Team:Kyoto/Notebook

From 2012.igem.org

(Difference between revisions)
(→March 6)
(→Florigen)
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=Florigen=
=Florigen=
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<div class="_kyoto-note">
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[[Team:Kyoto/Florigen/Note]]
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==August 2==
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'''Mutation of FT'''  <small>by Sato</small><br>
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FT gene has two BioBrick restriction enzyme sites, EcoR1 and Pst1 which is next to each other.<br>
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-
So we tried to delete both at once by using two primers with mutation.<br>
+
-
{|class="wikitable"
+
-
|+Inverse PCR
+
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!10xBufer!!2mM dNTPs!!primer fwd!!primer rev!!template!!polymerase!!MilliQ!!Total
+
-
|-
+
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|5||5||1.5||1.5||0.5||1||35.5||50
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-
|}
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94℃ 2min, (98℃ 10sec, 68℃ 4min)x4cycles, 4℃ Hold
+
-
 
+
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{|class="wikitable"
+
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|+Dpn1 Digestion
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!PCR product!!Dpn1
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|-
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|50||2
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|}  37℃,1h incubate
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+
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{|class="wikitable"
+
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|+Self-ligation
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!product!!MilliQ!!Ligase!!T4 Kinase!!Total
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|-
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|2||7||5||1||15
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-
|}
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16℃, 1h incubate
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-
 
+
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{|class="wikitable"
+
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|+Transformation
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!competent cell!!DNA
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-
|-
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|20||2
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|}
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Cells were stored on ice for 30min. <br>
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After 42℃ 60sec heat shock, cells were stored on ice for 2min.<br>
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Then cells were precultureed at 37℃ for 1hr, plated to Kanamycin plate.
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-
<br>
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<br>
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==August 13==
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'''Liquid culture'''<br>
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FT at 37°C, for overnight.
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-
 
+
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==August 14==
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'''Miniprep of FT'''<small> : by Sato, Takeuchi</small><br>
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The concentration was 81.5ng/uL<br><br>
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'''Restriction digestion and Electrophoresis'''<small> : by Sato</small><br>
+
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To check wheter mutation was succeed, we did restriction enzyme digestion.
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{|class="wikitable"
+
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!DNA(FT,80ng/uL)!!10xBuferH!!EcoR1!!Pst1!!MilliQ!!Total
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|-
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|5||2||1||-||12||20
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|-
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|5||2||-||1||12||20
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|}
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37°C 2h incubate<br>
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We did electrophoresis of three FT plasmids, non-cutted, EcoR1 cutted, Pst1 cutted.<br>
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However, we couldn't get any bands (data not shown.)<br>
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<br>
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'''Liquid culture'''<br>
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FT (4mL)
+
-
 
+
-
==August 15==
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'''Miniprep of FT'''<small> : by Sato, Takeuchi, Hyungcheol</small><br>
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We couldn't get enough concentration of plasmids.<br><br>
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'''Electrophoresis'''<small> : by Sato</small><br>
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We retried electrophoresis of three samples same as yesterday.<br>
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However, we couldn't get any bands as well.
+
-
 
