bp ladder----

Igku 9108.jpg
Igku 9109.jpg

LaneSampleEnzyme1Enzyme2
1100bp ladder----
2Pcon-pT181 attenuatorEcoRISpeI
3Pcon-pT181 attenuatorEcoRISpeI
4Pcon-pT181 attenuatorEcoRISpei
5Pcon-pT181 attenuatorEcoRISpeI
6100bp ladder----

Igku 91010.jpg
Igku 91011.jpg

LaneSampleEnzyme1Enzyme2
1Plux-RBS-GFP-DTEcoRISpeI
2Plux-RBS-GFP-DT EcoRISpeI
3Plux-RBS-GFP-DT EcoRISpeI
4Plux-RBS-GFP-DT EcoRISpeI
5100bp ladder----
6apt12-1R-DTXbaIPstI
7apt12-1R-DTXbaIPstI
8apt12-1R-DTXbaIPstI
9apt12-1R-DTXbaIPstI

Igku 91012.jpg
Igku 91013.jpg

Sep 11

Electrophoresis

LaneSample
11kb ladder
2Pbad/araC+RBS-luxI-DT-1
3Pbad/araC+RBS-luxI-DT-1
4Plac+RBS-lysis-DT
5Pcon+RBS-luxI-DT
61kb ladder

Igku 9111.jpg

PCR

Tatsui

pKaiBC(200p)10*buffer KOD plusdNTPsMgSO4primer mix(F&R)KOD plusMilliQtotal
0.52.52.51.51.50.51625
PreDenatureDenatureAnnealingExtensioncycle
　94°C　98°C　60°C　68°C30
2min　10sec　30sec　30sec　
pKaiBC(400p)10*buffer KOD plusdNTPsMgSO4primer mix(F&R)KOD plusMilliQtotal
12.52.51.51.50.515.525
PreDenatureDenatureAnnealingExtensioncycle
　94°C　98°C　60°C　68°C30
2min　10sec　30sec　30sec　
pKaiBC(800p)10*buffer KOD plusdNTPsMgSO4primer mix(F&R)KOD plusMilliQtotal
22.52.51.51.50.514.525
PreDenatureDenatureAnnealingExtensioncycle
　94°C　98°C　60°C　68°C30
2min　10sec　30sec　30sec　
pKaiBC(1ng)10*buffer KOD plusdNTPsMgSO4primer mix(F&R)KOD plusMilliQtotal
2.52.52.51.51.50.51425
PreDenatureDenatureAnnealingExtensioncycle
　94°C　98°C　60°C　68°C30
2min　10sec　30sec　30sec　

Electrophoresis

Tatsui

LaneSample
1100bp ladder
2pKaiBC 800pg 60°C
3pKaiBC 1ng 60°C
4pKaiBC 800pg 61°C
5pKaiBC 1ng 61°C
6pKaiBC 800pg 62°C
7pKaiBC 1ng 62°C
8100bp ladder

Igku 9112.jpg

colony PCR

Tatsui

Samplebase pair
9/9Pcon-lacZα(p3B4k5)712bp
9/9Pcon-spinach-DT(p3B4k5)605bp

Tatsui

PreDenatureDenatureAnnealingExtensioncycle
　94°C　94°C　55°C　68°C--
5min30s　30s　42s　30


Samplebase pair
Pbad\araC-RBS-luxI-DT
PreDenatureDenatureAnnealingExtensioncycle
　94°C　94°C　55°C　68°C--
5min30s　30s　2min　30

Electrophoresis

Tatsui

LaneSample
1100bp ladder
2Pcon-lacZα(pSB4K5)3
3Pcon-lacZα(pSB4K5)4
4Pcon-lacZα(pSB4K5)5
5Pcon-lacZα(pSB4K5)6
6Pcon-spinach-DT(pSB4K5)3
7Pcon-spinach-DT(pSB4K5)4
8Pcon-spinach-DT(pSB4K5)5
9Pcon-spinach-DT(pSB4K5)6
10100bp ladder

Igku 9113.jpg

LaneSample
11kbp ladder
2Pbad/araC-RBS-luxI-DT3
3Pbad/araC-RBS-luxI-DT4
4Pbad/araC-RBS-luxI-DT5
5Pbad/araC-RBS-luxI-DT6
61kbp ladder

Igku 9114.jpg

colony PCR

Samplebase pair
9/9Pcon+RBS-luxI-DT1079bp
9/10Pcon+sptamer12_1R-DT584bp
9/10DT+Pcon-pT181 attenuator 1781bp
9/10Fusion1 attenuator(p3B1C3)-1638bp
PreDenatureDenatureAnnealingExtensioncycle
　94°C　94°C　55°C　68°C--
50sec　30sec　30sec　1min　30

Gel Extraction

Igku 9115.jpg
Igku 9116.jpg
Igku 9117.jpg
Igku 9118.jpg

Ligation

Okazaki

stateVectorInserterLigation High ver.2
experiment9/8pSB4K5(EcoRI&SpeI)21.8ng/µL2.3 µL9/8Pcon-Spinach-DT(EcoRI&SpeI)10.5ng/µL1.7 µL2.0 µL
experment9/8pSB4K5(EcoRI&SpeI)21.8ng/µL2.3 µL9/8RBS-lacZα-DT(EcoRI&SpeI)4.4ng/µL 6.7µL3.5 µL
experiment9/8pSB4K5(EcoRI&SpeI)21.8ng/µL2.3 µL9/8Pcon-RBS-lacZα-DT(EcoRI&SpeI)5.6ng/µL1.9 µL2.1 µL
experiment9/6Plux 25ng/µL2 µL9/8 RBS-lysis1-DT(XbaI&PstI)6.6 µL3.5 µL
experiment9/6Plux 25ng/µL2 µL9/8 RBS-lysis3-DT(XbaI&PstI)4.7 µL3.4 µL
experiment9/10Pcon-pT181 attenuator(SpeI&PstI) 3.4µL9/8RBS-lacZα-DT(EcoRI&SpeI)4.5 µL3.5 µL
experiment9/6Pcon-RBS laxR-DT(EcoRI&XbaI) 26.7ng/µL1.9 µLPlax-RBS-GFP-DT5.5 µL3.5 µL
experiment9/9 Pcon-pT181 attenuator(SpeI&PstI)3.4 µL9/10 aptamer12_1R-DT(XbaI&PstI)1.7 µL2.6 µL

Transformation

Nakamoto Tatsui

NameSampleCompetent CellsTotalPlate
9/9 Fusion1 antisense(pSB2C3)2µL20µL22µLCP
9/9 Fusion6 antisense(pSB2C3)2µL20µL22µLCP
9/9 aptamer 12_P(pSB2C3)2µL20µL22µLCP
9/9 aptamer 12_1&micro(pSB2C3)2µL20µL22µLCP
9/9 Fusion2 attenuator(pSB2C3) 2µL20µL22µLCP
9/11 Pcon-spinach-DT(pSB4K5)2µL20µL22µLK
9/11 RBS-lacZα-DT(pSB4K5)2µL20µL22µLK
9/11 Pcon-RBS-lacZα-DT(pSB4K5)2µL20µL22µLK
Plux+RBS-lysis1-DT2µL20µL22µLCP
Plux+RBS-lysis3-DT2µL20µL22µLCP
Pcon-pT181attenuator+RBS-lacZα-DT2µL20µL22µLAmp
Pcon-iuxR+Plux-GFP2µL20µL22µLAmp
Pcon-pT181attenuator+apt12_1R-DT2µL20µL22µLAmp
Plac(BBa_R0011)2µL20µL22µLAmp

Electrophoresis

Tatsui

LaneSample
1100bp ladder
2Pcon+RBS luxI-DT2
3Pcon+RBS luxI-DT3
4Pcon+RBS luxI-DT4
5Pcon+RBS luxI-DT5
6Pcon+RBS luxI-DT6
7Pcon+apF12_1R-DT1
8Pcon+apF12_1R-DT2
9Pcon+apF12_1R-DT3
10Pcon+apF12_1R-DT4
11DT+Pcon-pT181attenuator1
12F1attenuator(pSB1C3)
13100bp ladder

Igku 9119.jpg

Restriction Enzyme Digestion

Nakamoto

DNAEcoRIXbaISpeIPstIbufferBSAMilliQTotal
pSB4K5(EcoRI+SpeI)9.4µL 1µL 0µL 1µL 0µL 3µL 3µL 12.6µL30 µL
pSB4K5(EcoRI+SpeI) negative0.5µL 0µL 0µL 0µL 0µL 1µL 1µL 7.5µL 10µL
RBS-lacZα-DT(EcoRI+SpeI) 10µL 1µL 0µL 1µL 0µL 3µL 3µL 12µL 30µL
RBS-lacZα-DT(EcoRI+SpeI) negative 0.5µL 0µL 0µL 0µL 0µL 1µL 1µL 7.5µL 10µL
Ptet-pT181 antisense(SteI+PstI) 10.8µL 0µL 0µL 1µL 1µL 3µL 3µL 12µL 30µL
Ptet-pT181 antisense(SteI+PstI) negative 0.5µL 0µL 0µL 0µL 0µL 1µL 1µL 7.5µL 10µL
Pcon-RBS-lacZα-DT(EcoRI+SpeI) 7.1µL 1µL 0µL 1µL 0µL 3µL 3µL 14.9µL 30µL
Pcon-RBS-lacZα-DT(EcoRI+SpeI) negative 0.4µL 0µL 0µL 0µL 0µL 1µL 1µL 7.6µL 10µL
RBS-lysis2-DT(XbaI+PstI) 22µL 0µL 1µL 0µL 1µL 3µL 3µL 0µL 30µL
RBS-lysis2-DT(XbaI+PstI) negative 1.4µL 0µL 0µL 0µL 0µL 1µL 1µL 6.6µL 10µL

Liquid Culture

Nakamoto

Samplemedium
8/16 Master4 Plax
8/25 Pλ-laxI-2 　

PCR

Hirano

8/29 Pλ-RBS-luxI-DT(76.5ng/µL)10x buffer for KOD plus-ver.2KOD-plusdNTPMgSO4F primer(RBS-luxI-DT-cloning-fwd)R primer(RBS-luxI-DT-cloning-rev)MilliQtotal
0.32.50.52.51.50.750.7516.225
PreDenatureDenatureAnnealingExtensioncycle
　94°C　94°C　65°C　68°C--
5min30sec30sec1min30
8/31 Pbad/araC-RBS-RFP(1)(67.6ng/µL)10x buffer for KOD plus-ver.2KOD-plusdNTPMgSO4F primer(Pbad/araC(pSBlc3)-cloning-fwd)R primer(Pbad/araC(pSBlc3)-cloning-rev)MilliQtotal
0.32.50.52.51.50.750.7516.225
PreDenatureDenatureAnnealingExtensioncycle
　94°C　94°C　54°C　68°C--
5min30sec30sec3min 20sec30

Liquid Culture

Hirano

Samplemedium
9/4 RBS-lysis1+DT 3plusgrow(+CP) 3ml 37°C
9/4 RBS-lysis2+DT 1plusgrow(+CP) 3ml 37°C
9/4 RBS-lysis3+DT 3plusgrow(+CP) 3ml 37°C

Sep 12

Miniprep

Nakamoto

DNAconcentration[µg/mL]260/280260/230
Pcon-pT181attenuator-DT293.6　1.88　1.84
Pcon-apt12-1R-DT303.8　1.75　1.64
Fusion1attenuator(pSB1c3)172.0　1.96　2.31
RBS-lisis1-DT235.11.591.60
RBS-lisis2-DT387.71.912.01
RBS-lisis3-DT387.91.912.09
Plux166.71.981.63

Electrophoresis

Nakamoto

LaneSample
1100bp ladder
29/11 P-RBS-lisis1-DT(Colony PCR prodution)

Igku 9121.png

Liquid Culture

Nakamoto

Samplemedium
9/10 Pcon-apt12-1R-DT1Plusgrow medium(+Amp)
9/10 DT-Pcon-pT181attenuator1Plusgrow medium(+CP)
9/10 Fusion1-attenuator(pSB1c3)Plusgrow medium(+CP)　

Electrophoresis

Tatsui
LaneSample
11kb ladder
2pSB4K5(EcoRI&SpeI)
3 pSB4K5 NC(EcoRI&SpeI)
4RBS-lacz-DT(EcoRI&SpeI)
5 EcoRI&SpeI NC( EcoRI&SpeI)
6Pcon-RBS-lacz-DT( EcoRI&SpeI)
7 Pcon-RBS-lacz-DT NC( EcoRI&SpeI)
81kb ladder

Igku 9122.png

2

LaneSample
11kb ladder
2Ptet-pT181antisense(SpeI&PstI)
3 Ptet-pT181antisense NC(SpeI&PstI)
4RBS-lysis2-DT(XbaI&PstI)
5 EcoRI&SpeI NC( EcoRI&SpeI)
6--
7--
81kb ladder

Igku 9123.png

Gel Extraction

Tatsui

LaneDNAEnzyme
11kb ladder--
2RBS-lacZα-DTEcoRI&SpeI
3RBS-lacZα-DTEcoRI&SpeI
4RBS-lacZα-DTEcoRI&SpeI
5----
6Pcon-RBS-lacZα-DTEcoRI&SpeI
7Pcon-RBS-lacZα-DTEcoRI&SpeI
8Pcon-RBS-lacZα-DTEcoRI&SpeI
91kb ladder--

Igku 9129.png
Igku 91210.png

Nameconcentration[µg/mL]260/280260/230
RBS-lacZα-DT(EcoRI&SpeI)9.81.500.39
Pcon-RBS-lacZα-DT(EcoRI&SpeI)11.21.470.08

Gel Extraction

Tatsui

LaneDNAEnzyme
11kb ladder--
2RBS-lysis2-DTXbaI&PstI
3RBS-lysis2-DTXbaI&PstI
4RBS-lysis2-DTXbaI&PstI
5----
61kb ladder--

Igku 9127.png
Igku 9128.png

Nameconcentration[µg/mL]260/280260/230
RBS-lysis2-DT(XbaI&PstI)5.41.430.77

Electrophoresis

No name


LaneSample
11kb ladder
2Pλ-RBS-luxI-DT(PCR product)
3Pbad/araC-RBS-RFP(PCR product)

Igku 9124.png

Colony PCR

Honda

Samplebase pair
9/11 apt12-1M(pSB1C3) 1513
9/11 apt12-1M(pSB1C3) 2513
9/11 Fusion3m2a09ttenuator(pSB1C3) 1609
9/11 Fusion6 antisense(pSB1C3)1431
9/11 aptamer 12-P(pSB1C3)1515
9/11 Fusion1 antisense(pSB1C3) 1420
9/11 Fusion1 antisense(pSB1C3) 2420
9/11 Plac(BBa-R0011) 1293
9/11 Plac(BBa-R0011) 2293
9/11 Plac(BBa-R0011) 3293
9/11 Plac(BBa-R0011) 4293
9/11 Pcon-luxR-Plux-GFP 12138
9/11 Pcon-RBS-lacZα-DT(pSB4K5) 1712
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s2min12s30cycles

Restriction Enzyme Digestion

Tatsui

9/5 Pcon EcoRISpeIXbaIPstI BufferBSAMilliQtotal
1cuts(PstI)5.70µL0µL0µL1µL3µL3µL17.3µL30µL
NC(PstI)1.90µL0µL0µL0µL1µL1µL6.1µL10µL
8/17 DTEcoRIBufferBSAMilliQtotal
1cut(EcoRI)10.61µL3µL3µL12.4µL30µL
NC(EcoRI)3.50µL1µL1µL4.5µL10µL

Colony PCR

Nakamoto and Tatsui

Samplebase pair
9/11 Pcon-pT181attenuator-RBS-lacZα-DT965
9/11 Plux-PBS-lysis1-DT 1613
9/11 Pcon-pT181attenuator-aptamer12-1R-DT 1859
9/11 Pcon-pT181attenuator-aptamer12-1R-DT 2859
9/11 Plux-RBS-lysis3-DT 11210
9/11 Plux-RBS-lysis3-DT 21210
Pcon-Spinach-DT(pSB4K5) 1605
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s1min12s30cycles

Transformation

Tatsui

NameSampleCompetent CellsPlate
9/7 pSB4K52µL20µLAmp

Electrophoresis

No name

1

LaneSample
1Fusion6 antisense
2Fusion1 antisense-1
3 Fusion1 antisense-2
4100bp ladder
5Plac(BBa-R0011) 1
6 Plac(BBa-R0011) 2
7 Plac(BBa-R0011) 3
8 Plac(BBa-R0011) 4

Igku 91211.png

2

LaneSample
11kb ladder
2aptamer12-1M 1
3 aptamer12-1M 2
4Fusion3m2 attenuator
5aptamer12-P1
6Plux-RBS-GFP-DT-Pcon-RBS-luxR-DT
7Pcon-RBS-lacZα=DT
8 1kb ladder

Igku 91212.png

Electrophoresis

No name

LaneSampleEnzyme
1Pcon-pT181attenuator-RBS-lacZα-DT 1--
2Pcon-pT181attenuator-RBS-lacZα-DT 2--
3Plux-RBS-lysis1-DT 1 --
4Pcon-pT181attenuator-aptamer12-1R-DT 1 --
5Pcon-pT181attenuator-aptamer12-1R-DT 2 --
6Plux-RBS-lysis3-DT 1--
71kbp ladder --
8 Plux-RBS-lysis3-DT 2--
9Pcon-spinach-DT(pSB4K5) 1--
109/5 PconPstI
119/5 Pcon NC--
128/17 DT EcoRI
138/17 DT --

Igku 91213.png

Liquid Culture

Nakamoto

Samplemedium
9/11Fusion6 antisense-1Plusgrow medium(+CP)
Plac(BBa-R0011)-3Plusgrow medium(+Amp)
Plac(BBa-R0011)-4Plusgrow medium(+Amp)
Fusion3m2 attenuator -1Plusgrow medium(+CP)　
Pλ-luxI-2Plusgrow medium(+Amp)　
9/11 aptamer12-1M-2Plusgrow medium(+CP)

Colony PCR

Nakamoto

Samplebase pair
9/11 Fusion1 antisense 3394
9/11 Fusion1 antisense 4394
9/11 aptamer12-P 2378
9/11 aptamer12-P 3378
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s36s30cycles


Electrophoresis

Nakamoto

LaneSampleEnzyme
1Fusion1 antisense 2--
2Fusion1 antisense 3--
31kb ladder --
4apt12-P 2 --
5apt12-P 3 --

Igku 91214.png

Restriction Enzyme Digestion

Tatsui

9/12 RBS-lysis2-DTXbaIPstIBufferBSAMilliQtotal
2 cuts5.2 1.0µL1.0µL3.0µL3.0µL16.8µL 30µL
NC0.3µL0µL0µL1.0µL1.0µL7.7µ10µL
9/12 RBS-lysis3-DTXbaI PstIBufferBSAMilliQtotal
2 cuts5.2 1.0µL1.0µL3.0µL3.0µL16.8µL 30µL
NC0.3µL0µL0µL1.0µL1.0µL7.7µ10µL
9/11 RBS-lysis1-DTXbaIPstIBufferBSAMilliQtotal
2 cuts8.51.0µL1.0µL3.0µL3.0µL13.5µL 30µL
NC0.4µL0µL0µL1.0µL1.0µL7.6µ10µL
8/20 J23100-RBS-luxR -DTEcoRI BufferBSAMilliQtotal
1cut5.81.0µL3.0µL3.0µL17.2µL 30µL
NC0.3µL0µL1.0µL1.0µL7.7µ10µL
9/10 pSB1C3EcoRI SpeIBufferBSAMilliQtotal
2cuts8.11.0µL1.0µL3.0µL3.0µL13.9µL 30µL
NC0.4µL0µL0µL1.0µL1.0µL7.6µ10µL
9/12 Plux PstI BufferBSAMilliQtotal
1cut12.01.0µL3.0µL3.0µL11.0micro;L 30µL
NC0.6µL0µL1.0µL1.0µL7.4µ10µL
9/10 pSB1C3XbaI PstIBufferBSAMilliQtotal
2cuts8.11.0µL1.0µL3.0µL3.0µL13.9µL 30µL

Electrophoresis

Tatsui

LaneSampleEnzyme1Enzymel2
19/12 RBS-lysis2-DT XbaIPstI
29/12 RBS-lysis2-DT NC ----
39/11 RBS-lysis1-DT XbaIPstI
49/11 RBS-lysis1-DT NC ----
5 9/11 RBS-lysis3-DT XbaIPstI
6 9/11 RBS-lysis3-DT NC1----
71kb ladder ----
8 100bp ladder----
9J23100-RBS-luxR-DT EcoRI--
10 J23100-RBS-luxR-DT ----
119/10 pSB1C3 EcoRISpeI
12 9/10 pSB1C3 NC-----
139/10 pSB1C3 XbaIPstI
149/12 PluxPstI--
159/12 Plux NC----

Igku 91215.png

Observation through a confocal microscope

We used the liquid culture media with 200μL 9/10Pcon-pT181antisense-spinach-DT& 200μL 9/10 Pcon-spinach-DT& 200μL 9/10 JM109(overnight culture).
We translated them into each 1.5mL tube.

