Template:Kyoto/Notebook/Sep 12

From 2013.igem.org

(Difference between revisions)
(→Sep 12)
(→Colony PCR)
 
(54 intermediate revisions not shown)
Line 6: Line 6:
!DNA||concentration[µg/mL]||260/280||260/230  
!DNA||concentration[µg/mL]||260/280||260/230  
|-
|-
-
|Pcon-pT181atte-DT||293.6　||1.88　||1.84
+
|Pcon-pT181attenuator-DT||293.6　||1.88　||1.84
|-
|-
|Pcon-apt12-1R-DT||303.8　||1.75　||1.64
|Pcon-apt12-1R-DT||303.8　||1.75　||1.64
|-
|-
-
|Fusion1atte(pSB1c3)||172.0　||1.96　||2.31
+
|Fusion1attenuator(pSB1c3)||172.0　||1.96　||2.31
|-
|-
|RBS-lisis1-DT||235.1||1.59||1.60
|RBS-lisis1-DT||235.1||1.59||1.60
Line 33: Line 33:
|-
|-
|}
|}
-
[[File:igku_xxxxxx.xxx]]<br>
+
[[File:igku_9121.png]]<br>
 +
 
===Liquid Culture===
===Liquid Culture===
<div class="experiment">
<div class="experiment">
Line 42: Line 43:
|9/10 Pcon-apt12-1R-DT1||Plusgrow medium(+Amp)
|9/10 Pcon-apt12-1R-DT1||Plusgrow medium(+Amp)
|-
|-
-
|9/10 DT-Pcon-pT181atte1||Plusgrow medium(+CP)
+
|9/10 DT-Pcon-pT181attenuator1||Plusgrow medium(+CP)
|-
|-
-
|9/10 Fusion1-atte(pSB1c3)||Plusgrow medium(+CP)　
+
|9/10 Fusion1-attenuator(pSB1c3)||Plusgrow medium(+CP)　
|}
|}
-
</div>v>
+
</div>
===Electrophoresis===
===Electrophoresis===
Line 52: Line 53:
<span class="author">Tatsui</span></div>
<span class="author">Tatsui</span></div>
-
1
 