+
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==August 16==
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'''Transformation'''<small> : by Takeuchi, Ota</small><br>
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{| class="wikitable"
+
-
!Name||Well||Sample||Competent Cells||Total||Plate||Colony
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-
|-
+
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|FT||-||1 &micro;L||10||11||LB (Kan+)||&#xD7;
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-
|-
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|pSB1C3||1-3-A||1||10||11||LB (CP+)||&#x25CB;
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|-
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|I719005||1-15-N||1||10||11||LB (Amp+)||&#x25CB;
+
-
|}
+
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==August 17==
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'''Transformation'''<small> : by Takeuchi</small><br>
+
-
{| class="wikitable"
+
-
!Name||Well||Sample||Competent Cells||MilliQ||Total||Plate||Colony
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-
|-
+
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|FT||-||2||2||18||22||LB (Kan+)||&#xD7;
+
-
|}
+
-
We found that mutation of FT was not successful.
+
-
==August 20==
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-
We decided to do PCR using FT specific primers before mutation.<br>
+
-
'''PCR of FT''' <small> : by Sato</small><br>
+
-
{|class="wikitable"
+
-
!10xBufer!!dNTPs!!MgSO4!!primer fwd!!primer rev!!template!!polymerase!!MilliQ!!Total
+
-
|-
+
-
|5||5||3||1||1||1(130ng/&micro;L)||1(KOD plus neo)||33||50
+
-
|}
+
-
94°C 2min,  (98°C 10sec, 68°C 15sec)x30cycles,  4°C Hold<br>
+
-
After the ethanol precipitation, we diluted in 30&micro;L of MilliQ.<br>
+
-
The concentration was 203ng/&micro;L.
+
-
<br>
+
-
<br>
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'''Electrophoresis'''[[File:Electrophoresis0821.png|400px|thumb|right]]
+
-
{| class="wikitable"
+
-
!Lane!!Name!!length(bp)
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-
|-
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|1||1kb ladder||-
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-
|-
+
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|2||FT||600
+
-
|}
+
-
<br>
+
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==August 21==
+
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'''Restriction digestion''' <small> : by Sato</small><br>
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-
{|class="wikitable"
+
-
!DNA(FT,203ng/&micro;L)!!10xBuferM!!Xba11!!Pst1!!MilliQ!!Total
+
-
|-
+
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|10||4||1||1||24||40
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-
|}
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37°C, 5h incubate<br>
+
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After the ethanol precipitation, we diluted in 30&micro;L of MilliQ.<br>
+
-
The concentration was 54,9ng/&micro;L.<br>
+
-
<br>
+
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'''Ligation''' <small> : by Sato</small><br>
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{| class="wikitable"
+
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!colspan="2"|Vector||colspan="2"|Insert||Ligation High Ver.2
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|-
+
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|pSB1C3||1||FT||10||5.5
+
-
|}
+
-
 
+
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'''Liquid culture'''<br>
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-
T7 promoter, pSB1C3 (4mL)
+
-
<br>
+
-
<br>
+
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==August 22==
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'''Miniprep'''<small> : by Sato</small><br>
+
-
{| class="wikitable"
+
-
!T7 promoter||pSB1C3
+
-
|-
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|85.3ng/&micro;L||82.93ng/&micro;L
+
-
|}
+
-
<br>
+
-
 
+
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==August 23==
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'''Ethanol Precipitation<br>'''
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<small>diluted in 20µL 79.3ng/µL</small><br>
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<br>mada dekite nai
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+
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+
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==August 24==
+
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'''Restriction enzyme processing'''<br>
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-
{|class="wikitable"
+
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!T7 promoter(85.3ng/µL)!!Spel!!Pstl!!buffer M!!MiliQ!!Total
+
-
|-
+
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|10||1||1||2||6||20
+
-
|}
+
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->purifying column 33.4ng/µL(dissolution 40µL)<br>
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-
{|class="wikitable"
+
-
!pSB1C3(82.9ng/µL)!!Xbal!!Spel!!buffer M!!MiliQ!!Total
+
-
|-
+
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|20||1||1||4||14||40
+
-
|}
+
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->gene clean2 39.9ng/µL(dissolution 40µL)<br>
+
-
<br>
+
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<<picture1,2>>
+
-
<br>
+
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'''Ligation'''<br>
+
-
{|class="wikitable"
+
-
!FT(600bp, 79.3ng/µL)!!pSB1C3(2000bp,39.9ng/µL)!!Ligation High Ver.2
+
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|-
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|3µL => 597fmol||2µL => 60fmol||2.5µL
+
-
|}
+
-
{|class="wikitable"
+
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!FT(600bp, 79.3ng/µL)!!T7(2100bp,33.4ng/µL)!!Ligation High Ver.2
+
-
|-
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|2.4µL => 478fmol||2µL => 48fmol||2.2µL
+
-
|}
+
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=> 16℃,1hr incubate
+
-
 
+
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==August 27==
+
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'''Colony PCR'''<br>
+
-
{|class="wikitable"
+
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!2X Quick Tag!!VF2!!VR!!MiliQ!!Total
+
-
|-
+
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|25||1||1||23||50
+
-
|}
+
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Lane1: 1kb ladder<br>
+
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Lane2~16: FT(pSB1C3) about 800bp<br>
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+
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'''FT(TOPO) PCR(re)'''<br>
+
-
 
+
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{|class="wikitable"
+
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!buffer!!dNTPs!!MgSO4!!primer f!!primer r!!Template(130ng/µL)!!KODplus neo!!MilliQ!!Total
+
-
|-
+
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|5||5||3||1||1||1||1||33||50
+
-
|}
+
-
 