We elminated each supernatant using a 5000xg centrifuge for 1minute,.
Then,we resuspended with 100µL M9(distilled water) and eliminated each supernatant using a 5000xg centrifuge for 1 minute.x2

Adding 100µL M9(in 20µM DFHBI), we resuspended the pret.

15min after incubating in shield light, we eliminated each supernatant using a 5000xg centrifuge for 1 minute.

We looked at the fluorescence of the pret.
We failed to observe it.

Liquid Culture

Hirano

Samplemedium
9/10 Pcon-pT181antisense-spinach-DTPlusgrow medium
9/10 Pcon-spinach-DTPlusgrow medium
9/1 spinach-DT(RNA master plate) Plusgrow medium

Sep 13

Miniprep

Nakamoto

DNAconcentration[µg/mL]260/280260/230
9/12 Fusion6 antisense245.91.881.41
9/12 Plux-3115.51.581.32
9/12 Plux-4125.31.931.44
9/12 Fusion3n2 attenuator-1231.91.811.31

Gel Extraction

Tatsui,Ashida

LaneDNAEnzyme
11kb ladder--
2RBS-lysis1-DTXbaI&PstI
3RBS-lysis1-DTXbaI&PstI
4RBS-lysis1-DTXbaI&PstI

Igku 9131.jpg
Igku 9132.jpg

LaneDNAEnzyme
11kb ladder--
3RBS-lysis2-DTXbaI&PstI
4RBS-lysis2-DTXbaI&PstI
5RBS-lysis2-DTXbaI&PstI

Igku 9133.jpg
Igku 9134.jpg

LaneDNAEnzyme
3RBS-lysis3-DTXbaI&PstI
4RBS-lysis3-DTXbaI&PstI
5RBS-lysis3-DTXbaI&PstI
61kb ladder--

Igku 9135.jpg
Igku 9136.jpg

LaneDNAEnzyme
21kb ladder--
3pSB1C3EcoRI&SpeI
4pSB1C3EcoRI&SpeI
5pSB1C3EcoRI&SpeI

Igku 9137.jpg
Igku 9138.jpg

LaneDNAEnzyme
11kb ladder--
2pSB1C3XbaI&PstI
3pSB1C3XbaI&PstI
4pSB1C3XbaI&PstI

Igku 9139.jpg
Igku 91310.jpg

Restriction Enzyme Digestion

Nakamoto

9/13 PlacEcoRISpeIXbaIPstI BufferBSAMilliQtotal
1cuts(PstI)160µL0µL0µL1µL3µL3µL7µL30µL
NC0.80µL0µL0µL0µL1µL1µL7.2µL10µL
9/10 Pcon attenuatorEcoRISpeIXbaIPstIBufferBSAMilliQtotal
1cut(PstI)100µL0µL0µL1µL3µL3µL13µL30µL
NC0.50µL0µL0µL0µL1µL1µL7.5µL10µL
9/12 Plux EcoRISpeIXbaIPstI BufferBSAMilliQtotal
1cuts(PstI)120µL0µL0µL1µL3µL3µL11µL30µL
NC0.60µL0µL0µL0µL1µL1µL7.4µL10µL
9/12 DT-EEcoRISpeIXbaIPstIBufferBSAMilliQtotal
1cut(XbaI)23.70µL0µL1µL0µL3µL3µL0µL30.7µL
NC1.20µL0µL0µL0µL1µL1µL6.8µL10µL
9/12 Pcon-RBS-luxR-bTE EcoRISpeIXbaIPstI BufferBSAMilliQtotal
1cuts(XbaI)22.40µL0µL1µL0µL3µL3µL0.6µL30µL
NC2.20µL0µL0µL0µL1µL1µL5.8µL10µL
9/12 Pcon-PEcoRISpeIXbaIPstIBufferBSAMilliQtotal
1cut(SpeI)210µL1µL0µL0µL3µL3µL12.0µL30µL
NC2.10µL0µL0µL0µL1µL1µL5.7µL10µL

M9 (EDTA)

Kanbayashi

1mM EDTA
volume10ml
Na2HPO4 300mg
KH2PO4150mg
NaCl25mg
NH4Cl50mg
Fe(3)-EDTA21mg　
5mM EDTA
volume10ml
Na2HPO4300mg
KH2PO4150mg
NaCl25mg
NH4Cl50mg
Fe(3)-EDTA105mg　

Gel Extraction

Nakamoto

LaneDNAEnzyme
11kb ladder--
3Pcon-PSpeI
4Pcon-PSpeI
5Pcon-PSpeI

Igku 91311.jpg
Igku 91312.jpg

Liquid Culture

Tatsui

Samplemedium
9/11 aptamer12_1M(pSB1C3)-1--
9/11 aptamer 12_P1--
9/11 Fusaion1 antisense-4--
9/12 pSB4K5-1--
9/12 pSB4K5-2--

Restriction Enzyme Digestion

Nakamoto

9/13 Plac-PEcoRISpeIXbaIPstI BufferBSAMilliQtotal
1cuts(SpeI)23.50µL0.5µL0µL0µL3µL3µL0µL30µL
NC80µL0µL0µL0µL1µL1µL0µL10µL
9/13 Pcon-pT181 attenuator-PEcoRISpeIXbaIPstIBufferBSAMilliQtotal
1cut(SpeI)23.50µL0.5µL0µL0µL3µL3µL0µL30µL
NC02.30µL0µL0µL0µL1µL1µL5.7µL10µL
9/13 Plux-P EcoRISpeIXbaIPstI BufferBSAMilliQtotal
1cuts(SpeI)23.80µL0.2µL0µL1µL3µL3µL0µL30µL
NC80µL0µL0µL0µL1µL1µL0µL10µL
9/5 RBS-lacZα-DTEcoRISpeIXbaIPstIBufferBSAMilliQtotal
2cut(XbaI&PstI)10.80µL0µL1µL1µL3µL3µL11.2µL30µL
NC0.50µL0µL0µL0µL1µL1µL7.5µL10µL

Electrophoresis

Hirano

LaneSample
1100bp ladder
2RBS-lacZα-DT(XbaI&PstI)
3RBS-lacZα-DT(NC)

Igku 91313.jpg

Ligation

No Name

stateVectorInserterLigation High ver.2
experiment9/13 Plac (SpeI&PstI)8.5µL9/13 pT181 antisense (XbaI&PstI)2.1 µL5.3 µL
experiment9/13 Plac (SpeI&PstI)8.5 µL9/10 aptamer 12_1R-DT (XbaI&PstI)1.8 µL5.15µL
experiment9/13 Plac (SpeI&PstI)8.5 µL9/2 pT181 attenuator(XbaI&PstI)2.3 µL10.4 µL
experiment9/13 Plux (SpeI&PstI)11.4 µL9/13 RBS-lysis1-DT (XbaI&PstI)5.0 µL8.2 µL
experiment9/13 Plux (SpeI&PstI)11.4 µL9/13 RBS-lysis2-DT (XbaI&PstI)7.0 µL9.2 µL
experiment9/13 Plux (SpeI&PstI)7.2 µL9/13 RBS-lysis3-DT (XbaI&PstI)6.0 µL6.6 µL
experiment9/13 Pcon-RBS-luxR-DT (EcoRI&XbaI)2.0 µL9/4 Plux-RBS-GFP-DT (EcoRI&SpeI)5.2 µL3.6 µL
experiment9/13 Pcon-RBS-luxR-DT (EcoRI&XbaI)2.0 µL9/10 aptamer 12_1R-DT (XbaI&PstI)1.6 µL1.8 µL
experiment9/12 Ptet-pT181 antisense (--)1.0 µL9/10 spinach-DT (--)2.4 µL1.7 µL

PCR

No name

genome DNABig DyeMilliQ5x buffertemp(400mg)SasA_fwd primerSasA_rev primertotal
9/7 tetR aptamer 12_1R(pSB1C3) 0.56.051.751.70.5010.5
genome DNABig DyeMilliQ5x buffertemp(400mg)SasA_fwd primerSasA_rev primertotal
9/6 pT181 antisense-1(pSB1C3) 0.56.051.751.70.5010.5
genome DNABig DyeMilliQ5x buffertemp(400mg)SasA_fwd primerSasA_rev primertotal
9/6 pT181 attenuator (pSB1C3)0.53.851.753.90.5010.5
genome DNABig DyeMilliQ5x buffertemp(400mg)SasA_fwd primerSasA_rev primertotal
8/27 Pλ-RBS-luxI-DT-10.52.551.755.20.5010.5
genome DNABig DyeMilliQ5x buffertemp(400mg)SasA_fwd primerSasA_rev primertotal
8/31 Pbad/araC-RBS-RFP-20.53.751.7540.5010.5
genome DNABig DyeMilliQ5x buffertemp(400mg)SasA_fwd primerSasA_rev primertotal
9/13 Plac(pSB1A2) 0.54.21.753.50.5010.5
genome DNABig DyeMilliQ5x buffertemp(400mg)SasA_fwd primerSasA_rev primertotal
8/20 J23100-RBS-lacZα-DT0.56.351.751.40.5010.5
genome DNABig DyeMilliQ5x buffertemp(400mg)SasA_fwd primerSasA_rev primertotal
9/7 Ptet-RBS-lacZα-DT0.56.051.751.70.5010.5
genome DNABig DyeMilliQ5x buffertemp(400mg)SasA_fwd primerSasA_rev primertotal
9/8 J23100-spinach-DT0.55.751.7520.5010.5
genome DNABig DyeMilliQ5x buffertemp(400mg)SasA_fwd primerSasA_rev primertotal
9/11 RBS-lysis1-DT0.56.051.751.70.5010.5
genome DNABig DyeMilliQ5x buffertemp(400mg)SasA_fwd primerSasA_rev primertotal
9/12 RBS-lysis2-DT0.56.751.751.00.5010.5
genome DNABig DyeMilliQ5x buffertemp(400mg)SasA_fwd primerSasA_rev primertotal
9/12 RBS-lysis3-DT0.56.751.751.00.5010.5
genome DNABig DyeMilliQ5x buffertemp(400mg)SasA_fwd primerSasA_rev primertotal
9/8 J23100-spinach-DT0.56.351.751.400.510.5
genome DNABig DyeMilliQ5x buffertemp(400mg)SasA_fwd primerSasA_rev primertotal
8/27 Pλ-RBS-luxI-DT-10.52.551.755.200.510.5
genome DNABig DyeMilliQ5x buffertemp(400mg)SasA_fwd primerSasA_rev primertotal
9/12 RBS-lysis2-DT0.56.751.751.000.510.5
genome DNABig DyeMilliQ5x buffertemp(400mg)SasA_fwd primerSasA_rev primertotal
9/11 RBS-lysis1-DT0.56.051.751.700.510.5
PreDenatureDenatureAnnealingExtensioncycle
96°C96°C50°C68°C--
2m10s5s4m30

PCR

Yoshida

genome DNAKOD plus10x bufferdNTPMgSO4Prefix-RBS-RpaB_fwd primerRpaB-GG4-suffix_rev primerMilliQtotal
RpaB0.52.52.51.50.750.7516.525
genome DNAKOD plus10x bufferdNTPMgSO4Prefix-PkaiBC_fwd primer ver.3PkaiBC-suffix_rev primer ver.3MilliQtotal
RpaB0.52.52.51.50.750.7516.525


PreDenatureDenatureAnnealingExtensioncycle
94°C98°C58°C68°C--
2m10s30s30s30

Gel Extraction

Nakamoto

LaneDNAEnzyme
1100bp ladder--
3RBS-lacZα-DTXbaI&PstI
4RBS-lacZα-DTXbaI&PstI
5RBS-lacZα-DTXbaI&PstI
6RBS-lacZα-DTXbaI&PstI

Igku 91318.jpg
Igku 91319.jpg

Transformation

No name

NameSampleCompetent CellsTotalPlate
9/13 Plac+pT181 antisense2µL20µL22µLAmp
9/13 Plac+apt12_1R2µL20µL22µLAmp
9/13 Plac+pT181 attenuator2µL20µL22µLAmp
9/13 Plux+pT181 antisense2µL20µL22µLCP
9/13 Plux+RBS-lysis2-DT2µL20µL22µLCP
9/13 Plux+RBS-lysis2-DT2µL20µL22µLCP
9/13 Pcon-RBS-lacR-DT+Plux-RBS-GFP-DT2µL20µL22µLAmp
9/13 Pcon-pT181 antisense+apt12_1R-DT2µL20µL22µLAmp
9/13 Ptet-pT181antisense+spinach-DT2µL20µL22µLCP
Pλ-luxI-12µL20µL22µLAmp

Sep 14

Restriction Enzyme Digestion

Hirano

9/14 pSB4K5SEcoRIXbaISpeIPstIBufferBBufferDBSAMilliQtotal
2cuts10µL1µL0µL1µL0µL3µL0µL3µL12µL30µL
NC0.5µL0µL0µL0µL0µL1µL0µL1µL7.5µL10µL
9/13 PlacXbaIPstIBufferHBSAMilliQtotal
1 cut9µL0µL1µL3µL3µL14µL30µL
NC1µL0µL0µL1µL1µL7µL10µL
9/14 PKaiBCEcoRIXbaISpeIPstIBufferBBSAMilliQtotal
2cuts11µL1µL0µL1µL0µL3µL3µL11µL30µL
NC4µL0µL0µL0µL0µL1µL1µL4µL10µL
9/14 RpaBEcoRIXbaISpeIPstIBufferDBSAMilliQtotal
2cuts13µL0µL1µL0µL1µL3µL3µL9µL30µL
NC2µL0µL0µL0µL0µL1µL1µL5µL10µL
8/17 RBS-GFP-DTEcoRIXbaISpeIPstIBufferDBSAMilliQtotal
1cut9µL0µL1µL0µL0µL3µL3µL14µL30µL
NC0.5µL0µL0µL0µL0µL1µL1µL7.5µL10µL
  • incubated 37°C 1hour

Liquid Culture

Hirano

Samplemedium
9/11 Plac(BBa-R0011)-(3)Plusgrow medium(+Amp)

Electrophoresis

Hirano

LaneSampleEnzyme1Enzyme2
11kb ladder----
2pSB4K5(1)EcoRISpeI
3 pSB4K5(1)----
4Plac(1A2)PstI--
5Plac(1A2)----
6PKaiBCEcoRISpeI
7PKaiBC----
8RpaB(1)XbaIPstI
9RpaB(1)----
10RBS-GFP-DTXbaI--
11RBS-GFP-DT----
121kb ladder----

Igku 0914 E1.jpg

LaneSampleEnzyme
11kb ladder--
2Plac(pSB1A2)PstI
3Plac(pSB1A2)--

Igku 0914 E2.jpg

Sequence PCR

No name

genome DNABig Dye5x buffertemp(400mg)primermilliQ
Pcon-RBS-luxR-DT0.51.750.90.56.85
Pcon-RBS-luxR-DT0.51.750.40.57.35
Pcon-RBS-tetR-DT0.51.7530.55.25
Pcon-RBS-tetR-DT0.51.7530.55.25
Plux0.51.752.40.55.35
Plux-RBS-GFP-DT0.51.751.90.55.85
Plux-RBS-GFP-DT0.50.51.90.55.85
Pcon-RBS-GFP-DT0.50.51.20.56.55
Pcon-RBS-GFP-DT0.50.51.20.56.55
RBS-lysis3-DT0.50.51.00.56.75
Pcon-antisense-Spinach-DT0.50.51.40.56.35
Pcon-RBS lacZα-DT0.50.51.40.56.35
Ptet-RBS lacZα-DT0.50.50.590.51.85
PreDenatureDenatureAnnealingExtensioncycle
96°C96°C50°C60°C--
2min10sec5sec4min30cycle

Gel Extraction

Tatsui

LaneDNAEnzyme
11kb ladder--
2pSB4K5EcoRI&SpeI
3pSB4K5EcoRI&SpeI
4pSB4K5EcoRI&SpeI

Igku 0914 G8.jpg
Igku 0914 G9.jpg

Restriction Enzyme Digestion

No name

9/14 Plac(1A2)EcoRIXbaISpeIPstIBufferBBSAMilliQtotal
2cuts23µL0µL0µL0.3µL0µL3µL3µL0µL30µL
9/14 RBS-GFP-DTEcoRIXbaISpeIPstIBufferHBSAMilliQtotal
2 cuts23µL1µL0µL0µL0µL3µL3µL0µL30µL

Ligation

Nakamoto and Hirano

stateVectorInserterLigation High ver.2
experiment9/13 Pcon-PT181attenuator(SpeI&PstI)1.79/13 RBS-lacZα-DT (XbaI & PstI)12.55
experiment9/13 pSB2C3 (XbaI & PstI)1.99/14 RpaB (XbaI & PstI)16.310.5
experiment9/13 pSB2C3 (XbaI & PstI)4.79/14 PkaiBC (EcoRI & SpeI)6.75.7
experiment9/14 RBS-GFP-DT (PstI&XbaI)0.79/14 PkaiBC (EcoRI & SpeI)2.61.6
experiment9/14 Plac (SpeI&PstI)2.49/14 RBS-lysis1-DT (XbaI & PstI)4.93.6
experiment9/14 Plac (SpeI&PstI)2.49/14 RBS-lysis2-DT (XbaI & PstI)7.04.7
experiment9/14 Plac (SpeI&PstI)2.49/14 RBS-lysis3-DT (XbaI & PstI)9.25.8