{| class="wikitable"
{| class="wikitable"
!Lane||Sample
!Lane||Sample
Line 58: Line 58:
|1||1kb ladder
|1||1kb ladder
|-
|-
-
|2||pSB4K5(EcoR1&Spe1)
+
|2||pSB4K5(EcoRI&SpeI)
|-
|-
-
|3|| pSB4K5 NC(EcoR1&Spe1)
+
|3|| pSB4K5 NC(EcoRI&SpeI)
|-
|-
-
|4||RBS-lacz-DT(EcoR1&Spe1)
+
|4||RBS-lacz-DT(EcoRI&SpeI)
|-
|-
-
|5|| EcoR1&Spe1 NC( EcoR1&Spe1)
+
|5|| EcoRI&SpeI NC( EcoRI&SpeI)
|-
|-
-
|6||Pcon-RBS-lacz-DT( EcoR1&Spe1)
+
|6||Pcon-RBS-lacz-DT( EcoRI&SpeI)
|-
|-
-
|7|| Pcon-RBS-lacz-DT  NC( EcoR1&Spe1)
+
|7|| Pcon-RBS-lacz-DT  NC( EcoRI&SpeI)
|-
|-
|8||1kb ladder
|8||1kb ladder
Line 74: Line 74:
|}
|}
-
[[File:igku_xxxxxx.xxx]]
+
[[File:igku_9122.png]]
</div>
</div>
Line 84: Line 84:
|1||1kb ladder
|1||1kb ladder
|-
|-
-
|2||Ptet-pT181anti(Spe1&Pst1)  
+
|2||Ptet-pT181antisense(SpeI&PstI)  
|-
|-
-
|3|| Ptet-pT181anti NC(Spe1&Pst1)
+
|3|| Ptet-pT181antisense NC(SpeI&PstI)
|-
|-
-
|4||RBS-lysis2-DT(Xba1&Pst1)
+
|4||RBS-lysis2-DT(XbaI&PstI)
|-
|-
-
|5|| EcoR1&Spe1 NC( EcoR1&Spe1)
+
|5|| EcoRI&SpeI NC( EcoRI&SpeI)
|-
|-
|6||--
|6||--
Line 100: Line 100:
|}
|}
-
[[File:igku_xxxxxx.xxx]]
+
[[File:igku_9123.png]]
===Gel Extraction===
===Gel Extraction===
Line 110: Line 110:
|1||1kb ladder||--
|1||1kb ladder||--
|-
|-
-
|2||RBS-lacZ-DT||EcoR1&Spe1
+
|2||RBS-lacZ&alpha;-DT||EcoRI&SpeI
|-
|-
-
|3||RBS-lacZ-DT||EcoR1&Spe1
+
|3||RBS-lacZ&alpha;-DT||EcoRI&SpeI
|-
|-
-
|4||RBS-lacZ-DT||EcoR1&Spe1
+
|4||RBS-lacZ&alpha;-DT||EcoRI&SpeI
|-
|-
|5||--||--
|5||--||--
|-
|-
-
|6||Pcon-RBS-lacZ-DT||EcoR1&Spe1
+
|6||Pcon-RBS-lacZ&alpha;-DT||EcoRI&SpeI
|-
|-
-
|7||Pcon-RBS-lacZ-DT||EcoR1&Spe1
+
|7||Pcon-RBS-lacZ&alpha;-DT||EcoRI&SpeI
|-
|-
-
|8||Pcon-RBS-lacZ-DT||EcoR1&Spe1
+
|8||Pcon-RBS-lacZ&alpha;-DT||EcoRI&SpeI
|-
|-
|9||1kb ladder||--
|9||1kb ladder||--
|}
|}
-
[[File:igku_Aug19electrophoresis3]]<br>
+
[[File:igku_9129.png]]<br>
-
[[File:igku_Aug19electrophoresis4]]<br>
+
[[File:igku_91210.png]]<br>
{| class="wikitable"
{| class="wikitable"
!Name||concentration[&micro;g/mL]||260/280||260/230