+
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{| class="wikitable"
+
-
!94℃||98℃||68℃||cycles
+
-
|-
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|2min||10sec||15sec||30
+
-
|}
+
-
<br>
+
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'''Liquid culture'''    <small>by Nobeyama</small><br>
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FT 4ml<br>
+
-
 
+
-
==August 28==
+
-
 
+
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'''Mutation of FT (re)'''<br><br>
+
-
inverse PCR<br>
+
-
{|class="wikitable"
+
-
!MilliQ!!buffer!!dNTP!!primer f!!primer r!!FT(130ng/µL)!!KODplus!!Total
+
-
|-
+
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|35.5||5||5||1.5||1.5||0.5||1||50
+
-
|}
+
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{| class="wikitable"
+
-
!94℃||98℃||68℃||cycles
+
-
|-
+
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|2min||10sec||4min||18
+
-
|}<br>
+
-
Lane1: 1kb ladder<br>
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-
Lane2: FT<br>
+
-
 
+
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'''Miniprep FT(TOPO''')<br>
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158ng/µL<br><br>
+
-
'''Tranformation'''<br>
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-
competent cell: 20<br>
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-
BBa.I746902  : 2<br>
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(plate 316f)<br>
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(GFP generator of pBAD/araC-mut3 GFP:6His-DT)<br>
+
-
 
+
-
==August 29==
+
-
'''Mutaion of FT(re;re)'''<br><br>
+
-
'''Inverse PCR'''<br><br>
+
-
first
+
-
{|class="wikitable"
+
-
!MilliQ!!buffer!!dNTP!!primer f!!primer r!!FT(130ng/µL)!!KODplus!!Total
+
-
|-
+
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|35.5||5||5||1.5||1.5||0.5||1||50
+
-
|}
+
-
second
+
-
{|class="wikitable"
+
-
!MilliQ!!buffer!!dNTP!!primer f!!primer r!!FT(52ng/µL)!!KODplus!!Total
+
-
|-
+
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|35||5||5||1.5||1.5||1||1||50
+
-
|}
+
-
{| class="wikitable"
+
-
!94℃||98℃||68℃||cycles
+
-
|-
+
-
|2min||10sec||4min||30
+
-
|}<br>
+
-
Lane1: 1kb ladder<br>
+
-
Lane2: first Template 130ng/µL<br>
+
-
Lane3: second Template 53ng/µL<br>
+
-
 
+
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{| class="wikitable"
+
-
!first sample||Dpnl||total
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|-
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|45µL||2µL||47µL
+
-
|}
+
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in 37℃, 1 hour<br>
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+
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'''Self-Ligation'''<br>
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-
 
+
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{| class="wikitable"
+
-
!PCR products||MilliQ||Ligation High||T4 kinase||total
+
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|-
+
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|2µL||7µL||5µL||1µL||15µL
+
-
|}<br>
+
-
in 16℃, 1.5hour incubate<br>
+
-
Transformation<br>
+
-
competent cell: 20<br>
+
-
DNA          : 2<br><br>
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-
Liquid culture(I746902): 3mL
+
-
 
+
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==August 30==
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'''Liquid culture(FT) 4mL x2'''<br>
+
-
 
+
-
==August 31==
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'''Miniprep(FT)'''<br>
+
-
 
+
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(1) 64.9ng/µL<br>
+
-
(2) 52.6ng/µL<br>
+
-
 
+
-
'''Restriction enzyme processing (Mutation check)'''<br>
+
-
{| class="wikitable"
+
-
!FT(52.6ng/µL)||bufferH||E.coli||Pst1||MilliQ||total
+
-
|-
+
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|1µL||5µL||0.5µL||0.5µL||3.5µL||10µL
+
-
|}<br>
+
-
in 37℃,1.5hour<br>
+
-
'''PCR(RBS primer)'''<br>
+
-
 
+
-
{|class="wikitable"
+
-
!buffer for KODplus neo!!dNTPs!!MgSO4!!primer f!!primer r!!Template(52.6ng/µL)!!KODplus neo!!MilliQ!!Total
+
-
|-
+
-
|5||5||3||1||1||1||1||33||50
+
-
|}<br>
+
-
 
+
-
{| class="wikitable"
+
-
!94℃||98℃||68℃||cycles
+
-
|-
+
-
|2min||10sec||15sec||30
+
-
|}<br>
+
-
 
+
-
Lane1: 1kb ladder<br>
+
-
Lane2: FT<br>
+
-
Lane3: FT(E.coli)<br>
+
-
Lane4: FT(Pst1)<br>
+
-
Lane5: FT PCR<br><br>
+
-
 