Samples were evaporeted used evaporator into about 7 µL

Colony PCR

No name

Samplebase pair
9/13 Plux＋RBS-lysis1-DT (1)~(3)--
9/13 Plux＋RBS-lysis2-DT (1)~(3)-
9/13 Plux＋RBS-lysis3-DT (1)~(2)-
Samplebase pair
9/13 Pcon-RBS-luxR-DT＋Plux-RBS-GFP-DT (1)~(4)--
9/13 Pcon-PT181attenuator＋aptamer121R-DT (1)~(2)-
9/13 Plac＋PT181attenuator (1)-
Samplebase pair
Dλ-lux(1) (1)~(2)--
NC --

Electrophoresis

No name

LaneSample
11kbp ladder
29/13 Plux+RBS-lysis1-DT1
39/13 Plux+RBS-lysis1-DT2
49/13 Plux+RBS-lysis1-DT3
59/13 Plux+RBS-lysis2-DT1
69/13 Plux+RBS-lysis2-DT2
79/13 Plux+RBS-lysis2-DT3
89/13 Plux+RBS-lysis3-DT1
99/13 Plux+RBS-lysis3-DT2
109/13 Pcon-RBS-luxR-DT+Plux-RBS-GFP-DT1
119/13 Pcon-RBS-luxR-DT+Plux-RBS-GFP-DT2
129/13 Pcon-RBS-luxR-DT+Plux-RBS-GFP-DT3
139/13 Pcon-RBS-luxR-DT+Plux-RBS-GFP-DT4
149/13 Ptet-pT181 antisense+Spinach-DT1
159/13 Pcon-pT181 attenuator+aptamer12-1R-PT1
169/13 Pcon-pT181 attenuator+aptamer12-1R-PT1
179/13 Plac+pT181 attenuator

Igku 0914 E5.jpg

LaneSample
11kbp ladder
2Pλ-lux(1)1
3 Pλ-lux(1)1
4NC

Igku 0914 E6.jpg

Liquid Culture

No name

Samplemedium
9/13 Pλ-luxI(1)-1Plusgrow medium(+Amp)

Transformation

Nakamoto

NameSampleCompetent CellsTotalPlate
9/13 RBS-lysisZα-DT(XbaI&PstI)＋9/13 Pcon-PT181 attenuator(SpeI&PstI)2µL20µL22µLAmp
9/13 SB2C3＋9/14 PzαB2µL20µL22µLCP
9/13 pSB2C3＋9/14 PkaiBC2µL20µL22µLCP
9/13 RBS-GFP-DT＋9/14 PkaiBC2µL20µL22µLCP
Plac＋RBS-lysis1-DT2µL20µL22µLAmp
Plac＋RBS-lysis2-DT2µL20µL22µLAmp
Plac＋RBS-lysis2-DT2µL20µL22µLAmp

Sequence PCR

Tatsui

genome DNAtempBig Dye5x bufferprimerMilliQ
9/6 PT181 attenuator(VR)3.90.51.750.53.85
8/17 RBS lacZα-DT2.10.51.750.55.65
9/6 PT181 attenuator(VR)3.90.51.750.53.85
PreDenatureDenatureAnnealingExtensioncycle
96°C50°C60°C4°C--
10sec5sec4min1min30cycle

Sep 15

Miniprep

No name

DNAconcentration[µg/mL]260/280260/230
Pλ-luxI225　1.59　0.83

Liquid Culture

No name

Samplemedium
9/13 Pλ-luxI-1
9/7 pSB4K5-1
9/11 Plac-3

Ligation

No name

stateVectorInserterLigation High ver.2
experiment9/13 Pcon(SpeI & PstI)3.6 µL9/12 RBS-lacZα-DT(XbaI & PstI)4.7 µL8 µL

Restriction Enzyme Digestion

No Name

DNAEcorISpeIBufferBSAMilliQtotal
9/7Pcon-RBS-tetR-DT (EcoRI & SpeI)15 µL1 µL1 µL3 µL3 µL6 µL30 µL
NC0.7 µL0 µL0 µL1 µL1 µL7.3 µL10 µL


DNAEcoRISpeIBufferBSAMilliQtotal
9/12 Pcon-pT181 attenuator-DT (EcoRI & SpeI)6.8 µL1 µL1 µL3 µL3 µL15.2 µL30 µL
NC0.3 µL0 µL0 µL1 µL1 µL7.7 µL10 µL


DNAEcoRISpeIBufferBSAMilliQtotal
9/12 Pcon-apt12_1R-DT(EcoRI & SpeI)6.6 µL1 µL1 µL3 µL3 µL15.4 µL30 µL
NC0.3 µL0 µL0 µL1 µL1 µL7.7 µL10 µL


DNAEcoRISpeIBufferBSAMilliQtotal
9/8 Pcon-spinach-DT (EcoRI & SpeI)7.6 µL1 µL1 µL3 µL3 µL15.4 µL30 µL
NC0.3 µL0 µL0 µL1 µL1 µL7.7 µL10 µL


DNAXbaIPstIBufferBSAMilliQtotal
9/11 Pcon-pT181 anticodone-spinach-DT(XbaI & PstI)7.2 µL1 µL1 µL3 µL3 µL14.8 µL30 µL
NC0.4 µL0 µL0 µL1 µL1 µL7.6 µL10 µL


DNAXbaIPstIBufferBSAMilliQtotal
8/17 RBS-lacZα-DT (XbaI & PstI)5.3 µL1 µL1 µL3 µL3 µL16.7 µL30 µL
NC0.5 µL0 µL0 µL1 µL1 µL7.5 µL10 µL


DNAEcoRIXbaIBufferBSAMilliQtotal
9/7 Ptet-RBS-LacZα-DT (EcoRI & XbaI)2.9 µL1 µL1 µL3 µL3 µL18.5 µL30 µL
NC1.5 µL0 µL0 µL1 µL1 µL7 µL10 µL


Electrophoresis

No Name

LaneSampleEnzyme1Enzyme2
1,14 100bp ladder
2 Pcon-RBS-tetR-DT EcoRI SpeI
3 Pcon-RBS-tetR-DT
4 Pcon-pT181 attenuator-DT EcoRI SpeI
5 Pcon-pT181 attenuator-DT
6 Pcon-apt12-1R-DT EcoRI SpeI
7 Pcon-apt12-1R-DT
8 Pcon-spinach-DT EcoRI SpeI
9 Pcon-spinach-DT
10 Pcon-pT181 antisense-spinach-DT XbaI PstI
11 Pcon-pT181 antisense-spinach-DT
12 RBS-lacZα-DT XbaI PstI
13 RBS-lacZα-DT
15 Ptet-RBS-lacZα-DT EcoRI XbaI
16 Ptet-RBS-lacZα-DT
17 1kbp ladder

Igku 9151.jpg

Gel Extraction

No Name 1

LaneDNAEnzyme
1 100bp ladder
2~4 Pcon-pT181 attenuator-DT EcoRI & SpeI
6~8 Pcon-apt12_1R-DT
10~12 Pcon-spinach-DT

Igku 9152.jpg
Igku 9153.jpg
2

LaneDNAEnzyme
1 100bp ladder
2~4 Pcon-pT181 anticodone-spinach-DT XbaI & PstI
6~8 RBS-lacZα-DT XbaI & PstI
10~12 Pcon-RBS-tetR-DT EcoRI & SpeI

Igku 9154.jpg
Igku 9155.jpg

Nameconcentration[µg/mL]260/280260/230
9/15 Ptet-RBS-lacZα-DT (EcoRI & SpeI) 1.9 1.08 0.77
9/15 Pcon-RBS-tetR-DT (EcoRI & SpeI) 5.9 1.30 0.17
9/15 RBS-lacZα-DT (XbaI & PstI) 1.0 0.85 0.44
Pcon-pT181 anticodone-spinach-DT (XbaI & PstI) 1.3 0.95 0.28
Pcon-spinach-DT 0.9 1.37 0.05
Pcon-apt12_1R-DT 1.7 1.42 0.29
Pcon-pT181 attenuator-DT (EcoRI & SpeI) 2.1 1.53 0.46

Ligation

No Name

stateVectorInserterLigation High ver.2
experiment9/13 pSB1C3 EcoRI & SpeI4.7 µL9/15 Pcon-RBS-tetR-DT EcoRI & SpeI1.9 µLxx µL
experiment9/13 pSB1C3 EcoRI & SpeI4.7 µL9/15 Pcon-spinach-DT EcoRI & SpeI4.7 µLxx µL
experiment9/13 pSB1C3 EcoRI & SpeI4.7 µL9/15 Pcon-apt12_1R-DT EcoRI & SpeI2.0 µLxx µL
experiment9/13 pSB1C3 EcoRI & SpeI4.7 µL9/15 Pcon-pT181 attenuator-DT EcoRI & SpeI3.0 µLxx µL
experiment9/13 pSB1C3 XbaI & PstI1.9 µL9/15 Pcon-pT181 anticodone-spinach-DT XbaI & PstI4.4 µLxx µL
experiment9/13 pSB1C3 EcoRI & SpeI4.7 µL9/3 pT181 attenuator EcoRI & SpeI3.8 µLxx µL
experiment9/6 Pcon SpeI & PstI1.4 µL9/15 RBS-lacZα-DT XbaI & PstI2.0 µLxx µL
experiment9/14 pSB 4k5 EcoRI & SpeI µL9/15 Pcon-spinach-DT EcoRI & SpeI9.4 µLxx µL
experiment9/15 Peet-RBS-lacZα-DT EcoRI & XbaI2.6 µLPcon-RBS-tetR-DT EcoRI & SpeI1.5 µLxx µL

Restriction Enzyme Digestion

No Name

DNAXbaIPstIBufferBSAMilliQtotal
9/13 pT181 attenuator XbaI & PstI6.3 µL1 µL1 µL3 µL3 µL15.7 µL30 µL
NC0.3 µL0 µL0 µL1 µL1 µL7.7 µL10 µL
DNAEcoRISpeIBufferBSAMilliQtotal
9/10 pSB1C3 EcoRI & SpeI8.1 µL1 µL1 µL3 µL3 µL13.9 µL30 µL
NC0.4 µL0 µL0 µL1 µL1 µL7.6 µL10 µL
DNAXbaIPstIBufferBSAMilliQtotal
9/14 pSB4k5 XbaI & PstI9.5 µL1 µL1 µL3 µL3 µL12.5 µL30 µL
NC0.5 µL0 µL0 µL1 µL1 µL7.5 µL10 µL
DNASpeIPstIBufferBSAMilliQtotal
9/11 DT SpeI & PstI10.4 µL1 µL1 µL3 µL3 µL11.6 µL30 µL
NC0.5 µL0 µL0 µL1 µL1 µL7.5 µL10 µL
DNASpeIPstIBufferBSAMilliQtotal
9/6 apt12_1R-DT SpeI & PstI7.9 µL1 µL1 µL3 µL3 µL14.1 µL30 µL
NC0.4 µL0 µL0 µL1 µL1 µL7.6 µL10 µL
DNASpeIPstIBufferBSAMilliQtotal
9/10 spinach-DT SpeI & PstI9.9 µL1 µL1 µL3 µL3 µL12.1 µL30 µL
NC0.5 µL0 µL0 µL1 µL1 µL7.5 µL10 µL

Liquid Culture

No name

Samplemedium
pSB1C3
DT
Spinach-DT 　

Sep 16

Liquid Culture

No name

Samplemedium
pSB1C3+P-KaiBC①Plusgrow medium(+CP)
pSB1C3+P-KaiBC②Plusgrow medium(+CP)

Colony PCR

No name

Samplebase pair
pSB1C3+P-KaiBC①283
pSB1C3+P-KaiBC②283
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
2min30s30s18s30cycles

Gel Extraction

No name

LaneDNAEnzyme
11kb ladder
2pT181 attenuatorXbaI+PstI
3pT181 attenuatorXbaI+PstI
4pT181 attenuatorXbaI+PstI
6pSB1C3EcoRI+SpeI
7pSB1C3EcoRI+SpeI
8pSB1C3EcoRI+SpeI
10pSB4K5XbaI+PstI
11pSB4K5XbaI+PstI
12pSB4K5XbaI+PstI

Igku 9161.png
Igku 9161a.png

Nameconcentration[µg/mL]260/280260/230
pT181 attenuator(XbaI+PstI)8.21.620.11
pSB1C3(EcoRI+SpeI)49.51.480.18
pSB4K5(XbaI+PstI)18.11.730.57
DT(SpeI+PstI)70.41.361.02
aptamer12-1R-DT(SpeI+PstI)42.01.820.47
spinach-DT(SpeI+PstI)36.81.790.61

Miniprep

No name

DNAconcentration[µg/mL]260/280260/230
Dx-lux1136.71.461.28
RBS-laczα-DT237.61.881.70
pSB4K5148.41.551.09
Pcon-KBS-tetR-DT126.61.731.36
9/11Plac1160.01.781.28
9/15Plac2144.01.731.81
9/14Plac332.51.512.33
Ptet-RBS-laczα-DT231.31.841.57

PCR

No name

DNA9/6pT181 antisense psB1C3(1)KOD plus10x bufferdNTPMgSO4GG1_1C3_fwd primerGG2_1C3_rev primerMilliQtotal
400pg10.52.52.51.50.750.7515.525
800pg20.52.52.51.50.750.7514.525
1000pg2.50.52.52.51.50.750.751425
nega00.52.52.51.50.750.7516.525
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C57°C68°C--
2min10s30s30s30cycles
DNA9/6 12-1R aptamer-DTKOD plus10x bufferdNTPMgSO4GG1_1C3_fwd primerGG1_1C3_rev primerMilliQtotal
400pg10.52.52.51.50.750.7515.525
800pg20.52.52.51.50.750.7514.525
1000pg2.50.52.52.51.50.750.751425
NC00.52.52.51.50.750.7516.525
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C57°C68°C--
2min10s30s30s30cycles

Electrophoresis

Yoshida

LaneSample
1100bp ladder
2antisense 1000pg
3antisense 800pg
4antisense 400pg
5tet aptamer-DT 1000pg
6tet aptamer-DT 800pg
7tet aptamer-DT 400pg
8NC

Igku 9162.png

Restriction Enzyme Digestion

Tatsui

9/16 Pcon-RBS-tetR-DT(µL)EcoRI(µL)SpeI(µL)XbaI(µL)PstI(µL)Buffer(µL)BSA(µL)MilliQ(µL)total(µL)
2 cuts15.80011336.230
NC0.80000117.210
9/16 pSB4K5(µL)EcoRI(µL)SpeI(µL)XbaI(µL)PstI(µL)Buffer(µL)BSA(µL)MilliQ(µL)total(µL)
2 cuts13.50011338.530
NC0.70000117.310
8/22 pSB1C3(µL)EcoRI(µL)SpeI(µL)XbaI(µL)PstI(µL)Buffer(µL)BSA(µL)MilliQ(µL)total(µL)
2 cuts18.51001333.530
NC0.90000117.110
8/22 pSB1C3(µL)EcoRI(µL)SpeI(µL)XbaI(µL)PstI(µL)Buffer(µL)BSA(µL)MilliQ(µL)total(µL)
2 cuts18.51100333.530
NC0.90000117.110
8/21 pT181 attenuator2(µL)EcoRI(µL)SpeI(µL)XbaI(µL)PstI(µL)Buffer(µL)BSA(µL)MilliQ(µL)total(µL)
2 cuts7.411003314.630
NC0.40000117.610

Ligation

Tatsui

stateVectorInserterLigation High ver.2
experimentPcon-pT181antisense-spinach-DT(XbaI+PstI)5.0µg/mLpSB4K5(XbaI+PstI)18.1µg/mLxx µg/mL
experimentpC181 attenuator(XbaI+PstI)8.2µg/mLpSB1C3(XbaI+PstI)26.0µg/mLxx µg/mL

Transformation

tatsui

NameSampleCompetent CellsTotalPlate
Pcon+RBS-laczα-DT µL µL µL
Pcon-pT181 attenuator-DT+pSB1C3 µL µL µL
pT181 attenuator(EcoRI&SpeI)+pSB1C3 µL µL µL
Pcon-aptamer12_1R-DT(EcoRI&SpeI)+pSB1C3 µL µL µL
Pcon-spinach-DT(EcoRI&SpeI)+pSB1C3 µL µL µL
Pcon-RBS-tetR-DT(EcoRI&SpeI)+pSB1C3 µL µL µL
Pcon-pT181 antisense-spinach-DT(XbaI&PstI)+pSB1C3 µL µL µL

Miniprep

tatsui

DNAconcentration[µg/mL]260/280260/230
9/15 pSBiC3111.71.791.36
9/15 spinach-DT113.91.571.38
9/15 DT77.02.051.92

PCR

tatsui

9/14 aptamer12-P(VF)Big Dye5×BufferprimerMilliQtotal
1.50.51.750.55.7510.5
9/14 aptamer12-1M(VF)Big Dye5×BufferprimerMilliQtotal
1.70.51.750.56.0510.5
9/14 Fusion1 attenuator(VF)Big Dye5×BufferprimerMilliQtotal
2.30.51.750.55.4510.5
9/14 Fusion1 antisense(VF)Big Dye5×BufferprimerMilliQtotal
1.30.51.750.56.4510.5
9/13 Fusion3m2 attenuator(VF)Big Dye5×BufferprimerMilliQtotal
1.70.51.750.56.0510.5
9/13 Fusion6 antisense(VF)Big Dye5×BufferprimerMilliQtotal
1.60.51.750.56.1510.5
9/12 plux(VR)Big Dye5×BufferprimerMilliQtotal
2.40.51.750.55.3510.5
9/9 Ptet antisense(VF)Big Dye5×BufferprimerMilliQtotal
2.20.51.750.55.5510.5
9/12 Pcon-pT181 attenuator-DT(VF)Big Dye5×BufferprimerMilliQtotal
1.40.51.750.56.3510.5
9/12 Pcon-pT181 attenuator-DT(VR)Big Dye5×BufferprimerMilliQtotal
1.40.51.750.56.3510.5
PreDenatureDenatureAnnealingExtensioncycle
96°C96°C50°C60°C--
2min10s5s4min30cycles

Sep 17

Liquid Culture

Tatsui and Hirano

Samplemedium
Spinach-DTLB medium(+CP)
Pcon-Spinach-DTLB medium(+Amp)　
Pcon-pT181 antisense-Spinach-DTLB medium(+Amp)　

PCR

Hirano

Pcon-tetR aptamer-DT(VF)BigDye5x bufferprimer(VF)MilliQtotal
1.30.51.750.50.4510.5
Pcon-tetR aptamer-DT(VR)BigDye5x bufferprimer(VR)MilliQtotal
1.30.51.750.50.4510.5
PreDenatureDenatureAnnealingExtensioncycle
96 °C96 °C50 °C60 °C--
2 min10 sec5 sec4 min30 cycles

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
11kbp ladder----
2Pcon-RBS-tetR-DTXbaIPstI
3Pcon-RBS-tetR-DT----
4pSB4K5XbaIPstI
5pSB4K5----
6pSB1C3EcoRISpeI
7pSB1C3EcoRIPstI
8pSB1C3----
9pT181 attenuatorEcoRISpeI
10pT181 attenuator----
111kbp ladder----