!Name||concentration[&micro;g/mL]||260/280||260/230
|-
|-
-
|RBS-lacZ-DT(EcoR1&Spe1)||9.8||1.50||0.39
+
|RBS-lacZ&alpha;-DT(EcoRI&SpeI)||9.8||1.50||0.39
|-
|-
-
|Pcon-RBS-lacZ-DT(EcoR1&Spe1)||11.2||1.47||0.08
+
|Pcon-RBS-lacZ&alpha;-DT(EcoRI&SpeI)||11.2||1.47||0.08
|-
|-
|}
|}
Line 148: Line 148:
|1||1kb ladder||--
|1||1kb ladder||--
|-
|-
-
|2||RBS-lysis2-DT||Xba1&Pst1
+
|2||RBS-lysis2-DT||XbaI&PstI
|-
|-
-
|3||RBS-lysis2-DT||Xba1&Pst1
+
|3||RBS-lysis2-DT||XbaI&PstI
|-
|-
-
|4||RBS-lysis2-DT||Xba1&Pst1
+
|4||RBS-lysis2-DT||XbaI&PstI
|-
|-
|5||--||--
|5||--||--
Line 159: Line 159:
|}
|}
-
[[File:igku_Aug19electrophoresis3]]<br>
+
[[File:igku_9127.png]]<br>
-
[[File:igku_Aug19electrophoresis4]]<br>
+
[[File:igku_9128.png]]<br>
{| class="wikitable"
{| class="wikitable"
!Name||concentration[&micro;g/mL]||260/280||260/230
!Name||concentration[&micro;g/mL]||260/280||260/230
|-
|-
-
|RBS-lysis2-DT(Xba1&Pst1)||5.4||1.43||0.77
+
|RBS-lysis2-DT(XbaI&PstI)||5.4||1.43||0.77
|-
|-
|}
|}
</div>
</div>
 +
 +
===Electrophoresis===
 +
<div class="experiment">
 +
<span class="author">No name </span></div>
 +
 +
 +
{| class="wikitable"
 +
!Lane||Sample
 +
|-
 +
|1||1kb ladder
 +
|-
 +
|2||P&lambda;-RBS-luxI-DT(PCR product)
 +
|-
 +
|3||Pbad/araC-RBS-RFP(PCR product)
 +
|}
 +
[[File:igku_9124.png]]
 +
 +
===Colony PCR===
<div class="experiment">
<div class="experiment">
<span class="author">Honda</span>
<span class="author">Honda</span>
-
{|===Colony PCR===
+
{| class="wikitable"
-
class="wikitable"
+
!|Sample||base pair
-
!Sample||base pair
+
|-
|-
|9/11 apt12-1M(pSB1C3) 1||513
|9/11 apt12-1M(pSB1C3) 1||513
Line 181: Line 198:
|9/11 Fusion3m2a09ttenuator(pSB1C3) 1||609
|9/11 Fusion3m2a09ttenuator(pSB1C3) 1||609
|--
|--
-
|9/11 Fusion6 antisense(pSB1C3)1||431-
+
|9/11 Fusion6 antisense(pSB1C3)1||431
|-
|-
|9/11 aptamer 12-P(pSB1C3)1||515
|9/11 aptamer 12-P(pSB1C3)1||515
Line 200: Line 217:
|-
|-
|9/11 Pcon-RBS-lacZ&alpha;-DT(pSB4K5) 1||712
|9/11 Pcon-RBS-lacZ&alpha;-DT(pSB4K5) 1||712
-
|
 