+
-
'''PCR(re)'''<br>
+
-
 
+
-
{|class="wikitable"
+
-
!buffer!!dNTPs!!MgSO4!!primer f!!primer r!!Template!!KOD neo!!MilliQ!!Total
+
-
|-
+
-
|5||5||3||1||1||1||1||33||50
+
-
|}<br>
+
-
{| class="wikitable"
+
-
!94℃||98℃||68℃||cycles
+
-
|-
+
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|2min||10sec||15sec||35
+
-
|}<br>
+
-
 
+
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Lane1: 1kb ladder<br>
+
-
Lane2: FT<br>
+
-
 
+
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'''PCR(re)'''<br>
+
-
 
+
-
{|class="wikitable"
+
-
!buffer!!dNTPs!!MgSO4!!primer f!!primer r!!Template!!KOD neo!!MilliQ!!Total
+
-
|-
+
-
|5||5||2||1||1||1||1||34||50
+
-
|}<br>
+
-
{| class="wikitable"
+
-
!94℃||98℃||68℃||cycles
+
-
|-
+
-
|2min||10sec||10sec||30
+
-
|}<br>
+
-
 
+
-
==September 2==
+
-
 
+
-
'''PCR(re;re)'''<br><br>
+
-
 
+
-
first
+
-
{|class="wikitable"
+
-
!buffer!!dNTPs!!MgSO4!!primer f!!primer r!!Template(1ng/µL)!!KODplus neo!!MilliQ!!Total
+
-
|-
+
-
|5||5||3||1||1||1||1||33||50
+
-
|}<br>
+
-
 
+
-
second
+
-
{|class="wikitable"
+
-
!buffer!!dNTPs!!MgSO4!!primer f!!primer r!!Template(10ng/µL)!!KODplus neo!!MilliQ!!Total
+
-
|-
+
-
|5||5||3||1||1||1||1||33||50
+
-
|}<br>
+
-
{| class="wikitable"
+
-
!94℃||98℃||68℃||cycles
+
-
|-
+
-
|2min||10sec||10sec||25
+
-
|}<br>
+
-
 
+
-
Lane1: first Template 1ng<br>
+
-
Lane2: second Template 10ng<br>
+
-
Lane3: 1kb ladder<br>
+
-
 
+
-
refine first Template => 132ng/µL<br>
+
-
</div>
+
=Golden Gate Assembly=
=Golden Gate Assembly=

Revision as of 05:18, 7 September 2012

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Contents

Secretion

February 7

Preculture by_???
We started preculture at 12:10.

February 8

March 1

March 2

March 3

March 4

March 5

March 6

Restriction

GFPEcoR1Spe1BufferMBSAMilliQtotal
120.50.530.513.530

March 7

Electrophoresis
Electrophoresis0307.JPG

1. 1kb ladder 2µL
2. pspA (PCR product)2.5µL + Loading Dye 0.5µL
3. GFP 30µL + Loading Dye 6µL
4. 1kb ladder 2µL

  • The GFP was gel extracted on 3/6 and concentrated by Vacuum in 150µg/mL

Ligation

torApSB1C3Ligation High Ver.2total
3339
pspApSB1C3Ligation High Ver.2total
4239

at 4℃, for overnight

  • torA→31.8ng/µL×3µL=95.4ng=0.529pmol
  • pSB1C3→19.4ng/µL×3µL=58.2ng=0.042pmol
  • pspA→49.3ng/µL×4µL=197.2ng=0.308pmol
  • pSB1C3→19.4ng/µL×2µL=38.8ng=0.029pmol

March 8

Restriction

pSB4K5EcoR1Spe1BufferMBSAMilliQtotal
200.20.230.26.430

at 37℃ for 1 hour.
→Purification : 36.6ng/µL

Ligation

KilpSB4K5Ligation High Ver.2total
101516

at 4℃ for overnight

  • Kil→37.7ng/µL×10µL=377ng=879fmol
  • pSB4K5→36.6ng/µL×1µL=36.6ng=86fmol

Liquid culture
Lacp + pSB3C5 -1, 2

Transformation

torApspAcompetent celltotal
101011
011011

We use commercially available competent cells in this time.