Igku 9171.jpg

Ligation

No Name

stateVectorInserterLigation High ver.2
experiment8/28 Plux (SpeI & PstI)5.0 µL9/13 RBS-lysis1-DT (XbaI & PstI)3.5 µL3.5 µL
experiment8/28 Plux (SpeI & PstI)5.0 µL9/13 RBS-lysis2-DT (XbaI & PstI)3.6 µL3.5 µL
experiment8/28 Plux (SpeI & PstI)11.0 µL9/13 RBS-lysis3-DT (XbaI & PstI)9.5 µL3.5 µL
experiment9/14 Plac (SpeI & PstI)2.4 µL9/16 pT181 attenuator-DT (XbaI & PstI)4.8 µL3.5 µL
experiment9/14 Plac (SpeI & PstI)2.4 µL9/3 pT181 antisense (XbaI & PstI)2.0 µL2.2 µL
experiment9/14 Plac (SpeI & PstI)2.4 µL9/10 aptamer 12_1R-DT (XbaI & PstI)1.8 µL2.1 µL
experiment9/13 pSB1C3 (XbaI & PstI)2.0 µL9/15 Pcon-PT181 antisense-Spinach-DT (XbaI & PstI)12.0 µL3.5 µL
experiment9/16 pSB1C3 (EcoRI & SpeI)1.0 µL9/15 Pcon-Spinach-DT (EcoRI & SpeI)4.1 µL2.5 µL
experiment9/16 pSB1C3 (EcoRI & SpeI)1.0 µL9/15 Pcon-RBS-tetR-DT (EcoRI & SpeI)2.0 µL1.5 µL
experiment9/16 pSB1C3 (EcoRI & SpeI)1.0 µL9/15 Pcon-PT181 attenuator-DT (EcoRI & SpeI)2.4 µL1.7 µL
experiment9/16 pSB1C3 (EcoRI & SpeI)1.0 µL9/15 Pcon-aptamer 12_1R-DT (EcoRI & SpeI)2.0 µL1.5 µL
experiment9/13 pSB1C3 (XbaI & PstI)2.0 µL9/16 Pcon-pT181-attenuator (XbaI & PstI)4.4 µL3.2 µL

Gel Extraction

No Name

LaneDNAEnzyme
11kbp ladder--
2pT181 attenuator -(2)EcoRI & SpeI
3
4

Igku 9172.jpg
Igku 9173.jpg

Nameconcentration[µg/mL]260/280260/230
pT181 attenuator (EcoI & SpeI)9.21.150.12
LaneDNAEnzyme
11kbp ladder--
2pSB1C3EcoRI & SpeI
3
4

Igku 9174.jpg
Igku 9175.jpg

Nameconcentration[µg/mL]260/280260/230
pSB1C310.01.840.40
LaneDNAEnzyme
11kbp ladder--
2pSB4K5XbaI & PstI
3
4

Igku 9176.jpg
Igku 9177.jpg

Nameconcentration[µg/mL]260/280260/230
pSB4K57.61.810.32
LaneDNAEnzyme
11kbp ladder--
2Pcon-RBS-tetR-DTXbaI & PstI
3
4

Igku 9178.jpg
Igku 9179.jpg

Nameconcentration[µg/mL]260/280260/230
Pcon-RBS-tetR-DT8.02.401.14
LaneDNAEnzyme
11kbp ladder--
2pSB1C3EcoRI & PstI
3
4

Igku 91710.jpg
Igku 91711.jpg

Nameconcentration[µg/mL]260/280260/230
pSB1C39.12.170.35

Mutation PCR

Kinose

pC KaiABC(100pg/µL)Prime STAR(2x)Primer(KaiA-BsaI MuT-Fwd)Primer(KaiA-BsaI MuT-Rev)MilliQtotal
1.025.01.01.022.050.0
1.525.01.01.021.550.0
2.025.01.01.021.050.0
PreDenatureDenatureAnnealingExtensioncycle
96 °C98 °C65 °C72 °C--
5 min10 s15 s40 s30 cycles

Sep 18

Restriction Enzyme Digestion

No name

9/18Plux SpeIPstIBufferBSAMilliQtotal
2cuts3.2µL1µL1µL3µL3µL18.8µL 30µL
NC1.7µL0µL0µL1µL1µL6.3µL 10µL
9/18Pcon-pT181antisense-spinach-DT②XbaIPstIBufferBSAMilliQtotal
2cuts7.5µL1µL1µL3µL3µL14.5µL 30µL
NC1.4icro;L0µL0µL1µL1µL6.6µL 10µL
9/16 Plac②SpeIPstIBufferBSAMilliQtotal
2cuts13.8µL1µL1µL3µL3µL14.5µL 30µL
NC0.7µL0µL0µL1µL1µL7.3µL 10µL
9/16 Spinach-DTXbaIPstIBufferBSAMilliQtotal
2cuts17.6µL1µL1µL3µL3µL4.4µL 30µL
NC0.9µL0µL0µL1µL1µL7.1µL 10µL
9/8 Pcon J23100SpeIPstIBufferBSAMilliQtotal
2cuts5.7µL1µL1µL3µL3µL16.3µL 30µL
NC0.3µL0µL0µL1µL1µL7.7µL 10µL
9/16 RBS-lacZα-DTXbaIPstIBufferBSAMilliQtotal
2cuts8.4µL1µL1µL3µL3µL13.6µL 30µL
NC0.4µL0µL0µL1µL1µL7.6µL 10µL
9/11 Pcon-pT181antisense-spinach-DT&#9312:XbaIPstIBufferBSAMilliQtotal
2cuts7.2µL1µL1µL3µL3µL14.8µL 30µL
NC0.4µL0µL0µL1µL1µL7.6µL 10µL
9/6 apt12-1R-DTXbaIPstIBufferBSAMilliQtotal
2cuts7.9µL1µL1µL3µL3µL14.1µL 30µL
NC0.4µL0µL0µL1µL1µL7.6µL 10µL
9/3 pT181antisenseXbaIPstIBufferBSAMilliQtotal
2cuts6.3µL1µL1µL3µL3µL15.7µL 30µL
NC0.3µL0µL0µL1µL1µL7.7µL 10µL
9/14 pSB4K5EcoRISpeIBufferBSAMilliQtotal
2cuts7.3µL1µL1µL3µL3µL14.7µL 30µL
NC0.4µL0µL0µL1µL1µL7.6µL 10µL
8/31 Plux-RBS-GFP-DT EcoRISpeIBufferBSAMilliQtotal
2cuts9.2µL1µL1µL3µL3µL12.8µL 30µL
NC0.5µL0µL0µL1µL1µL7.5µL 10µL
9/12 RBS-lysis3-DT XbaIPstIBufferBSAMilliQtotal
2cuts5.2µL1µL1µL3µL3µL16.8µL 30µL
NC0.3µL0µL0µL1µL1µL7.6µL 10µL
9/18 Pcon-pT181attenuatorEcoRISpeIBufferBSAMilliQtotal
2cuts14.3µL1µL1µL3µL3µL7.7µL 30µL
NC0.7µL0µL0µL1µL1µL7.3µL 10µL

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
1Plae② SpeIPstI
2RBS-lysis3-DTXbaIPstI
3Plux-RBS-GFP-DTEcoRISpeI
4PSB4K5EcoRISpeI
5PconSpeIPstI
6PluxSpeIPstI
71kbp ladder----
8Plae② ----
9RBS-lysis3-DT----
10Plux-RBS-GFP-DT----
11PSB4K5----
12Pcon----
13Plux----

Igku 9183.jpg

Electrophoresis

Okazaki

LaneSampleEnzyme1Enzyme2
1100bp ladder -- --
2Pcon pT181antisense spinach-DT②XbaIPstI
3Spinach-DTXbaIPstI
4RBS-lacZα-DT XbaIPstI
5Pcon pT181antisense-Spinach-DT① XbaIPstI
6aptamer12-1R-DTXbaIPstI
7pT181attenuatorXbaIPstI
8Pcon attenuatorEcoRISpeI

Igku 9181.jpg

LaneSampleEnzyme1Enzyme2
1100bp ladder -- --
2Pcon pT181antisense spinach-DT②XbaIPstI
3Spinach-DTXbaIPstI
4RBS-lacZα-DTXbaIPstI
5Pcon pT181antisense-Spinach-DT①XbaIPstI
6apt12-1R-DTXbaIPstI
7pT181attenuatorXbaIPstI
8Pcon attenuatorEcoRISpeI
LaneSampleEnzyme1Enzyme2
1100bp ladder -- --
2Pcon pT181antisense spinach-DT②----
3Spinach-DT----
4RBS-lacZα-DT----
5Pcon pT181antisense-Spinach-DT①----
6apt12-1R-DT----
7pT181attenuator----
8Pcon attenuator----

Igku 9182.jpg

Gel Extraction

No name

LaneDNAEnzyme
11kbp ldder--
2Plac②SpeI+PstI
3Plac②SpeI+PstI
4----
5RBS-lysis3-DTXbaI+PstI
6RBS-lysis3-DTXbaI+PstI
7----
8Plux-RBS-GFP-DTEcoRI+SpeI
9Plux-RBS-GFP-DTEcoRi+SpeI
10----
11PSB4KSEcoRI+SpeI
12PSB4KSEcoRI+SpeI

Igku 9188.jpg
Igku 9189.jpg

LaneDNAEnzyme
11kbp ldder--
2PconSpeI+PstI
3 PconSpeI+PstI
4----
5PluxSpeI+PStI
6 PluxSpeI+PStI

Igku 9184.jpg
Igku 9185.jpg

LaneDNAEnzyme
1Pcon PT181 antisense spinach-DTXbaI+PstI
2 Pcon PT181 antisense Spinach-DTXbaI+PstI
3100bp ladder --
4Pcon PT181 antisense Spinach-DTXbaI+PstI
5 Pcon PT181 antisense Spinach-DTXbaI+PstI
6 1kbp ladder--

Igku 9186.jpg
Igku 9187.jpg

LaneDNAEnzyme
1100bp ladder --
2apt12-1R-DTXbaI+PstI
3apt12-1R-DTXbaI+PstI
4----
5PT181 attenuatorXbaI+PstI
6PT181 attenuatorXbaI+PstI

Igku 91810.jpg
Igku 91811.jpg

LaneDNAEnzyme
1100bp ladder --
2Pcoa attenuatorEcoRI+PstI
3Pcoa attenuatorEcoRI+PstI
4----
5RBS-lacZα-DTXbaI+PstI
6RBS-lacZα-DT XbaI+PstI

Igku 91812.jpg
Igku 91813.jpg

Sep 19

Sep 20

Ligation

Okazaki, Kanaoka, Tatsui and Hirano

stateVectorInserterLigation High ver.2
experiment9/18 Plac (SpeI & PstI)3.0 µL9/18 RBS-lysis3-DT (XbaI & PstI)12.4 µL3.5 µL
experiment9/12 Ptet-pT181 antisense1.0 µL9/18 spinach-DT11.9 µL3.5 µL
experiment9/13 Pcon-RBS-luxR-DT2.1 µL9/10 Plux-RBS-GFP-DT5.6 µL3.5 µL
experiment9/13 Pcon-pT181 attenuator (SpeI & PstI)2.0 µL9/18 aptamer 12-1R-DT (XbaI & PstI)6.3 µL3.5 µL
experiment9/18 Plac ②(SpeI & PstI)3.0 µL9/18 aptamer 12-1R-DT(XbaI & PstI)4.5µLx3.5 µL

Transformation

Tatsui

NameSampleCompetent CellsPlate
9/17 pSB1C3-Pcon-pT181 attenuator-DT2 µL20 µLCP
9/17 pSB1C3-Pcon-aptamer 12_1R-DT2 µL20 µLCP
9/17 pSB1C3-Pcon-antisense-spinach-DT2 µL20 µLCP
9/17 pSB1C3-Pcon-RBS-tetR-DT x22 µL20 µLCP
9/14 pSB1C3-PkaiBC2 µL20 µLCP
9/14 pSB1C3-RpaB2 µL20 µLCP
9/16 pSB1C3-pT181 attenuator(EcoRI&SpeI) 2 µL20 µLCP
9/17 pSB1C3-pT181 attenuator(XbaI&PstI) 2 µL20 µLCP
9/17 pSB1C3-Pcon-spinach-DT2 µL20 µLCP
9/14 RBS-GFP-DT-PkaiBC2 µL20 µLCP
Plux-RBS-lysis3-DT2 µL20 µLCP
9/20 Ptet-pT181 antisense-spinach-DT2 µL20 µLCP
9/20 Pcon-spinach-DT(pSB4K5) 2 µL20 µLKan
9/20 Pcon-pT181 antisense-spinach-DT(pSB4K5) 2 µL20 µLKan
9/20 KaiABC2 µL20 µLKan
9/20 KaiABC2 µL20 µLKan
9/20 Plac-RBS-lysis3-DT2 µL20 µLAmp
9/20 Plac-aptamer 12_1R2 µL20 µLAmp
9/20 Plux-RBS-GFP-DT-Pcon-RBS-luxR-DT2 µL20 µLAmp
Plac-aptamer12_1R-DT2 µL20 µLAmp
9/20 Plac-aptamer12_1R-DT2 µL20 µLAmp
9/20Pcon-pT181attenuator-aptamer-DT2 µL20 µLAmp
9/17 Plac-pT181-attenuatorx22 µL20 µLAmp
9/15 Pcon-RBS-lacZα-DT2 µL20 µLAmp
9/14 Pcon-pT181attenuator-RBS-lacZα-DT2 µL20 µLAmp
PUC192 µL20 µLAmp

PCR

Tatsui

Samplebase pair
pT181 attenuator(pSB1C3)--
Pcon-RBS-lacZα-DT--
PreDenatureDenatureAnnealingExtensioncycle
94 °C94 °C55 °C68 °C--
5 min30 s30 s3 min30 cycles

Electrophoresis

No name

LaneSampleEnzyme
1Pcon-RBS-lacZα-DT--
21kb ladder--
3pSB1C3-pT181attenuator --

Sep 21

Electrophoresis

no name

LaneSampleEnzyme1Enzyme2
1Pcon-RBS-lacZα-DT----
21kbp ladder----
3pSB1C3+pT181 attenuator----

Igku 9211.jpg

Liquid Culture

No name

Samplemedium
9/2 spinach-DT-2--
8/16 Plux-4--
8/27 Plux-RBS-GFP-DT-1--
9/5 Pcon-pT181 attenuator-1--

Master Plate

No name

NumberUse LB plate
18/27 Pλ-luxI-1(+Amp)
29/10 Plac(BBa-R0011)-3(+Amp)
39/5 Pcon-RBS-tetR-DT-1(+Amp)
49/5 Pcon-pT181 antisense-1(+Amp)
59/15 Pcon-pT181 attenuator(+Amp)
69/- Pcon-apt12_1R-DT-1(+Amp)
79/5 Pcon-spinach-DT-3(+Amp)
88/27 Pcon-RBS-GFP-DT-Pcon-RBS-luxR-DT(+Amp)
NumberUse LB plate
19/4 pT181 antisense(pSB1C3)-1(+CP)
29/11 Fusion1 antisense(pSB1C3)-4(+CP)
39/11 Fusion6 antisense(pSB1C3)-1(+CP)
49/4 pT181 attenuator(pSB1C3)-1(+CP)
59/10 Fusion1 attenuator(pSB1C3)-1(+CP)
69/11 Fusion3n2 attenuator(pSB1C3)-1(+CP)
79/4 aptamer12_1R(pSB1C3)-1(+CP)
89/11 aptamer12_P(pSB1C3)-3(+CP)
99/11 aptamer12_1M(pSB1C3)-1(+CP)
NumberUse LB plate
18/29 Pbad/araC-RBS-RFP-DT-1(+CP)
29/4 RBS-lysis3-DT-3(+CP)
39/10 Pcon-pT181 attenuator-DT-1(+CP)
49/5 Ptet-pT181 antisense-1(+CP)
59/4aptamer 12_1R-DT-1(+CP)
69/5 Ptet-RBS-lacZα-DT-4(+CP)
78/10 RBS-lacI-DT-2(+CP)
88/29 Plux-RBS-GFP-DT-1(+CP)

Restriction Enzyme Digestion

Noname

8/17 RBS-DTEcoRISpeIXbaIPstI BufferBSAMilliQtotal
2cuts(XbaI&PstI)8.10µL0µL1µL1µL3µL3µL13.9µL30µL
NC0.40µL0µL0µL0µL1µL1µL7.6µL10µL
8/17 RBS-DTEcoRISpeIXbaIPstIBufferBSAMilliQtotal
2cut(EcoRI&XbaI)16.21µL1µL0µL0µL3µL3µL5.8µL30µL
9/15 Pcon-pT181attenuatorEcoRISpeIXbaIPstI BufferBSAMilliQtotal
2cuts(EcoRI&PstI)121µL0µL0µL1µL3µL3µL10µL30µL
NC0.60µL0µL0µL0µL1µL1µL7.4µL10µL
8/20 Pcon=RBS-GFP-DT-1EcoRISpeIXbaIPstIBufferBSAMilliQtotal
2cut(EcoRI&XbaI)61µL0µL1µL0µL3µL3µL0µL14µL
NC0.30µL0µL0µL0µL1µL1µL7.3µL10µL
incubate 37°C 1hour

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
1RBS-GFP-DTXbaIPstI
2RBS-GFP-DT----
3RBS-GFP-DTEcoRIXbaI
41kbp ladder----
5Pcon-pT181attenuatorSpeIPstI
6Pcon-pT181attenuator----
7Pcon-RBs-GFP-DTEcoRIXbaI
8Pcon-RBs-GFP-DT----

Igku 9212.jpg

PCR

Nakamoto

Samplebase pair
Pcon-apt12_1R-DT(E-1A)50
Pcon-spinach-DT(E-1A)402
Pcon-pT181 attenuator(E-1A)400
DT(1-3)65
tetR aptamer 12_1R-DT(1-3)283
Pcon-pT181 attenuator(1-SA)303
spinach-DT(2-1)338
Pcon-pT181 attenuator(E-0)403
Pcon-pT181 attenuator(2-SA)403
spinach-DT(3-2)395
Ptet-PT181 anisense 400
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C57°C68°C--
2min10s30s35s30cycles

Gel Extraction

No name

LaneDNAEnzyme
11kb ladder--
3RBS-GFP-DTXbaI&PstI
4RBS-GFP-DTXbaI&PstI
5RBS-GFP-DTXbaI&PstI

Igku 9213.jpg
Igku 9214.jpg

Nameconcentration[µg/mL]260/280260/230
RBS-GFP-DT(XbaI&PstI)14.81.740.47
RBS-GFP-DT(EcoRI&SpeI)88.41.851.49
Pcon-RBS-GFP-DT(EcoRI&XbaI)47.51.821.12

Sep 22

Colony PCR

Tatsui

Samplebase pair
9/20 Plac-PT181 attenuator(1~4)664
9/20 Plac-PT181 antisense(1~6)369
9/20 Pcon-PT181 attenuator-aptamer-DT(1~6)859
9/20 Pcon-PT181 antisense-spinach-DT(1~8)723

Igku 0903 E1.jpg
Igku 0922 E2.jpg

Electrophoresis

Nakamoto

LaneSample
1Pcon-apt12_1R-DT(E-1A)
2Pcon-spinach-DT(E-1A)
3Pcon-pT181 attenuator(E-1A)
4DT(1-3)
5tetR aptamer 12_1R-DT(1-3)
6100bp ladder
7Pcon-pT181 attenuator(1-SA)
8spinach-DT(2-1)
9Pcon-pT181 attenuator(E-0)
10Pcon-pT181 attenuator(2-SA)
11spinach-DT(3-2)
12Ptet-PT181 anisense