|}
|}
{| class="wikitable"
{| class="wikitable"
Line 217: Line 233:
! ||9/5 Pcon ||EcoRI||SpeI||XbaI||PstI|| Buffer||BSA||MilliQ||total
! ||9/5 Pcon ||EcoRI||SpeI||XbaI||PstI|| Buffer||BSA||MilliQ||total
|-
|-
-
|1cuts(P)||0&micro;L||0&micro;L||0&micro;L||1&micro;L||3&micro;L||3&micro;L||17.3&micro;L||30&micro;L
+
|1cuts(PstI)||5.7||0&micro;L||0&micro;L||0&micro;L||1&micro;L||3&micro;L||3&micro;L||17.3&micro;L||30&micro;L
|-
|-
-
|NC(P)||0&micro;L||0&micro;L||0&micro;L||0&micro;L||1&micro;L||1&micro;L||6.1&micro;L||10&micro;L
+
|NC(PstI)||1.9||0&micro;L||0&micro;L||0&micro;L||0&micro;L||1&micro;L||1&micro;L||6.1&micro;L||10&micro;L
|-
|-
|}
|}
{| class="wikitable"
{| class="wikitable"
-
! ||8/17 DT||EcoR1||Buffer||BSA||MilliQ||total
+
! ||8/17 DT||EcoRI||Buffer||BSA||MilliQ||total
|-
|-
-
|1cut||1&micro;L||3&micro;L||3&micro;L||12.4&micro;L||30&micro;L|
+
|1cut(EcoRI)||10.6||1&micro;L||3&micro;L||3&micro;L||12.4&micro;L||30&micro;L
|-
|-
-
|NC||0&micro;L||1&micro;L||1&micro;L||4.5&micro;L||10&micro;L|
+
|NC(EcoRI)||3.5||0&micro;L||1&micro;L||1&micro;L||4.5&micro;L||10&micro;L
|}
|}
 +
===Colony PCR===
===Colony PCR===
<div class="experiment">
<div class="experiment">
-
<span class="author">Nakamoto,Tatsui</span>
+
<span class="author">Nakamoto and Tatsui</span>
{| class="wikitable"
{| class="wikitable"
!Sample||base pair
!Sample||base pair
|-
|-
-
|9/11 Pcon-pT181atte-RBS-lacZ&alpha;-DT||965
+
|9/11 Pcon-pT181attenuator-RBS-lacZ&alpha;-DT||965
|-
|-
|9/11 Plux-PBS-lysis1-DT 1||613
|9/11 Plux-PBS-lysis1-DT 1||613
Line 243: Line 260:
|9/11 Pcon-pT181attenuator-aptamer12-1R-DT 2||859
|9/11 Pcon-pT181attenuator-aptamer12-1R-DT 2||859
|-
|-
-
|9/11 Plux\RBS-lysis3-DT 1||1210
+
|9/11 Plux-RBS-lysis3-DT 1||1210
|-
|-
-
|9/11 Plux\RBS-lysis3-DT 2||1210
+
|9/11 Plux-RBS-lysis3-DT 2||1210
|-
|-
|Pcon-Spinach-DT(pSB4K5) 1||605
|Pcon-Spinach-DT(pSB4K5) 1||605
Line 257: Line 274:
|}
|}
</div>
</div>
 +
===Transformation===
===Transformation===
<div class="experiment">
<div class="experiment">
-
<span class="author">tatsui</span>
+
<span class="author">Tatsui</span>
{| class="wikitable"
{| class="wikitable"
!Name||Sample||Competent Cells||Plate
!Name||Sample||Competent Cells||Plate
|-
|-
-
|}
+
|9/7 pSB4K5||2&micro;L||20&micro;L||Amp
-
</div>
+
-
 