Igku 0922 E3.jpg
Igku 0922 E4.jpg

Miniprep

Honda

DNAconcentration[µg/mL]260/280260/230
9/21 spinach-DT197.91.671.54
9/21 Plux125.91.711.27
9/21 Plux-RBS-GFP-DT-(1)168.01.621.73
9/21 Pcon-PT181 attenuator-(1)368.31.571.62
9/21 RBS-GFP-DT532.81.751.78
9/21 Pcon-RBS-GFP-DT845.22.021.70

Colony PCR

Honda

Samplebase pair
9/20 Plux-RBS-GFP-DT-Pcon-RBS-luxR-DT-(1~2)283
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s2min10s30cycles

Igku 0922 E5.jpg
Igku 0922 E6.jpg

Gel Extraction

No name

LaneDNAEnzyme
1100bp ladder--
2~4Pcon-aptamer12-1R-DT(E-1A)--
6~8Pcon-spinach-DT(E-1A)--
10~12Pcon-attenuator PT181(E-1A)--
LaneDNAEnzyme
1100bp ladder--
2~4DT(1-3)--
6~8aptamer12-1RDT(1-3)--
10~12Pcon-PT181 attenuator(1-5A)--
Nameconcentration[µg/mL]260/280260/230
Pcon-aptamer12-1R-DT(E-1A)31.21.870.30
Pcon-spinach-DT(E-1A)25.61.880.99
Pcon-attenuator PT181(E-1A)25.31.990.86
DT(1-3)22.11.820.20
aptamer12-1RDT(1-3)33.51.990.05
Pcon-PT181 attenuator(1-5A)36.91.120.96

Igku 0922 G1.jpg
Igku 0922 G2.jpg
Igku 0922 G3.jpg
Igku 0922 G4.jpg

Restriction Enzyme Digestion

No name

9/16 PlacXbaISpeIPstIBufferBBSAMilliQtotal
2cuts12.5µL0µL1µL1µL3µL3µL9.5µL30µL
NC0.8µL0µL0µL0µL1µL1µL7.4µL10µL
9/22 PluxXbaISpeIPstIBufferBBSAMilliQtotal
2cuts16µL0µL1µL1µL3µL3µL14µL30µL
NC0.8µL0µL0µL0µL1µL1µL7.2µL10µL
9/20 RBS-tetR-2R-DT XbaISpeIPstIBufferDBSAMilliQtotal
2cuts8.5µL1µL0µL1µL3µL3µL13.5µL30µL
NC0.4µL0µL0µL0µL1µL1µL7.6µL10µL

Igku 0922 E7.jpg

Gel Extraction

No name

LaneDNAEnzyme
1100bp ladder--
2~4Pcon-attenuator PT181(E-0)--
LaneDNAEnzyme
1100bp ladder--
2~4Pcon-attenuator PT181(E-0)--
LaneDNAEnzyme
2100bp ladder--
4~6Pcon-attenuator PT181(2-SA)--
LaneDNAEnzyme
1100bp ladder--
3~5spinach-DT(3-2)--
8~10Ptet-antisense PT181(1-3)--
Nameconcentration[µg/mL]260/280260/230
Pcon-attenuator PT181(E-0)2.41.800.17
Pcon-attenuator PT181(2-SA)10.81.850.54
spinach-DT(3-2)36.71.800.75
Ptet-antisense PT181(1-3)16.81.740.78

Igku 0922 G5.jpg
Igku 0922 G6.jpg
Igku 0922 G7.jpg
Igku 0922 G8.jpg
Igku 0922 G9.jpg
Igku 0922 G10.jpg

Colony PCR

No name

Samplebase pair
9/20 Pcon-PT181 attenuator-aptamer12-1R-DT-(7~14)859

Colony PCR

Tatsui

Samplebase pair
PconPT181antisense-spinach-DT(9~15)723

Igku 0922 E8.jpg

Ligation

No name

stateVectorInserterLigation High ver.2
experiment9/13 Pcon-PT181 attenuator (SpeI & PstI)1.7 µL9/18 aptamer-1R-DT (XbaI & PstI)5.4µL 3.5µL
experiment9/12 Plac(SpeI & PstI)2.4 µL9/18 aptamer-1R-DT (XbaI & PstI)6.1µL3.5 µL
experiment9/13 Ptet(SpeI & PstI)1.7 µL9/10 Plux-RBS-GFP-DT7.0 µL4.4 µL
experiment9/21 RBS-GFP-DT (EcoRI & XbaI)0.6 µL9/18 aptamer 12-1R-DT (XbaI & PstI)5.6µL3.1 µL
experiment9/13 PSB1C3(XbaI & PstI)1.9 µL9/16 PT181 attenuator(XbaI & PstI)4.6µL3.3 µL

Liquid Culture

No name

Samplemedium
9/20 Plac-antisense-1--
9/20 Plac-attenuator-1--
9/20 Plac-antisense-1--
9/20 Plac-attenuator-1--

digestion

No name

9/8 Pcon-PT181 antisenseEcoRISpeI BufferMMilliQtotal
2cuts8.3µL1µL1µL3µL16.7µL30µL
NC0.4µL0µL0µL1µL8.3µL10µL
9/7 Pcon-RBS-tetR-DTEcoRIXbaISpeIPstIBufferDBSAMilliQtotal
2cut14µL1µL1µL0µL0µL3µL3µL18.2µL30µL
NC0.7µL0µL0µL0µL0µL1µL1µL7.3µL10µL
9/22 RBS-GFP-DTEcoRIXbaISpeIPstI BufferDBSAMilliQtotal
2cuts3.8µL1µL1µL0µL0µL3µL3µL18.2µL30µL
NC0.2µL0µL0µL0µL0µL1µL1µL7.8µL10µL
9/6 aptamer12-1R-DTEcoRIXbaISpeIPstIBufferDBSAMilliQ
2cut8µL1µL1µL0µL0µL3µL3µL16µL
NC0.4µL0µL0µL0µL0µL1µL1µL7.6µL
incubate 37°C 1hour

Igku 0922 E9.jpg

Gel Extraction

No name

LaneDNAEnzyme
11kbp ladder--
3~5Pcon-PT181 antisense (EcoRI&SpaI)--

Igku 0922 G11.jpg
Igku 0922 G12.jpg

Transformation

No name

NameSampleCompetent CellsPlate
9/20 Plac-aptamer12-1R-DT(XbaI&PstI)2 µL20 µLAmp
9/20 pT181 attenuator(1C3)(EcoRI&SpeI)2 µL20 µLCP
9/17 pT181 attenuator(1C3)(XbaI&PstI)2 µL20 µLCP
9/20 Pcon-pT181 attenuator(SpaI&PstI)＋aptamer12-1R-DT(XbaI&PstI)2 µL20 µLAmp
kaiABC2 µL20 µLAmp
9/22 pSB1C3-RpaB(XbaI&PstI)2 µL20 µLAmp
9/22 pSB1C3-pT181 attenuator(XbaI&PstI) 2 µL20 µLAmp
9/22 Ptet(SpaI&PstI)＋RBS-GFP-DT(XbaI&PstI)2 µL20 µLCP
9/22 RBS-GFP-DT＋Pcon-attenuator pT181 (EcoRI&SpeI)2 µL20 µLCP
9/22 pT181 attenuator(XbaI&PstI)(1C3)2 µL20 µLCP

PCR

No name

Samplebase pair
Pcon-RBS-tetR-DT(1-2(A))--
Pcon-RBS-tetR-DT(3-2(A))--
Pcon-RBS-GFP-DT(1-S(A))--
Pcon-RBS-GFP-DT(2-S(A))--
Pcon-tetR-DT(E-2(A))--
PreDenatureDenatureAnnealingExtensioncycle
94 °C94 °C57 °C68 °C--
2 min10 s30 s1min5s30 cycles

Igku 0922 E10.jpg

Electrophoresis

No name

LaneSample
11kbp ladder
2Pcon-RBS-tetR-DT(1-2Ⓐ)
3Pcon-RBS-tetR-DT(3-2Ⓐ)
4Pcon-RBS-GFP-DT(1-SⒶ)
5Pcon-RBS-GFP-DT(2-SⒶ)
6Pcon-tetR-DT(E-2Ⓐ)

Igku 0922 E10.jpg

Sep 23

Transformation

No name

NameSampleCompetent CellsPlate
9/14 pSB1C3＋PkaiBC2 µL20 µL--
9/14 pSB1C3＋RpaB2 µL20 µL--
9/14 pkaiBC＋Rbs-GFP-DT2 µL20 µL--

Gel Extraction

No Name

LaneDNAEnzyme
1 1kbp ladder
3-4 Pcon-RBS-tetR-PT

Igku Sep23 Gel Extraction1.jpg
Igku Sep23 Gel Extraction2.jpg

Nameconcentration[µg/mL]260/280260/230
Pcon-PT181 anti EcoRI+SpeI46.7 1.06 0.93
Pcon-RBS-tetR-DT EcoRI+XbaI119.3 0.99
RBS-8FP-DT EcoRI+XbaI87.7 1.00 0.98
ap42_1R-DT EcoRI+XbaI68.8 1.32 1.03
Pcon-RBS-tetR-DT 1-2 A203.0 1.66 1.79
Pcon-RBS-tetR-DT 3-2 A67.7 2.64 1.28
Pcon-RBS-GFP-DT 1-S A185.6 1.88 2.04
Pcon-RBS-GFP-DT 2-S A298.6 1.68 1.55
Pcon-tetR-DT E-2 A202.4 1.57 1.66


PCR

No name

genome DNAKOD plus10x bufferdNTPMgSO4SasA_fwd primerSasA_rev primerMilliQtotal
9/8 Pcon-pT181 anti 0.52.52.51.50.750.7515.5 25
8/20 Ptet(1) 0.52.52.51.50.750.7515.5 25
9/16 Plac 2 0.52.52.51.50.750.7515.5 25
PreDenatureDenatureAnnealingExtensioncycle
94 °C98 °C57 °C68 °C--
2min 10sec 30sec 20sec 30cycles

Ligation

Yoshida

stateVectorInserterLigation High ver.2
experiment9/21 Pcon-RBS-GFP-DT EcoRI+XbaI47.5 µg/mL9/15 Pcon-RBS-tetR-DT EcoRI+SpeI59 µg/mL2.3 µg/mL
experiment9/21 Pcon-RBS-GFP-DT EcoRI+XbaI47.5 µg/mL9/15 Pcon-apt12_1R-DT EcoRI+SpeI17 µg/mL2.4 µg/mL
experiment9/21 Pcon-RBS-GFRDT EcoRI+XbaI2.1 µg/mL9/15 Pcon-DT181 attenuator-DT21 µg/mL3.0 µg/mL
experiment9/22 apt 12_1R-DT EcoRI+XbaI67.8 µg/mL9/18 Pcon-DT181 attenuator EcoRI+SpeI5.7 µg/mL3.5 µg/mL
experiment9/22RBS-GFP-DT EcoRI+XbaI87.7 µg/mL9/18 Pcon-DT18 attenuator EcoRI+SpeI6.7 µg/mL3.5 µg/mL
experiment9/13 DT EcoRI+XbaI25.4 µg/mL9/22 Pcon-DT181 antisense EcoRI+SpeI46.8 µg/mL2.3 µg/mL


PCR

No name

genome DNAKOD plus10x bufferdNTPMgSO4SasA_fwd primerSasA_rev primerMilliQtotal
Pcon-attenuator-DT (E-1A) 0.52.52.51.50.750.7577.5 25
Pcon-attenuator-DT (2-1A) 0.52.52.51.50.750.7577.5 25
Pcon-antisense-spinach-DT (1-2A) 0.52.52.51.50.750.7577.5 25
Pcon-antisense-spinach-DT (E-1A) 0.52.52.51.50.750.7577.5 25
PreDenatureDenatureAnnealingExtensioncycle
94 °C98 °C57 °C68 °C--
2min 10s 30s 45s 30cycle


Miniprep

No name

DNAconcentration[µg/mL]260/280260/230
9/22 Plac-DT181 attenuator-1204.0 2.00 1.95
9/22 Plac-DT181 attenuator-2195.3 1.84 1.62
9/22 Plac-DT181 antisense-1188.2 1.87 2.15
9/22 Plac-DT181 antisense-2165.0 1.95 2.66

Electrophoresis

Hirano

LaneSampleEnzyme1Enzyme2
1,6100bp ladder(Promega)----
2Pcon-attenuator-DT (E-1A)----
3Pcon-attenuator-DT (2-1A)----
4Pcon-antisense-spinach-DT (1-2A)----
5Pcon-antisense-spinach-DT (E-1A)----

Igku 9232.png

Gel Extraction

Hirano

Nameconcentration[µg/mL]260/280260/230
Pcon-pT181 attenuator-DT(E-1A)166.7 1.51 0.99
Pcon-pT181 attenuator-DT(2-1A)122.1 1.75 1.20
Pcon-pT181 antisense-spinach-DT(1-2A)99.1 1.86 1.81

Electrophoresis

Hirano

LaneSampleEnzyme1Enzyme2
1100bp ladder----
29/8 Pcon-pT181 antisense(E-1A)----
38/20 Ptet(1)(2-S)----
49/16 Plac2(E-1A)----

Igku 9231.png

Transformation

No Name

NameSampleCompetent CellsTotalPlate
pSB1C32µL20µL22µL


Ligation

No Name

stateVectorInserterLigation High ver.2
experiment9/19 Plac SpeI+PstI24.6 µg/mL9/18 ape12_1R-DT XbaI+PstI4.5 µg/mL2 µg/mL
experiment9/21 RBS-GFP-DT EcoRI+XbaI88.4 µg/mL9/15 Pcon-pT181 attenuator EcoRI+SpeI21 µg/mL2 µg/mL
experiment9/13 PSB1C3 XbaI+PstI26.0 µg/mL9/16 pT181 attenuator XbaI+PstI8.2 µg/mL2 µg/mL


Colony PCR

Tatsui

Samplebase pair
9/22 Pcon-PT181 attenuator12-1R-DT(1~8)--
9/22 Pcon-PT181 attenuator(1~4)--
9/20 Plac＋aptamer12-1R-DT(1~4)--
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s54s30cycle

Sep 24

Electrophoresis

No name

LaneSample
19/20 Pcon-pT181aptamer12-1R-DT 1
29/20 Pcon-pT181aptamer12-1R-DT 2
39/20 Pcon-pT181aptamer12-1R-DT 3
49/20 Pcon-pT181aptamer12-1R-DT 4
59/20 Pcon-pT181aptamer12-1R-DT 5
69/20 Pcon-pT181aptamer12-1R-DT 6
79/20 Pcon-pT181aptamer12-1R-DT 7
89/20 Pcon-pT181aptamer12-1R-DT 8
9100bp ladder
109/22 Pcon-pT181attenuator-aptamer12-1R-DT 1
119/22 Pcon-pT181attenuator-aptamer12-1R-DT 2
129/22 Pcon-pT181attenuator-aptamer12-1R-DT 3
139/22 Pcon-pT181attenuator-aptamer12-1R-DT 4
149/20 Plac-aptamer12-1R-DT 1
159/20 Plac-aptamer12-1R-DT 2
169/20 Plac-aptamer12-1R-DT 3
179/20 Plac-aptamer12-1R-DT 4

Igku 0924 E1.jpg

Transformation

No name

NameSampleCompetent CellsTotalPlate
9/23 pSB1C3-pT181attenuator 2 µL20 µL 22 µLLB (+CP)
RBS-GFP-DT+Pcon-pT181attenuator 2 µL20 µL 22 µLLB (+CP)
9/23 Plac-+spinach-DT2 µL20 µL 22 µLLB (+Amp)
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 2 µL20 µL 22 µLLB (+Amp)
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 2 µL20 µL 22 µLLB (+Amp)
9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 2 µL20 µL 22 µLLB (+Amp)
9/23 Pcon-pT181antisense-DT 2 µL20 µL 22 µLLB (+CP)
9/23 Pcon-pT181attenuator-RBS-GFP-DT 2 µL20 µL 22 µLLB (+CP)
9/23 Pcon-pT181attenuator-aptamer12-1R-DT 2 µL20 µL 22 µLLB (+CP)
PUC192 µL20 µL 22 µLLB (+Amp)
Ptet-RBS-lacZα-DT 2 µL20 µL 22 µLLB (+CP)
pSB1C32 µL20 µL 22 µLLB (+CP)

Liquid Culture

No name

Samplemedium
aptamer12-1R-DT(Master plate) LB(+CP)

Restriction Enzyme Digestion(PCR product)

Hirano

Concentrate PCR product by evaporator until volume become less than 1µL, add all sulution and pipetting.
10*cut smart bufferBsaI-HFMilliQtotal
9/22 Pcon-pT181 attenuator (E-0) 2.0 µL 0.3 µL 17 µL 19.3µL
9/22 Pcon-pT181 attenuator (2-3A) 2.0 µL 0.3 µL 17 µL 19.3µL
9/22 spinach-DT(3-2) 2.0 µL 0.3 µL 17 µL 19.3µL
9/22 Ptet-pT181 antisense (1-3) 2.0 µL 0.3 µL 17 µL 19.3µL
9/22 Pcon-tet aptamer-DT (E-1A) 2.0 µL 0.3 µL 17 µL 19.3µL
9/22 Pcon-spinach-DT (E-1A) 2.0 µL 0.3 µL 17 µL 19.3µL
9/22 Pcon-pT181 attenuator (E-1A) 2.0 µL 0.3 µL 17 µL 19.3µL
9/22 DT (1-3) 2.0 µL 0.3 µL 17 µL 19.3µL
9/22 tet aptamer-DT (1-3) 2.0 µL 0.3 µL 17 µL 19.3µL
9/22 Pcon-pT181 attenuator (1-SA) 2.0 µL 0.3 µL 17 µL 19.3µL
9/22 Pcon-RBS-tetR-DT (1-2A) 2.0 µL 0.3 µL 17 µL 19.3µL
9/22 Pcon-RBS-tetR-DT (3-2A) 2.0 µL 0.3 µL 17 µL 19.3µL
9/22 Pcon-RBS-tetR-DT (E-2A) 2.0 µL 0.3 µL 17 µL 19.3µL
9/22 Pcon-RBS-GFP-DT (1-2A) 2.0 µL 0.3 µL 17 µL 19.3µL
9/22 Pcon-RBS-GFP-DT (2-SA) 2.0 µL 0.3 µL 17 µL 19.3µL
9/23 Pcon-pT181 attenuator-DT (E-1A) 2.0 µL 0.3 µL 17 µL 19.3µL
9/23 Pcon-pT181 attenuator-DT (0-1A) 2.0 µL 0.3 µL 17 µL 19.3µL
9/23 Pcon-pT181 antisense-spinach-DT (1-2A) 2.0 µL 0.3 µL 17 µL 19.3µL
9/23 Pcon-pT181 antisense (1-2A) 2.0 µL 0.3 µL 17 µL 19.3µL
9/23 Ptet-1 (2-S) 2.0 µL 0.3 µL 17 µL 19.3µL
9/23 Plac-2 (E-1A) 2.0 µL 0.3 µL 17 µL 19.3µL
Incubate 37°C 12hour

Column Refining

Kojima

Nameconcentration[µg/mL]260/280260/230
8/22 Pcon-pT181 attenuator(E-0)7.81.700.03
8/22 Pcon-pT181 attenuator(2-SA)17.31.930.03
8/22 spinach-DT (3-2)22.41.930.09
8/22 Ptet-PT181 anisense (1-3)18.31.580.14
8/22 Pcon-aptamer12_1R-DT (E-1A)8.22.100.05
8/22 Pcon-spinach-DT(E-1A)5.60.910.05
8/22 Pcon-pT181 attenuator(E-1A)18.51.800.12
8/22 DT(1-3)7.31.130.04
8/22 tetR aptamer 12_1R-DT(1-3)14.61.740.06
8/22 Pcon-pT181 attenuator(1-SA)7.91.570.04
8/22 Pcon-RBS-tetR-DT(1-2Ⓐ)41.61.810.25
8/22 Pcon-RBS-tetR-DT(3-2Ⓐ)30.21.750.15
8/22 Pcon-RBS-tetR-DT(E-2Ⓐ)36.91.750.56
8/22 Pcon-RBS-GFP-DT(1-SⒶ)43.51.830.33
8/22 Pcon-RBS-GFP-DT(2-SⒶ)64.61.820.30
8/23 Pcon-pT181 attenuator-DT (E-1A)31.21.830.09
8/23 Pcon-pT181 attenuator-DT (0-1A)64.51.800.43
8/23 Pcon-pT181 antisense-spinach-DT (1-2A)36.41.880.21
8/23 Pcon-pT181 antisense-spinach-DT (E-1A)27.91.920.16
8/23 Ptet(1)(2-S)37.01.910.16
9/23 Plac-2(E-1A) 21.21.820.13

Transformation

No name

NameSampleCompetent CellsTotalPlate
9/23 Pcon-attenuator-aptamer12-1R-DT 2 µL20 µL 22 µLLB (+CP)
9/23 pT181attenuator(1C3) 2 µL20 µL 22 µLLB (+CP)
9/23 Pcon-pT181antisense-DT2 µL20 µL 22 µLLB (+CP)
9/23 Pcon-pT181attenuator-RBS--GFP-DT 2 µL20 µL 22 µLLB (+CP)

Liquid Culture

No name

Samplemedium
pSB1C3 (Master plate-1) LB(+CP)

Restriction Enzyme Digestion

Kojima

8/21 pT181attenuator (330 µg/mL) XbaIPstIbuffer BSAMilliQtotal
2 cuts6.1113315.930
NC0.300117.710
9/22 Pcon-pT181attenuator (368.3 µg/mL) EcoRISpeIbuffer BSAMilliQtotal
2 cuts5.4113019.630
NC0.300108.710
9/16 pSB1C3EcoRISpeIbuffer BSAMilliQtotal
2 cuts1811307.030
NC0.900108.110

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
11kb ladder--
2pT181attenuator XbaIPstI
3 pT181attenuator ----
4Pcon-pT181attenuator EcoRISpeI
5 Pcon-pT181attenuator ----
6pSB1C3 EcoRISpeI
7 pSB1C3 ----

Igku 0924 E2.jpg

Miniprep

No name

DNAconcentration[µg/mL]260/280260/230
9/24 aptamer12-1R-DT144.71.031.21

Gel Extraction

Kojima

LaneDNAEnzyme1Enzyme2
1100kb ladder----
2pT181attenuatorXbaIPstI
3 pT181attenuatorXbaIPstI
4 pT181attenuatorXbaIPstI
5------
6Pcon-pT181attenuatorEcoRISpeI
7 Pcon-pT181attenuatorEcoRISpeI
8 Pcon-pT181attenuatorEcoRISpeI
9------
10pSB1C3EcoRISpeI
11 pSB1C3EcoRISpeI
12 pSB1C3EcoRISpeI

Igku 0924 G1.jpg
Igku 0924 G2.jpg

Nameconcentration[µg/mL]260/280260/230
pT181attenuator(XbaI&PstI)10.1 2.02 0.43
Pcon-pT181attenuator(EcoRI&SpeI)10.3 1.81 0.32
pSB1C3(EcoRI&SpeI)10.02.810.02

Colony PCR

Nakamoto

Samplebase pair
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 11414
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 21414
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 31414
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 41414
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 51414
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 61414
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 71414
9/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 81414
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 11631
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 21631
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 31631
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 41631
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 51631
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 61631
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 71631
9/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 81631
9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 12042
9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 22042
9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 32042
9/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 42042
9/23 Pcon-pT181antisense-DT 1508
9/23 Pcon-pT181antisense-DT 2508
9/23 Pcon-pT181antisense-DT 3508
9/23 Pcon-pT181antisense-DT 4508
9/23 Pcon-pT181antisense-DT 5508
9/23 Pcon-pT181antisense-DT 6508
9/23 Pcon-pT181antisense-DT 7508
9/23 Pcon-pT181antisense-DT 8508
PreDenatureDenatureAnnealingExtensioncycle
94 °C94 °C55 °C68 °C--
5 min30 sec30 sec2min30 cycles
LaneSample
19/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 1
29/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 2
39/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 3
49/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 4
59/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 5
69/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 6
79/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 7
89/23 Pcon-aptamer12-1R-DT+Pcon-RBS-GFP-DT 8
91kb ladder
109/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 1
119/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 2
129/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 3
139/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 4
149/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 5
159/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 6
169/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 7
179/23 Pcon-pT181attenuator-DT+Pcon-RBS-GFP-DT 8

Igku 0924 E3.jpg

LaneSample
19/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 1
29/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 2
39/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 3
49/23 Pcon-RBS-tetR-DT+Pcon-RBS-GFP-DT 4
59/23 Pcon-pT181antisense-DT 1
69/23 Pcon-pT181antisense-DT 2
71kb ladder
89/23 Pcon-pT181antisense-DT 3
99/23 Pcon-pT181antisense-DT 4
109/23 Pcon-pT181antisense-DT 5
119/23 Pcon-pT181antisense-DT 6
129/23 Pcon-pT181antisense-DT 7
139/23 Pcon-pT181antisense-DT 8
14--

Igku 0924 E4.jpg

Restriction Enzyme Digestion

Nakamoto

9/24 aptamer12-1R-DT EcoRIXbaI bufferBSAMilliQtotal
2cuts17.5µL1.0µL 1.0µL 3.0µL 3.0µL 4.5µL 30µL
NC0.9µL 0µL 0µL 1.0µL 1.0µL 7.1µL 10µL
9/20 PtetSpeIPstI bufferBSAMilliQtotal
2cuts9.5µL1.0µL 1.0µL 3.0µL 0µL 15.5µL 30µL
NC0.5µL 0µL 0µL 1.0µL 0µL 8.5µL 10µL
9/22 RBS-GFP-DTEcoRI SpeI bufferBSAMilliQtotal
2cuts3.8µL1.0µL 1.0µL 3.0µL 0µL 21.2µL 30µL
NC0.2µL 0µL 0µL 1.0µL 0µL 8.8µL 10µL
9/22 Pcon-RBS-GFP-DTEcoRI XbaI bufferBSAMilliQtotal
2cuts2.4µL1.0µL 1.0µL 3.0µL 3.0µL 19.6µL 30µL
NC0.1µL 0µL 0µL 1.0µL 1.0µL 7.9µL 10µL
9/22 Pcon-pT181attenuator 1 EcoRI XbaI bufferBSAMilliQtotal
2cuts5.4µL1.0µL 1.0µL 3.0µL 3.0µL 16.6µL 30µL
NC0.3µL 0µL 0µL 1.0µL 1.0µL 7.7µL 10µL
9/22 RBS-GFP-DTXbaI PstI bufferBSAMilliQtotal
2cuts3.8µL1.0µL 1.0µL 3.0µL 3.0µL 18.2µL 30µL
NC0.2µL 0µL 0µL 1.0µL 1.0µL 7.8µL 10µL

Ligation

Nakamoto

stateVectorInserterLigation High ver.2
experiment9/22 aptamer12-1R-DT(EcoRI&XbaI) 1.4 µL9/24 Pcon-pT181attenuator(EcoRI&SpeI)26 µL3.5 µL
experiment9/4 Ptet(SpeI&PstI)2.0 µL9/21 RBS-GFP-DT(XbaI&PstI)9.0 µL3.5 µL
  • incubated at 37 °C for 1 hour.

colony PCR

Kojima

Samplebase pair
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 52042bp
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 62042bp
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 72042bp
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 82042bp
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 92042bp
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 102042bp
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 112042bp
9/23 Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 122042bp
9/23 Pcon-pT181antisense-DT 9508bp
9/23 Pcon-pT181antisense-DT 10508bp
9/23 Pcon-pT181antisense-DT 11508bp
9/23 Pcon-pT181antisense-DT 12508bp
9/23 Pcon-pT181antisense-DT 13508bp
9/24 Pcon-pT181attenuator-RBS-GFP-DT 11527bp
9/24 Pcon-pT181attenuator-RBS-GFP-DT 21527bp
9/24 Pcon-pT181attenuator-RBS-GFP-DT 31527bp
9/24 Pcon-pT181attenuator-RBS-GFP-DT 41527bp
9/24 Pcon-pT181attenuator-aptamer12-1R-DT 1859bp
9/24 Pcon-pT181attenuator-aptamer12-1R-DT 2859bp
9/24 Pcon-pT181attenuator-aptamer12-1R-DT 3859bp
9/24 Pcon-pT181attenuator-aptamer12-1R-DT 4859bp
9/24 pT181attenuator(pSB1C3) 1390bp
9/24 pT181attenuator(pSB1C3) 2390bp
9/24 pT181attenuator(pSB1C3) 3390bp
9/24 pT181attenuator(pSB1C3) 4390bp

Tatsui

PreDenatureDenatureAnnealingExtensioncycle
　94°C　94°C　55°C　68°C--
5min30s　30s　2min30cycle

Miniprep

No name

DNAconcentration[µg/mL]260/280260/230
pSB1C3183.9　1.91　1.59
Pcon-pT181attenuator-aptamer12-1R-DT231.71.822.35
Plac-aptamer12-1R-DT208.91.981.79

Electrophoresis

Nakamoto

LaneSampleEnzyme1Enzyme2
11kb ladder----
29/24 aptamer12-1R-DTEcoRIXbaI
3 9/24 aptamer12-1R-DT -- --
49/24 Ptet SpeI PstI
59/24 Ptet ----
69/24 RBS-GFP-DT EcoRISpeI
79/24 RBS-GFP-DT -- --
89/24 Pcon-RBS-GFP-DT EcoRIXbaI
89/24 Pcon-RBS-GFP-DT ----
99/24 Pcon-RBS-GFP-DT ----
109/24 Pcon-pT181attenuator 1 EcoRIXbaI
119/24 Pcon-pT181attenuator 1 ----
129/24 RBS-GFP-DT XbaIPstI
139/24 RBS-GFP-DT ----
141kb ladder----

Igku 0924 E5.jpg

Gel Extraction

No name 1

LaneDNAEnzyme1Enzyme2
11kb ladder----
2aptamer12-1R-DTEcoRIXbaI
3aptamer12-1R-DTEcoRIXbaI
4aptamer12-1R-DTEcoRIXbaI
5-- -- --
6PtetSpeIPstI
7PtetSpeIPstI
8PtetSpeIPstI
9------
10RBS-GFP-DTEcoRISpeI
11RBS-GFP-DTEcoRISpeI
12RBS-GFP-DTEcoRISpeI

Igku 0924 G3.jpg
Igku 0924 G4.jpg

LaneDNAEnzyme1Enzyme2
11kb ladder----
2Pcon-RBS-GFP-DTEcoRIXbaI
3Pcon-RBS-GFP-DTEcoRIXbaI
4Pcon-RBS-GFP-DTEcoRIXbaI
5------
6Pcon-pT181attenuatorEcoRIXbaI
7Pcon-pT181attenuatorEcoRIXbaI
8Pcon-pT181attenuatorEcoRIXbaI
9------
10RBS-GFP-DTXbaIPstI
11RBS-GFP-DTXbaIPstI
12RBS-GFP-DTXbaIPstI

Igku 0924 G5.jpg
Igku 0924 G6.jpg

Electrophoresis

No name
1

LaneSample
11kb ladder
2Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 5
3Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 6
4Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 7
5Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 8
6Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 9
7Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 10
81kb ladder


2

LaneSample
11kb ladder
2Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 11
3Pcon-RBS-tetR-DT-Pcon-RBS-GFP-DT 12
4Pcon-pT181attenuator-RBS-GFP-DT 1
5Pcon-pT181attenuator-RBS-GFP-DT 2
6Pcon-pT181attenuator-RBS-GFP-DT 3
7Pcon-pT181attenuator-RBS-GFP-DT 4
81kb ladder

Igku 0924 E6.jpg


3

LaneSample
1Pcon-pT181antisense-DT 9
2 Pcon-pT181antisense-DT 10
3 Pcon-pT181antisense-DT 11
4 Pcon-pT181antisense-DT 12
5 Pcon-pT181antisense-DT 13
6100bp ladder
7Pcon-pT181attenuator-aptamer12-1R-DT 1
8 Pcon-pT181attenuator-aptamer12-1R-DT 2


4

LaneSample
1Pcon-pT181attenuator-aptamer12-1R-DT 3
2 Pcon-pT181attenuator-aptamer12-1R-DT 4
3 100bp ladder
4 pT181attenuator(pSB1C3) 1
5 pT181attenuator(pSB1C3) 2
6 pT181attenuator(pSB1C3) 3
7 pT181attenuator(pSB1C3) 4

Igku 0924 E7.jpg

Ligation

Hirano

stateVectorInserter1Inserter2Inserter3Inserter4(NEB)T4ligase(NEB)BuffermilliQtotal
experimentRBS-GFP-DT(pSB1C3)1µLtetR-aptamer12-1R-DT(1-3)1 µLPtet(2-S)0.3µLPcon-tetR-aptamer12-1R-DT(3-2A)1.1µLPlac(E-1A)0.5µL0.5µL0.5µL0.2 µL5µL
experimentRBS-GFP-DT(pSB1C3)1µLPcon-pT181attenuator(E-1A)1 µLPcon-tetR-aptamer12-1R-DT(1-2A)1µLPtet(2-S)0.3µL-- -- 0.5µL0.5µL0.7 µL5µL
experimentRBS-GFP-DT(pSB1C3)1µLPcon-tetRaptamer12-1R-DT(E-1A)2µLPcon-tetRaptamer12-1R-DT(1-2A)1µLPtet(2-S)0.3µL----0.5µL0.5µL0 µL5.3µL
experimentRBS-GFP-DT(pSB1C3)1µLPcon-spinach-DT(E-1A)3µLPcon-tetRaptamer12-1R-DT(1-2A)1µLPtet(2-S)0.3µL----0.5µL0.5µL0 µL6.3µL
experimentRBS-GFP-DT(pSB1C3)1µLPcon-pT181antisense-DT(E-1A)0.5µLDT(1-3)2µLPtet(2-S)0.3µLPcon-tetRaptamer12-1R-DT(3-2A)1.1µL0.5µL0.5µL0.1 µL5µL
experimentRBS-GFP-DT(pSB1C3)1µLPcon-pT181attenuator(E-1A)1µLtetRaptamer12-1R-DT(1-3)1µLPtet(2-S)0.3µLPcon-tetRaptamer12-1R-DT(3-2A)1.1µL0.5µL0.5µL0µL5.4µL
experimentRBS-GFP-DT(pSB1C3)1µLPtet(2-S)0.3µLPcon-tetRaptamer12-1R-DT(E-2A)1µL--------0.5µL0.5µL1.7µL5µL
experimentRBS-GFP-DT(pSB1C3)1µLPcon-GFP-DT(2-SA)0.5µL1µL----------0.5µL0.5µL1.5µL5µL
experimentRBS-GFP-DT(pSB1C3)1µLPcon-tetRaptamer12-1R-DT(E-1A)2µLPcon-pT181attenuator(1-SA)2.1µL--------0.5µL0.5µL0µL6.1µL
experimentRBS-GFP-DT(pSB1C3)1µLPcon-pT181attenuator-DT(E-1A)1µLPcon-pT181attenuator(1-SA)2.1µL--------0.5µL0.5µL0µL5.1µL
experimentRBS-GFP-DT(pSB1C3)1µLPcon-tetR12-1R-DT(E-1A)2µLPcon-GFP-DT(2-SA)0.5µLspinach-DT(3-2)1µLPtet-pT181antisense(1-3)1µL0.5µL0.5µL0µL6.5µL
experimentRBS-GFP-DT(pSB1C3)1µLPcon-tetRaptamer12-1R-DT(E-1A)2µLPcon-pT181antisense(1-2A)0.7µLPcon-pT181attenuator(2-SA)2.1µL----0.5µL0.5µL0µL6.8µL
experimentRBS-GFP-DT(pSB1C3)1µLPcon-tetRaptamer12-1R-DT(E-1A)2µLPcon-pT181attenuator(2-SA)2.1µLSpinach-DT(3-2)1µLPtet-pT181antisense(1-3)1µL0.5µL0.5µL0µL8.1µL


Concentrate total (more than 5µL) by evaporater until volume become less than 5 µL

PCR

No name

genome DNAKOD plus10x bufferdNTPMgSO4F primerR primerKOD-plusMilliQtotal
Pcon antisense(1C3-GGE-fwd,1A2-GG2-rev)1µL2.5µL2.5µL1.5µL0.75µL0.75µL0.5µL15.525µL
DT-(1C3-GG1-fwd,1C3-GG2-rev)1µL2.5µL2.5µL1.5µL0.75µL0.75µL0.5µL15.525µL
Spinach-DT-(1C3-GG1-fwd,1C3-GG2-rev)1µL2.5µL2.5µL1.5µL0.75µL0.75µL0.5µL15.525µL
Pcon-antisense-spinach-DT(1C3-GGE-fwd,1A2-GG1-rev) 1µL2.5µL2.5µL1.5µL0.75µL0.75µL0.5µL15.525µL
Ptet(1C3-GGE-fwd,1C3-GG0-rev) 1µL2.5µL2.5µL1.5µL0.75µL0.75µL0.5µL15.525µL
Pcon attenuator(1C3-GGE-fwd,1C3-GG0-rev) 1µL2.5µL2.5µL1.5µL0.75µL0.75µL0.5µL15.525µL
DT(1C3-GG2-fwd,1C3-GG1-rev)1µL2.5µL2.5µL1.5µL0.75µL0.75µL0.5µL15.525µL
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C57°C68°C--
2min10sec30sec36sec30

Sep 25

Electrophoresis

nakamoto

LaneSampleEnzyme1Enzyme2
1100bp ladder----
2Pcon-antisense (1C3-66E-fwd A2-662 rev)----
3DT (1C3-661-fwd 1C3 662 rev)----
4spinach-DT(1C3-661-fwd 1C3 662 rev)----
5Pcon-antisense-spinach-DT(1C3-66E fwd A2-661 rev)----
6100bp ladder----

Igku 9251a.jpg

LaneSampleEnzyme1Enzyme2
2100bp ladder----
3Ptet (1C3 66E fwd-1C3 660 rev)----
4Pcon-attenuator(1C3-660 fwd A2-661 rev)----
5DT(1C3 662 fwd-1C3 661 rev)----
6Pcon-attenuator-aptamer-DT(1C3 66E fwd-A2-661-rev)----
7100bp ladder----