+
-
{| class="wikitable"
+
-
!Lane||Sample
+
|-
|-
-
|1||1kb ladder
 
-
|-
 
-
|2||P&lambda-RBS-luxI-DT(PCR product)
 
-
|-
 
-
|3||Pbad/araC-RBS-RFP(PCR product)
 
|}
|}
-
 
-
[[File:igku_xxxxxx.xxx]]
 
-
 
</div>
</div>
Line 304: Line 310:
|}
|}
-
[[File:igku_xxxxxx.xxx]]
+
[[File:igku_91211.png]]
</div>
</div>
Line 328: Line 334:
|8|| 1kb ladder
|8|| 1kb ladder
|}
|}
 +
[[File:igku_91212.png]]<br>
 +
 +
===Electrophoresis===
 +
<div class="experiment">
 +
<span class="author">No name </span>
 +
{| class="wikitable"
 +
!Lane||Sample||Enzyme
 +
|-
 +
|1||Pcon-pT181attenuator-RBS-lacZ&alpha;-DT 1||--
 +
|-
 +
|2||Pcon-pT181attenuator-RBS-lacZ&alpha;-DT 2||--
 +
|-
 +
|3||Plux-RBS-lysis1-DT 1 ||--
 +
|-
 +
|4||Pcon-pT181attenuator-aptamer12-1R-DT 1 ||--
 +
|-
 +
|5||Pcon-pT181attenuator-aptamer12-1R-DT 2 ||--
 +
|-
 +
|6||Plux-RBS-lysis3-DT 1||--
 +
|-
 +
|7||1kbp ladder ||--
 +
|-
 +
|8|| Plux-RBS-lysis3-DT 2||--
 +
|-
 +
|9||Pcon-spinach-DT(pSB4K5) 1||--
 +
|-
 +
|10||9/5 Pcon||PstI
 +
|-
 +
|11||9/5 Pcon NC||--
 +
|-
 +
|12||8/17 DT ||EcoRI
 +
|-
 +
|13||8/17 DT ||--
 +
|}
 +
</div>
 +
[[File:igku_91213.png]]
 +
 +
===Liquid Culture===
 +
<div class="experiment">
 +
<span class="author">Nakamoto</span>
 +
{| class="wikitable"
 +
!Sample||medium
 +
|-
 +
|9/11Fusion6 antisense-1||Plusgrow medium(+CP)
 +
|-
 +
|Plac(BBa-R0011)-3||Plusgrow medium(+Amp)
 +
|-
 +
|Plac(BBa-R0011)-4||Plusgrow medium(+Amp)
 +
|-
 +
|Fusion3m2 attenuator -1||Plusgrow medium(+CP)　
 +
|-
 +
|P&lambda;-luxI-2||Plusgrow medium(+Amp)　
 +
|-
 +
|9/11 aptamer12-1M-2||Plusgrow medium(+CP)
 +
|}
 +
</div>
 +
 +
===Colony PCR===
 +
<div class="experiment">
 +
<span class="author">Nakamoto</span>
 +
{| class="wikitable"
 +
!|Sample||base pair
 +
|-
 +
|9/11 Fusion1 antisense 3||394
 +
|-
 +
|9/11 Fusion1 antisense 4||394
 +
|-
 +
|9/11 aptamer12-P 2||378
 +
|--
 +
|9/11 aptamer12-P 3||378
 +
|}
 +
{| class="wikitable"
 +
!PreDenature||Denature||Annealing||Extension||cycle
 +
|-
 +
|94&deg;C||94&deg;C||55&deg;C||68&deg;C||--
 +
|-
 +
|5min||30s||30s||36s||30cycles
 +
|}
 +
 +
<br>
 +
 +
===Electrophoresis===
 +
<div class="experiment">
 +
<span class="author">Nakamoto</span>
 +
{| class="wikitable"
 +
!Lane||Sample||Enzyme
 +
|-
 +
|1||Fusion1 antisense 2||--
 +
|-
 +
|2||Fusion1 antisense 3||--
 +
|-
 +
|3||1kb ladder ||--
 +
|-
 +
|4||apt12-P 2 ||--
 +
|-
 +
|5||apt12-P 3 ||--
 +
|}
 +
</div>
 +
[[File:igku_91214.png]]
 +
 +
===Restriction Enzyme Digestion===
 +
<div class="experiment">
 +
<span class="author">Tatsui </span>
 +
{| class="wikitable"
 +
! ||9/12 RBS-lysis2-DT||XbaI||PstI||Buffer||BSA||MilliQ||total
 +
|-
 +
|2 cuts||5.2 ||1.0&micro;L||1.0&micro;L||3.0&micro;L||3.0&micro;L||16.8&micro;L ||30&micro;L
 +
|-
 +
|NC||0.3&micro;L||0&micro;L||0&micro;L||1.0&micro;L||1.0&micro;L||7.7&micro;||10&micro;L
 +
|}
 +
 +
{| class="wikitable"
 +
! ||9/12 RBS-lysis3-DT||XbaI ||PstI||Buffer||BSA||MilliQ||total
 +
|-
 +
|2 cuts||5.2 ||1.0&micro;L||1.0&micro;L||3.0&micro;L||3.0&micro;L||16.8&micro;L ||30&micro;L
 +
|-
 +
|NC||0.3&micro;L||0&micro;L||0&micro;L||1.0&micro;L||1.0&micro;L||7.7&micro;||10&micro;L
 +
|}
 +
 +
{| class="wikitable"
 +
! ||9/11 RBS-lysis1-DT||XbaI||PstI||Buffer||BSA||MilliQ||total