Igku 9251b.jpg

Colony PCR

Nakamoto

Samplebase pair
9/23 Pcon-PT181 attenuator-aptamer12-1R-DT-(5~12)859
9/23 Pcon-PT181 attenuator-RBS-GFP-DT-(5~8)1527
9/23 Pcon-PT181 attenuator-pSB1C3-(5~8)601
PreDenatureDenatureAnnealingExtensioncycle
94 °C94 °C55 °C68 °C--
2 min30 s30 s1min5s30 cycles


Electrophoresis

no name

LaneSampleEnzyme1Enzyme2
1Pcon-PT181 attenuator-aptamer12-1R-DT-5----
2Pcon-PT181 attenuator-aptamer12-1R-DT-6----
3Pcon-PT181 attenuator-aptamer12-1R-DT-7----
4Pcon-PT181 attenuator-aptamer12-1R-DT-8----
5Pcon-PT181 attenuator-aptamer12-1R-DT-9----
6Pcon-PT181 attenuator-aptamer12-1R-DT-10----
7Pcon-PT181 attenuator-aptamer12-1R-DT-11----
8Pcon-PT181 attenuator-aptamer12-1R-DT-12----
91kbp ladder----
10Pcon-PT181 attenuator-RBS-GFP-DT-5----
11Pcon-PT181 attenuator-RBS-GFP-DT-6----
12Pcon-PT181 attenuator-RBS-GFP-DT-7----
13Pcon-PT181 attenuator-RBS-GFP-DT-8----
14Pcon-PT181 attenuator-pSB1C3-5----
15Pcon-PT181 attenuator-pSB1C3-6----
16Pcon-PT181 attenuator-pSB1C3-7----
17Pcon-PT181 attenuator-pSB1C3-8----

Igku 9252.jpg

Transformation

kojima and nakamoto

NameSampleCompetent CellsTotalPlate
Plac+tet aptamer12+Pcon tetR-DT+Ptet+RBS-GFP-DT1µL10µL11µL--
Pcon-pT181attenuator-DT+Pcon-tetR-DT+Ptet+RBS-GFP-DT1µL10µL11µL--
Pcon-tet aptamer12-DT+Pcon-tetR-DT+Ptet+RBS-GFP-DT1µL10µL11µL--
Pcon-Spinach-DT+Pcon-tetR-DT+Ptet+RBS-GFP-DT1µL10µL11µL--
Pcon-pT181antisense+DT+Pcon-tetR-DT+Ptet+RBS-GFP-DT1µL10µL11µL--
Pcon-pT181attenuator+tet aptamer12-DT+Pcon-tetR-DT+Ptet+RBS-GFP-DT1µL10µL11µL--
Pcon tetR-DT+Ptet+RBS-GFP-DT1µL10µL11µL--
Pcon tetR-DT+Pcon-GFP-DT+RBS-GFP-DT1µL10µL11µL--
Pcon-tet aptamer12-DT+Pcon-pT181attenuator+RBS-GFP-DT1µL10µL11µL--
Pcon-pT181attenuator-DT+Pcon-pT181attenuator+RBS-GFP-DT1µL10µL11µL--
Pcon-tet aptamer12-DT+Ptet-pT181antisense+Spinach-DT+Pcon-RBS-GFP-DT+RBS-GFP-DT1µL10µL11µL--
Pcon-tet aptamer12-DT+Pcon-pT181antisense+Spinach-DT+Pcon-pT181attenuator+RBS-GFP-DT1µL10µL11µL--
Pcon-tet aptamer12-DT+Ptet-pT181antisense+Spinach-DT+Pcon-pT181attenuator+RBS-GFP-DT1µL10µL11µL--

NameSampleCompetent CellsTotalPlate
aptamer12-DT(EcoRI+XbaI)+Pcon-pT181attenuator(EcoRI+SpeI)2µL20µL22µL--
Ptet(SpeI+PstI)+RBS-GFP-DT(XbaI+PstI))2µL20µL22µL--

Ligation

nakamoto

stateVectorInserterLigation High ver.2
experiment9/25 aptamer12_1R-DT(EcoRI&XbaI)4.5 µLPcon-pT181 attenuator(EcoRI&SpeI)7.7 µL6.1 µL
experiment9/17 pSB1C3(EcoRI&SpeI)2.0 µL9/3 pT181 attenuator(EcoRI&SpeI)14 µL8 µL
experiment9/25 Pcon-pT181 attenuator(EcoRI&XbaI)4.5 µL9/25 RBS-GFP-DT(EcoRI&SpeI)19.3 µL11.9 µL
experiment9/25 Pcon-RBS-GFP-DT(EcoRI&XbaI)4.2 µLPcon-RBS-tetR-DT(EcoRI&SpeI)20 µL12.1 µL

Restriction Enzyme Digestion

nakamoto>

9/24 Pcon-attenuator-aptamer-DT(µL)EcoRI(µL)SpeI(µL)Buffer(µL)MilliQ(µL)total(µL)
2 cuts8.611316.430
NC0.40018.610
9/11 DT(µL)EcoRI(µL)XbaI(µL)Buffer(µL)BSA(µL)MilliQ(µL)total(µL)
2 cuts10.4113311.630
NC0.500117.510

Liquid Culture

Kojima

Samplemedium
DT --
pT181attenuator --
Pcon-RBS-tetR-DT --

Gel Extraction

Kojima

LaneDNAEnzyme
1100bp ladder--
2Pcon-pT181antisense--
3
4
5----
6DT--
7
8
9----
10Spinach-DT--
11
12

Igku 9253.jpg
Igku 9254.jpg

LaneDNAEnzyme
1100bp ladder--
3Pcon-antisense-spinach-DT(E-1A)--
4Pcon-antisense-spinach-DT(E-1A)--
5Pcon-antisense-spinach-DT(E-1A)--

Igku 9255.jpg
Igku 9256.jpg

LaneDNAEnzyme
7100bp ladder--
9Ptet(E-0)--
10Ptet(E-0)--
11Ptet(E-0)--

Igku 9257.jpg
Igku 9258.jpg

LaneDNAEnzyme
13100bp ladder--
15Pcon-attenuator(0-1A)--
16Pcon-attenuator(0-1A)--
17Pcon-attenuator(0-1A)--

Igku 9259.jpg
||-- Igku 92510.jpg

LaneDNAEnzyme
19100bp ladder--
21Pcon-attenuator-aptamer-DT(E-1A)--
22Pcon-attenuator-aptamer-DT(E-1A)--
23Pcon-attenuator-aptamer-DT(E-1A)--

Igku 92511.jpg
Igku 92512.jpg

Nameconcentration[µg/mL]260/280260/230
DT(2-1)24.91.730.37
Ptet(E-0)12.81.590.90
Pcon-antisense(E-2A)16.51.721.07
Pcon-attenuator-aptamer-DT(E-1A)22.11.750.94
Pcon-antisense-spinach-DT(E-1A)31.01.781.15
spinach-DT(1-2)23.41.851.11
Pcon-attenuator(0-1A)22.51.760.46
DT(1-2)25.11.781.21

Electrophoresis

LaneSampleEnzyme1Enzyme2
11kbp ladder----
2Pcon-pT181 attenuator-aptamer 12_1R-DTEcoRISpeI
3Pcon-pT181 attenuator-aptamer 12_1R-DT----
4DTEcoRIXbaI
5DT----

Igku 92513.jpg

Gel Extraction

Nakamoto

LaneDNAEnzyme
1DTEcoRI+XbaI
2DTEcoRI+XbaI
3Pcon-pT181 attenuator-aptamer12_1R-DTEcoRI+SpeI
4Pcon-pT181 attenuator-aptamer12_1R-DTEcoRI+SpeI
5Pcon-pT181 attenuator-aptamer12_1R-DTEcoRI+SpeI

Igku 92514.jpg
Igku 92515.jpg

Nameconcentration[µg/mL]260/280260/230
Pcon-pT181 attenuator-aptamer12_1R-DT(EcoRI&SpeI)11.81.800.46
DT(EcoRI+XbaI)42.61.840.98

Liquid Culture

No name

Samplemedium
Pcon-pT181anntisense-spinach-DT(4k5)-

Ligation

Nakamoto and Tatsui

stateVectorInserterLigation High ver.2
experiment9/24 pSB1C3(EcoRI+Spel)10µL9/25 PconpT181attenuator-aptamer12-1R-DT(EcoRI+Spel)5.9µL8µL

PCR

Hirano

800pg/μL DTKOD plus10x bufferdNTPMgSO41C3_GG2_fwdiC3_GG1_revMilliQtotal
10.52.52.51.50.750.7515.525
PreDenatureDenatureAnnealingExtensioncycle
94°C98°C57°C68°C--
2min10s30s24s30cycles

Transformation

Tatsui

NamePlate
aptamer12-1R-DTCP
pSB1C3(EcoRI+SpeI)+pT181attenuator(EcoRI+Spal)CP
Pcon-pT181attenuator(EcoRI+XbaI)+RBS-GFP-DT(EcoRI+SpeI)Amp
Pcon-RBS-GFP-DT(EcoRI+XbaI)+Pcon-RBS-tetR-DT(EcoRI+SpeI)Amp
pSB1C3(EcoRI+SpeI)+Pcon-pT181attenuator-aptamer12-1R-DT(EcoRI+SpeI)CP

Electrophoresis

Hirano

LaneSampleEnzyme1Enzyme2
1100bp ladder----
2DT(2-1)----

Igku 92516.jpg

Column Refining

Nameconcentration[µg/mL]260/280260/230
8/25 DT(2-1)------

Restriction Enzyme Digestion

Concentrate PCR product by evaporator until volume become less than 3µL, add all sulution and pipetting.
9/25 DT(2-1)10*cut smart BufferBsaI-HFMilliQtotal
3.0 µL2.0 µL0.3 µL14.7 µL20.0 µL

Incubate 37°C 12hour

Liquid Culture

Hirano

Samplemedium
Pcon-GFP-DTLB(Amp)
RBS-GFP-DTLB(CP)
Pcon-spinach-DTLB(Amp)
Pcon-tetRaptamer-DTLB(Amp)
Pcon-attenuator-DTLB(Amp)
spinach-DTLB(CP)
Pcon-antisense-spinach-DTLB(Amp)

Plating

Hirano

Mix Liquid Culture 1mL and LB(+Amp), incubate 37°C 30min
OD600-1.445
SampleUse plateThe number of plate
Pcon-GFP-DTM9(+Amp)3
Pcon-GFP-DTLB(+Amp)3

</div> incubate 37°C

Colony PCR

No name

Samplebase pair
9/24 GGA-1(1~8)3177
9/24 GGA-2(1~8)3189
9/24 GGA-3(1~8)2989
9/24 GGA-4(1~8)2939
9/24 GGA-7(1)2489
GGA-16(1)2717
RBS-GFP-DT--
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s3min15s30cycles

RNA Extraction

Nakamoto
sumple 0.25mL
Add 1mLISOGEN-LS
Lysis or homogenization
Store for 5 min, at room temperature
Add 0.2mL Chloroform
Shake vigorously for 15sec.
Store for 2~3min.at room temperature
Centrifuge 12K*g for 15min. at 4°C
Extract aqueous phase
Add 0.5mL isopropanol
Store for 5~10min. at room temperature
Centrifuge 12K*g for 10min. at 4°C
Remove aqueous phase
Add at least 1mL 70% ethanol
Vortex
Centrifuge 7.5K*g for 5min. at 4°C
Remove aqueous phase
Dry briefly
Add ddH2O, TE(pH8.0) or 0.5% SDS
Dissolve
Total RNA solution

DNAconcentration[µg/mL]260/280260/230
Pcon-RBS-GFP-DT27831.951.48

Add 91.2µL, Store at freezer
Total 250µL</div>

cDNA　Synthesis

Nakamoto

volume7 µL
7*cDNA Wipcout Buffer1 µL
Template RNA(9/25 Pcon-RBS-GFP-DT)3 µL
RNase-frec water4 µL

Incubate 42°C 2min, then on ice immediately
Add

5*Quantiscript Reverse Transcriptase0.5 µL
5*Quantiscript RT Buffer2 µL
RT Primer Mix0.5µL

Incubate 42°C 15min, then incubate 95°C 3min

Electrophoresis

Nakamoto

LaneSampleEnzyme1Enzyme2
1100bp ladder----
2Pcon-RBS-GFP-DT(RNA)----

Igku 92517.jpg

Ligation

Tatsui

stateVectorInserterLigation High ver.2
experiment9/13 Pcon-pT181-attenuator(SpeI&PstI)3.5µL9/25 RBS-GFP-DT(XbaI&PstI)13.5µL8.5 µL

Sep 26

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
1GGA1-1----
2GGA1-2----
3GGA1-3----
4GGA1-4----
5GGA1-5----
6GGA1-6----
7GGA1-7----
8GGA1-8----
91kbp ladder----
10GGA2-1----
11GGA2-2----
12GGA2-3----
13GGA2-4----
14GGA2-5----
15GGA2-6----
16GGA2-7----
17GGA2-8----

Igku 0926 E1.jpg

LaneSampleEnzyme1Enzyme2
1GGA3-1----
2GGA3-2----
3GGA3-3----
41kbp ladder----
5GGA3-4----
6GGA3-5----
7GGA3-6----
8GGA3-7----
LaneSampleEnzyme1Enzyme2
1GGA3-8----
2GGA4-1----
3GGA4-2----
4GGA4-3----
51kbp ladder----
6GGA4-4----
7GGA4-5----
8GGA4-6----

Igku 0926 E2.jpg


LaneSampleEnzyme1Enzyme2
1GGA4-7----
2GGA4-8----
31kbp ladder----
4GGA7-1----
5GGA16-1----
6NC(RBS-GFP-DT)----

Igku 0926 E3.jpg

Ligasion(Golden Gate Assenbly)

No name

stateVectorInserter1Inserter2Inserter3Inserter4Inserter5(NEB)T4 ligase(NEB)10*T4 ligaseMilliQ
experimentRBS-GFP-DT1.0 µLPcon-pT181 antisense(E-2A)0.6µLDT(2-1)0.5µLPcon-pT181 attenuator(1-SA)2.1 µL--------0.5 µL0.5 µLup to 5 µL
experimentpSB1C3(EcoRI&SpeI)1.8 µLPcon-pT181 antisense(E-2A)0.9 µLDT(2-1)0.5 µLPcon-GFP-DT(1-SA)1.5 µL--------0.5 µL0.5 µLup to 5 µL
experimentRBS-GFP-DT1.0 µLPtet(E-0)0.7µLPcon pT181 attenuator(0-1A)0.3µLtet aptamer(1-3)0.3 µLPcon-tetR-DT(3-2A)1.1 µLPtet(2-5)0.3 µL0.5 µL0.5 µLup to 5 µL
experimentRBS-GFP-DT1.0 µLPtet(E-0)0.7 µLPcon-pT181 attenuator-DT(0-1A)1.0 µLPcon-tetR-DT(1-2A)1.0 µLPtet(2-5)0.3µL----0.5 µL0.5 µLup to 5 µL
experimentRBS-GFP-DT1.0 µLPcon-pT181 attenuator-tetR-DT(E-1A)1.0 µLPcon antisense-spinach-DT(1-2A)0.7µLPcon-pT 181 attenuator(2-SA)2.1 µL--------0.5 µL0.5 µLup to 5 µL
experimentRBS-GFP-DT1.0 µLPcon antisense(E-1A)0.5 µLspinach-DT(1-2)1.0 µLPcon-GFP-DT(1-2)0.8 µL--------0.5 µL0.5 µLup to 5 µL
experimentpSB1C3(EcoRI&SpeI)1.8 µLPcon-pT181 antisense(E-1A)0.8µLspinach-DT(1-2)1.0µLPcon-GFP-DT(2-SA)0.8 µL--------0.5 µL0.5 µLup to 5 µL
experimentpSB1C3(EcoRI&SpeI)1.8 µLPcon-tet aptamer-DT(E-1A)3.0 µLPtet-pT181 antisense(1-3)1.5 µLspinach-DT(3-2)1.5 µLPcon-GFP-DT(2-5A)0.8 µL----0.5 µL0.5 µLup to 5 µL

Liquid Culture

No name

Samplemedium
Pcon-GFP-DTAmp
RBS-GFP-DTCP
Pcon-pT181 attenuator-DTAmp
Pcon-tet aptamer-DTAmp
Pcon-spinach-DTAmp
Pcon-pT181 antisense-spinach-DTAmp
spinach-DTCP

BSAI Digestion

No name

DNAMilliQBSAI-HF10*cut smarttotal
Pcon7 µL10.7 µL0.3 µL2 µL20 µL
RBS-luxR-DT10 µL7 µL0.3 µL2 µL20 µL

Igku 0926 E4.jpg

Colony PCR

Tatsui

Samplebase pair
GGA-1 (9~16)3177
GGA-2 (9~16)3189
GGA-3 (9~21)2989
GGA-4 (9~16)2939
GGA-6 (1)3359
GGA-16(2~4)2939
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s3min30s30cycles

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
1GGA-1 (9)----
2GGA-1 (10)----
3GGA-1 (11)----
4GGA-1 (12)----
5GGA-1 (13)----
6GGA-1 (14)----
7GGA-1 (15)----
8GGA-1 (16)----
91kbp ladder----
10GGA-2 (9)----
11GGA-2 (10)----
12GGA-2 (11)----
13GGA-2 (12)----
14GGA-2 (13)----
15GGA-2 (14)----
16GGA-2 (15)----
17GGA-2 (16)----

Igku 0926 E6.jpg

LaneSampleEnzyme1Enzyme2
1GGA-4 (9)----
2GGA-4 (10)----
3GGA-4 (11)----
4GGA-4 (12)----
5GGA-4 (13)----
6GGA-4 (14)----
7GGA-4 (15)----
8GGA-4 (16)----
91kbp ladder----
10GGA-6 (1)----
11GGA-16 (2)----
12GGA-16 (3)----
13GGA-16 (4)----

Igku 0926 E7.jpg

LaneSampleEnzyme1Enzyme2
1GGA-3 (9)----
2GGA-3 (10)----
3GGA-3 (11)----
4GGA-3 (12)----
51kbp ladder----
6GGA-3 (13)----
7GGA-3 (15)----
8GGA-3 (14)----
LaneSampleEnzyme1Enzyme2
1GGA-3 (17)----
2GGA-3 (18)----
3GGA-3 (19)----
41kbp ladder----
5GGA-3 (20)----
6GGA-3 (21)----
7GGA-3 (16)----

Igku 0926 E8.jpg

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
1RBS-GFP-DT(RNA)----
21kbp ladder----
3Pcon-RBS-GFP-DT(RNA)----

Igku 0926 E9.jpg

Liquid Culture

Hirano

Samplemedium
Pcon-GFP-DT--
RBS-GFP-DT--
Pcon-tet aptamer-DT--
Pcon-spinach-DT--
Pcon-pT181 antisense-spinach-DT--
spinach-DT--

Colony PCR

Hirano

Samplebase pair
9/25 Pcon-pH181 attenuator+RBS-GFP-DT-1--
9/25 Pcon-pH181 attenuator+RBS-GFP-DT-2--
9/25 Pcon-pH181 attenuator+RBS-GFP-DT-3--
9/25 Pcon-pH181 attenuator+RBS-GFP-DT-4--
9/24 Ptet-RBS-GFP-DT-1--
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s1min40s30cycles