 +
|-
 +
|2 cuts||8.5||1.0&micro;L||1.0&micro;L||3.0&micro;L||3.0&micro;L||13.5&micro;L ||30&micro;L
 +
|-
 +
|NC||0.4&micro;L||0&micro;L||0&micro;L||1.0&micro;L||1.0&micro;L||7.6&micro;||10&micro;L
 +
|}
 +
 +
{| class="wikitable"
 +
! ||8/20 J23100-RBS-luxR -DT||EcoRI ||Buffer||BSA||MilliQ||total
 +
|-
 +
|1cut||5.8||1.0&micro;L||3.0&micro;L||3.0&micro;L||17.2&micro;L ||30&micro;L
 +
|-
 +
|NC||0.3&micro;L||0&micro;L||1.0&micro;L||1.0&micro;L||7.7&micro;||10&micro;L
 +
|}
 +
{| class="wikitable"
 +
! ||9/10 pSB1C3||EcoRI ||SpeI||Buffer||BSA||MilliQ||total
 +
|-
 +
|2cuts||8.1||1.0&micro;L||1.0&micro;L||3.0&micro;L||3.0&micro;L||13.9&micro;L ||30&micro;L
 +
|-
 +
|NC||0.4&micro;L||0&micro;L||0&micro;L||1.0&micro;L||1.0&micro;L||7.6&micro;||10&micro;L
 +
|}
 +
 +
{| class="wikitable"
 +
! ||9/12 Plux ||PstI ||Buffer||BSA||MilliQ||total
 +
|-
 +
|1cut||12.0||1.0&micro;L||3.0&micro;L||3.0&micro;L||11.0micro;L ||30&micro;L
 +
|-
 +
|NC||0.6&micro;L||0&micro;L||1.0&micro;L||1.0&micro;L||7.4&micro;||10&micro;L
 +
|}
 +
 +
{| class="wikitable"
 +
! ||9/10 pSB1C3||XbaI ||PstI||Buffer||BSA||MilliQ||total
 +
|-
 +
|2cuts||8.1||1.0&micro;L||1.0&micro;L||3.0&micro;L||3.0&micro;L||13.9&micro;L ||30&micro;L
 +
 +
|}
 +
 +
<div>
 +
 +
===Electrophoresis===
 +
<div class="experiment">
 +
<span class="author">Tatsui </span>
 +
{| class="wikitable"
 +
!Lane||Sample||Enzyme1||Enzymel2
 +
|-
 +
|1||9/12 RBS-lysis2-DT ||XbaI||PstI
 +
|-
 +
|2||9/12 RBS-lysis2-DT NC ||--||--
 +
|-
 +
|3||9/11 RBS-lysis1-DT ||XbaI||PstI
 +
|-
 +
|4||9/11 RBS-lysis1-DT NC ||--||--
 +
|-
 +
|5|| 9/11 RBS-lysis3-DT ||XbaI||PstI
 +
|-
 +
|6|| 9/11 RBS-lysis3-DT NC1||--||--
 +
|-
 +
|7||1kb ladder ||--||--
 +
|-
 +
|8|| 100bp ladder||--||--
 +
|-
 +
|9||J23100-RBS-luxR-DT ||EcoRI||--
 +
|-
 +
|10|| J23100-RBS-luxR-DT ||--||--
 +
|-
 +
|11||9/10 pSB1C3 ||EcoRI||SpeI
 +
|-
 +
|12|| 9/10 pSB1C3 NC||--||---
 +
|-
 +
|13||9/10 pSB1C3 ||XbaI||PstI
 +
|-
 +
|14||9/12 Plux||PstI||--
 +
|-
 +
|15||9/12 Plux NC||--||--
 +
|}
 +
[[File:igku_91215.png]]<br>
 +
</div>
 +
 +
====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).
 +
<br>
 +
We translated them into each 1.5mL tube. <br>
 +
 +
We elminated each supernatant using a 5000xg centrifuge for 1minute,. <br>
 +
Then,we resuspended with 100&micro;L M9(distilled water) and eliminated each supernatant using a 5000xg centrifuge for 1 minute.x2<br>
 +
 +
Adding 100&micro;L M9(in 20&micro;M DFHBI), we resuspended the pret. <br>
 +
 +
15min after incubating in shield light, we eliminated each supernatant using a 5000xg centrifuge for 1 minute. <br>
 +
 +
We looked at the fluorescence of the pret.
 +
<br>
 +
We failed to observe it.
 +
 +
===Liquid Culture===
 +
<div class="experiment">
 +
<span class="author">Hirano</span>
 +
{| class="wikitable"
 +
!Sample||medium
 +
|-
 +
|9/10 Pcon-pT181antisense-spinach-DT||Plusgrow medium
 +
|-
 +
|9/10 Pcon-spinach-DT||Plusgrow medium
 +
|-
 +
|9/1 spinach-DT(RNA master plate)|| Plusgrow medium
 +
|}
 +
</div>

Latest revision as of 20:49, 27 September 2013

Contents

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
Retrieved from "http://2013.igem.org/Template:Kyoto/Notebook/Sep_12"