Ligasion(Golden Gate Assenbly)

Hirano

stateVectorInserter1Inserter2Inserter3Inserter4Inserter5
experimentGGA2Pcon-attenuator(E-1A)Pcon-tetR-DT(1-2A)Ptet(2-S)RBS-GFP-DT(EcoRI&XbaI)
experimentGGA3Pcon-tetap-DT(E-1A)Pcon-tetR-DT(1-2A)Ptet(2-S)RBS-GFP-DT(EcoRI&XbaI)
experimentGGA4Pcon-spi-DT(E-1A)Pcon-tetR-DT(1-2A)Ptet(2-S)RBS-GFP-DT(EcoRI&XbaI)
experimentGGA5Pcon-antisense(E-1A)DT(1-3)Pcon-tetR-DT(3-2A)Ptet(2-S)RBS-GFP-DT(EcoRI&XbaI)
experimentGGA6Pcon-attenuator(E-1A)tetap-DT(1-3)Pcon-tetR-DT(3-2A)Ptet(2-S)RBS-GFP-DT(EcoRI&XbaI)
experimentGGA7Pcon-tetR-DT(E-2A)Ptet(2-S)RBS-GFP-DT(EcoRI&XbaI)
PreDenatureDenatureAnnealingExtensioncycle
37°C16°C50°C80°C--
3min4min5min5min25cycles

RNA Extraction

Nakamoto

Sample
Pcon-Spinach-DT
Pcon-tetRaptamer-DT
Pcon-attenuator-DT
Spinach-DT
Pcon-antisense-Spinach-DT

Liquid Culture

Kozima

Sample
Pcon-attemuater-DT
Pcon-aptamer-DT
Pcon-spinach-DT
Pcon-RBS-tetR-DT
Pcon-antisense-spinach-DT(Master 1)
Pcon-antisense

Colony PCR

Nakamoto Kozima

Samplebase pair
9/25 9/19pSB1C3&9/3PT181 attenuator(1~3)601bp
9/25 aptamer12_1R-DT&9/24Pcon-pT181 attenuator(1~4)859bp
Pcon-attenuator-aptamer-DT(1)859bp
PreDenatureDenatureAnnealingExtensioncycle
　94°C　94°C　55°C　65°C--
5min30sec　30sec　54sec　30

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
11kbp ladder----
29/25 9/23 1C3&9/3attenuator----
39/25 9/23 1C3&9/3attenuator----
49/25 9/23 1C3&9/3attenuator----
59/25aptamer-DT&9/24Pcon-attenuator----
69/25aptamer-DT&9/24Pcon-attenuator----
79/25aptamer-DT&9/24Pcon-attenuator----
89/24 Pcon attenuator-aptamer-DT(1)----
99/25aptamer-DT&9/24Pcon-attenuator----

Igku 0926 E10.jpg

Plating

Hirano

SampleUse plate
9/26 GGA2 LB(CP)
9/26 GGA3 LB(CP)
9/26 GGA4LB(CP)
9/26 GGA5 LB(CP)
9/26 GGA6 LB(CP)
9/26 GGA7 LB(CP)
9/26 GGA11 LB(CP)
9/26 GGA312LB(CP)
9/26 GGA13 LB(CP)
9/26 GGA14LB(CP)
9/26 GGA15 LB(CP)
9/26 GGA20LB(CP)
9/26 GGA23 LB(CP)
9/26 GGA24LB(CP)
9/26 Ptet+RBS-GFP-DT LB(CP)

incubate 37°C

Restriction Enzyme Digestion

Kojima

9/24 pSB1C3EcoRISpeIbufferMilliQtotal
2cuts11µL1µL1µL3µL14µL30µL
NC0.5µL0µL0µL1µL8.5µL10µL
9/24 Pcon-pT181attenuator-aptamer12-1R-DTEcoRISpeIbufferMilliQtotal
2cuts8.7µL1µL1µL3µL16.3µL30µL
NC0.4µL0µL0µL1µL8.6µL10µL
9/24 pT181attenuatorEcoRISpeIbufferMilliQtotal
2cuts3.1µL1µL1µL3µL21.9µL30µL
NC0.2µL0µL0µL1µL8.8µL10µL
8/17 RBS-GFP-DTEcoRIBSA bufferMilliQtotal
1cut16.4µL1µL3µL3µL6.6µL30µL
NC0.4µL0µL1µL1µL7.6µL10µL
9/17 pSB4K5EcoRIPstIBSA bufferMilliQtotal
2cuts8.5µL1µL1µL3µL3µL13.5µL30µL
NC0.4µL0µL0µL1µL1µL7.6µL10µL
9/14 pSB4K5EcoRIPstIBSA bufferMilliQtotal
2cuts7.3µL1µL1µL3µL3µL14.7µL30µL
NC0.4µL0µL0µL1µL1µL7.6µL10µL
9/22 Pcon-pT181attenuator-DTEcoRISpeI bufferMilliQtotal
2cuts5.4µL1µL1µL3µL19.6µL30µL
NC0.3µL0µL0µL1µL8.7µL10µL
8/7 RBS-GFP-DT 1XbaIPstIBSA bufferMilliQtotal
2cuts8.1µL1µL1µL3µL3µL13.9µL30µL
NC0.4µL0µL0µL1µL1µL7.6µL10µL
9/26 RBS-GFP-DT 1XbaI BSA bufferMilliQtotal
1cut8.1µL1µL3µL3µL14.9µL30µL
9/16 Pλ-luxIEcoRIPstIBSA bufferMilliQtotal
2cuts14.7µL1µL1µL3µL3µL7.3µL30µL
NC0.7µL0µL0µL1µL1µL7.3µL10µL
9/21 PtetEcoRISpeI bufferMilliQtotal
2cuts14.2µL1µL1µL3µL10.8µL30µL
NC0.7µL0µL0µL1µL8.3µL10µL
9/24 Pcon-pT181attenuator-aptamer12-1R-DTXbaIPstIBSA bufferMilliQtotal
2cuts8.7µL1µL1µL3µL3µL13.3µL30µL
NC0.4µL0µL0µL1µL1µL7.6µL10µL

Electrophoresis

Kojima

LaneSample Enzyme1 Enzyme2
1pSB1C3EcoRISpeI
2pSB1C3----
3Pcon-pT181attenuator-aptamer12-1R-DTEcoRISpeI
4Pcon-pT181attenuator-aptamer12-1R-DT----
51kb ladder----
6pT181attenuatorEcoRISpeI
7pT181attenuator----
8RBS-GFP-DTEcoRI--
9RBS-GFP-DT----

Igku Sep26 Electrophoresis(E11) 1.jpg

Sep 27

Gel Extraction

Kojima

LaneDNAEnzyme
11kbp ladder--
2pSB1C3EcoRI+SpeI
3
4
6Pcon-attenuator-aptmer-DTEcoRI+SpeI
7
8
10Pcon-attenuator-aptmer-DTEcoRI+SpeI
11
12

Igku 9272.jpg
Igku 9273.jpg

Nameconcentration[µg/mL]260/280260/230
Pcon-attenuator-DT(EcoRI+SpeI)6.81.980.43
pT181-attenuator(EcoRI+SpeI)7.51.840.06
pSB1C3(EcoRI+SpeI)17.71.970.71

Gel Extraction

No name

LaneDNAEnzyme
11kbp ladder--
3pSB4K5EcoRI+PstI
4pSB4K5EcoRI+PstI
5pSB4K5EcoRI+PstI
7pSB6A1EcoRI+PstI
8pSB6A1EcoRI+PstI
9pSB6A1EcoRI+PstI

Igku 9274.jpg
Igku 9275.jpg

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
1pSB4K5EcoRIPstI
2pSB4K5--
31kbp ladder--
4Pcon-attenuatorEcoRISpeI
5Pcon-attenuator--


LaneSampleEnzyme1Enzyme2
1RBS-GFP-DTXbaIPstI
2RBS-GFP-DT--
3RBS-GFP-DTEcoRIXbaI
41kbp ladder--
5PtetEcoRISpeI
6Ptet--

Igku 0927 G5.jpg

Liquid Culture

No name

Samplemedium
Pcon-GFPAmp
RBS-GFP-DTCP
Pcon-attenuatorAmp
Pcon-attenuator-aptamer-DTAmp
Pcon-tetR-DTAmp

Ligation

No name

stateVectorInserterLigation High ver.2
experiment9/17 pSB1C3 (EcoRI+SpeI) 10.0ng/µL10µL9/26 pT181 attenuator(EcoRI+SpeI) 7.5ng/µL1.3µL3.5 µL
experiment9/25 DT (EcoRI+XbaI) 42.8ng/µL2.3µL9/22 Pcon-pT181 antisense(EcoRI+SpeI) 46.7ng/µL1.5µL1.9 µL
experiment9/27 pSB4K5(EcoRI+PstI) 12.9ng/µL7.8µL9/10 Pcon-attenuator(EcoRI+SpeI) 13.8ng/µL8.0µL3.5 µL
experiment9/27 pSB4K5(EcoRI+PstI) 12.9ng/µL7.8µL9/25 RBS-GFP-DT(XbaI+PstI) 13.9ng/µL12µL3.5 µL
experiment9/8 pSB4K5 (EcoRI+SpeI) 17.6ng/µL5.7 µL8/21 Pcon-GFP-DT(EcoRI+SpeI) 11ng/µL12.6µL3.5 µL

Liquid Culture

Hirano

Samplemedium
Pcon-spinach-DTplusgrow Amp
Pcon-tetRaptamer-DTplusgrow Amp　
Pcon-antisense-spinach-DTplusgrow Amp
spinach-DTplusgrow CP

RNA Extraction

Nakamoto

Nameconcentration[µg/mL]260/280260/230
9/26 Pcon-GFP-DT423.01.831.46
9/26 Pcon-GFP-DT K 298.71.821.50

Electrophoresis

No name

LaneSample
19/27 Pcon-GFP-DT(RNA)
21kb ladder
39/27 Pcon-GFP-DT K(RNA)

Igku 9271.jpg

Miniprep

Tatsui

DNA
Pcon-aptamer-DT
Pcon-spinach-DT
Pcon-antisense
Pcon-tetR-DT
Pcon-attenuator-DT
Pcon-antisense-spinach-DT

Transformation

Hirano

Name1Name2Sample1(µL)Sample2(µL)Competent Cells(µL)Total(µL)
Pcon-attenuator+RBS-GFP-DT(4K5)Pcon-attenuator-DT(1A2)213033
Pcon-attenuator+RBS-GFP-DT(4K5)Pcon-antisense-spinach-DT(1A2)213033
Pcon-attenuator+RBS-GFP-DT(4K5)Pcon-tetR aptamer-DT(1A2)213033
Pcon-RBS-GFP-DT(4K5)Pcon-attenuator-DT(1A2)213033
Pcon-RBS-GFP-DT(4K5)Pcon-antisense-spinach-DT(1A2)213033
Pcon-RBS-GFP-DT(4K5)Pcon-tetR aptamer-DT(1A2)213033
Pcon-attenuator+RBS-GFP-DT(4K5)--2--2022
Pcon-RBS-GFP-DT(4K5)--2--2022
attenuator(1C3)--2--2022

Miniprep

No name

DNAconcentration[µg/mL]260/280260/230
Pcon-RBS－GFP-DT137.61.951.78
RBS-GFP-DT77.02.061.96
Pcon-PT181 attenuator231.01.891.89
Pcon-antisense-Spinach-DT(2)130.01.561.62
Pcon-aptamer-DT218.81.921.72
Pcon-attenuator-DT180.71.951.78
Pcon-Spinach-DT177.61.961.06
Pcon-antisense-Spinach-DT(1)181.11.961.92
Pcon-antisense152.92.021.96
Pcon-RBS+ezR-DT164.11.931.91

Restriction Enzyme Digestion

No name

9/27 RBS-GFP-DTXbaIPstIBufferBSAMilliQtotal
2 cuts12µL1µL1µL3µL3µL20µL40µL
NC1.3µL0µL0µL1µL1µL6.7µL10µL
9/27 Pcon-tetR-DTEcoRIPstIBufferMilliQtotal
2 cuts12.2µL1µL1µL3µL12.8µL 30µL
NC0.6µL0µL0µL1µL8.4µL10µL

RNA Extraction

Nakamoto

Nameconcentration[µg/mL]260/280260/230
9/27 Pcon-RBS-GFP-DT686.61.991.34
9/27 Pcon-RBS-GFP-DT (+High salt)127.61.760.27

DNA sythesis

No name

genome DNADNA volumegenome DNA wipeout bufferRNase freewaterRTRT bufferPrimer Mixtotal
9/27 Pcon-RBS-GFP-DT0.8µL1µL5.2µL 0.5µL 2µL0.5µL 10µL
9/27 pcon-RBS-GFP-DT(+high salt)2.5µL1µL3.5µL 0.5µL 2µL 0.5µL 10µL
9/27 Pcon-RBS-GFP-DT(revised concentration) 0.5µL1µL5.5µL0.5µL2µL0.5µL10µL

Electrophoresis

No name

LaneSample
11kb ladder
2Pcon-RBS-GFP-DT
3 Pcon-RBS-GFP-DT

Igku 0927 E2.jpg

Liquid Culture

No name

Samplemedium
Pcon spinach DT plusgrow 2µL(Amp)
Pcon aptamer DT plusgrow 2µL(Amp)
Pcon antisense Spinach DT plusgrow 2µL(Amp)
Spinach DTplusgrow 4µL(CP)　

RT PCR

No name

genome DNAKOD plus10x buffer for KOD-plus-ver.22nM dNTPs25mM MgSO4F primerR primerMilliQtotal
0.512.52.51.50.750.7515.525
PreDenatureDenatureAnnealingExtensioncycle
　94°C　98°C　49.8°C　68°C--
2min　10sec　30sec　10sec　30

Electrophoresis

No name

LaneSample
1Pcon-GFP-DT(GFP)
2Pcon-GFP-DT(internal)
3Pcon-GFP-DT(HS)(GFP)
4Pcon-GFP-DT(internal)
LaneSample
1Pcon-GFP-DT(GFP)
2Pcon-GFP-DT(internal)
3100bp ladder

Igku 927E4.jpg

Digesting cut check

No name

LaneSample
1RBS-GFP-DT(XbaI&PstI)
2RBS-GFP-DT NC(XbaI&PstI)
31kbp ladder
4Pcon-RBS-tetR-DT(EcoRI&SpeI)
5Pcon-RBS-tetR-DT NC(EcoRI&SpeI)

Igku 927 E3.jpg

Gel Extraction

No name

LaneDNAEnzyme
11kbp ladder--
3RBS-GFP-DTXbal+PstI
4RBS-GFP-DTXbal+PstI
5RBS-GFP-DTXbal+PstI
71kbp ladder--
9Pcon-tetR-DTEcoRI+SpeI
10Pcon-tetR-DTEcoRI+SpeI
11Pcon-tetR-DTEcoRI+SpeI

Igku 0927 G7.jpg
Igku 0927 G8.jpg
Igku 0927 G9.jpg
Igku 0927 G10.jpg

RNA Extraction

Nakamoto

05min15min20min23min28min33min36min
aptamer generator0.3480.4080.5500.865
Pcon-spinach-DT0.2500.2400.3050.4260.4710.5540.6680
GFP generator0.3890.4200.5160.884
antisense-spinach0.3710.4380.5370.733
Spinach-DT0.0840.1100.1340.4330.2690.3260.4090.742
Nameconcentration[µg/mL]260/280260/230
Spinach-DT83.91.630.17
Pcon-tetRaptamer-DT425.12.100.64
Pcon-RBS-GFP-DT262.42.010.84
Pcon-antisense-Spinach-DT489.12.050.68
Pcon-Spinach-DT266.62.010.66

cDNA Synthesis

Nakamoto

9/27 Spinach-DT9/23 Pcon-tetRaptamer-DT9/23 Pcon-RBS-GFP-DT9/23 Pcon-antisense-spinach-DT9/23 Pcon-Spinach-DT
RNA11111
gDNA wipeout61.21.91.01.9
RNase-free water04.84.15.04.1
RT0.50.50.50.50.5
RT Buffer22222
Primer mix0.50.50.50.50.5
total10µL10µL10µL10µL10µL

Sep 28

Electrophoresis

Tatsui

LaneSample
1100bp ladder
2Spinach-DT+GAPDH+A1
3Spinach-DT+Spinach+C1
4Spinach-DT+gfp+E1
5Spinach-DT+aptamer+G1
6Pcon-tetR aptamer-DT+GADPH+A3
7Pcon-tetR aptamer-DT+Spinach+C3
8Pcon-tetR aptamer-DT+gfp+E3
9Pcon-tetR aptamer-DT+aptamer+G3
10Pcon-RBS-GFP-DT+GADPH+A5
11Pcon-RBS-GFP-DT+Spinach+C5
12Pcon-RBS-GFP-DT+gfp+E5
13Pcon-RBS-GFP-DT+aptamer+G5
14100bp ladder

File:Igku xxxxxx.xxx

LaneSample
1Pcon-antisense-Spinach-DT+GAPDH+A7
2Pcon-antisense-Spinach-DT+Spinach+C7
3Pcon-antisense-Spinach-DT+gfp+E7
4Pcon-antisense-Spinach-DT+aptamer+G7
5100bp ladder
6Pcon-Spinach-DT+GAPDH+A9
7Pcon-Spinach-DT+Spinach+C9
8Pcon-Spinach-DT+gfp+E9
9Pcon-Spinach-DT+aptamer+G9

File:Igku xxxxxx.xxx

Colony PCR

Samplebase pair
9/27 pT181 attenuatorC(C3)1330
9/27 pT181 attenuatorC(C3)2330
9/27 Pcon antisense-DT1372
9/27 Pcon antisense-DT2372
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s24s
Samplebase pair
9/27 Pcon-RBS-GFP-DT (4&5)1158
9/27 Pcon-RBS-GFP-DT (4&5)1158
9/27 Pcon-RBS-GFP-DT (4&5)1158
9/27 Pcon-RBS-GFP-DT (4&5)1158
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s1min24s

Electrophoresis

No name

LaneSample
1100bp ladder
2Pcon-tetR aptamer-DT+GADPH+A3
3Pcon-antisense-Spinach-DT+GADPH+A7
4Spinach-DT+GADPH+A1
5Pcon-RBS-GFP-DT+GADPH+A5
6Pcon-Spinach-DT+GADPH+A9


LaneSample
1100bp ladder
2Pcon-tetR aptamer-DT+gfp+E3
3Pcon-antisense-Spinach-DT+gfp+E7
4Spinach-DT+gfp+E1
5Pcon-RBS-GFP-DT+gfp+E5
6Pcon-Spinach-DT+gfp+E9

Igku 0928 E1.jpg

LaneSample
1100bp ladder
2Pcon-tetR aptamer-DT+Spinach+C3
3Pcon-antisense-Spinach-DT+gfp+C7
4Spinach-DT+Spinach+C1
5Pcon-RBS-GFP-DT+Spinach+C5
6Pcon-Spinach-DT+Spinach+C9


LaneSample
1100bp ladder
2Pcon-tetR aptamer-DT+aptamer+G3
3Pcon-antisense-Spinach-DT+aptamer+G7
4Spinach-DT+aptamer+G1
5Pcon-RBS-GFP-DT+aptamer+G5
6Pcon-Spinach-DT+aptamer+G9

Igku 0928 E2.jpg

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