March 9

Restriction

tatABCDXba1Pst1BufferMBSAMilliQtotal
100.20.230.316.330

at 37℃ for 1hour
→purification : 75.0μg/mL (1.11 260/280 , 0.81 260/230)

Miniprep
lacP + pSB3C5 1 80.5μg/mL (1.78 260/280 , 2.00 260/230)
lacP + pSB3C5 2 107.2μg/mL (1.83 260/280 , 1.90 260/230)

Colony PCR

Quick TaqVFVRMilliQtotal
25112350

94℃ 2min
94℃ 30sec
55℃ 30sec
68℃ 6sec
25cycles

Ligation

tatABCDconstP J23107Ligation High Ver.2total
5139

tatABCD : 227fmol
constP J23107 : 21fmol

Transformation

pspAtorAKilcompetent cell
110010
201010
300110

Miniprep
4mL of plusgrow which had been cultured for overnight.
pSB4K5 : 80.5μg/mL

March 10

Screening PCR

Quick TaqVFVRMilliQtotal
25112350
Electrophoresis0310.JPG

Then we did electrophoresis to confirm.
1.1kb ladder
2.kil (649bp)
3,4,5, pspA (969bp)
6,1kb ladder

1, 100bp ladder
2,3,4, torA signal

Restriction

LacP-pSB3C5Spe1Pst1BufferMBSAMilliQtotal
100.20.220.27.420

for 2.5 hours at 37℃
→purification 29.0ng/μL

torAXba1Pst1BufferMBSAMilliQtotal
100.20.220.27.420

for 2.5 hours at 37℃
→purification 91.8ng/μL

Ligation

torALacp-pSB3C5Ligation High Ver.2total
4239

torA : 929fmol
Lacp-pSB3C5 : 284fmol
for overnight at 4℃

March 11

Miniprep
We used 3μL of plus grow that we had cultured for overnight.
torA : 62.8ng/μL

Screening PCR

Quick TaqVFVRMilliQtotal
25112350

Electrophoresis

Electrophoresis031101.JPG

1. 1kb ladder
2〜11. pspA (969bp)
12. 1kb ladder

The results were shown as photograph in the right.

It seemed that there were shorter sample than expected sample, so we did electrophoresis with pspA which was product of PCR and pspA which had already cut with EcoR1 and Spe1.





Electrophoresis031102.JPG

1.1kb ladder
2. pspA (PCR product)
3, pspA (Eco, Spe)
4〜6, pspA (colony PCR)
7,1kb ladder

The results were shown as photograph in the right.

March 12

Transformation

DT(1ng/μL)DT(0.1ng/μL)KillacP-torAMilliQcompetent celltotal
100002021
010002021
005005051
000505051
000012021

Restriction

pspAEcoR1Spe1BufferMBSAMilliQtotal
100.50.530.315.730

at 37℃ for 4 hours
→ We did purification and got 48.3ng/μL pspA.

Ligation

pspApSB1C3MilliQLigation High Ver.2total
42039
22026
02226

March 13

Miniprep pSB1C3 74.2µg/mL 1.65 (260/280) 1.31 (260/230)

March 14

Restriction

pSB1C3EcoR1Spe1BufferMBSAMilliQtotal
200.20.240.415.240

We did gel extraction and got 47.2ng/µL pSB1C3 but we did not cut out RFP.

Ligation

torApSB1C3Ligation High Ver.2total
4239
MilliQpSB1C3Ligation High Ver.2total
4239

at 16℃, for 1 hour

  • torA : 0.707pmol
  • pSB1C3 : 0.068pmol

Liquid culture
We cultured Lacp-pSB3C5 1,2,3,4,5 (CP tolerance)on culture with Amp.
→Only 4 which did not be cultured succeeded.

March 15

Liquid culture
We cultured Lacp-pSB3C5 6,7,8,9,10 on culture with ampicillin.
→6,8,9,10 were succeeded.

Restriction

GFPEcoR1Spe1Buffer2BSAMilliQtotal
100.50.530.515.530
DTEcoR1Xba1Buffer2BSAMilliQtotal
100.50.530.515.530

for 2 hours at 37℃.

Miniprep
pspA (pSB1C3) 40.5ng/µL

Ligation

pspADTLigation High Ver.2total
51.539.5
  • pspA : 385fmol
  • DT : 36fmol

Transformation

J23107-tatABCDDT (0.1ng/µL)DT (0.01ng/µL)pspA-DTcompetent cells on 3/15total
20002022
02002022
00202022
00022022

Screening PCR
Kil, pspA and torA

Quick TaqPrimer-rPrimer-fMilliQtotal
25112350

March 16

Miniprep
torA (pSB1C3) 68.8ng/µL
Kil (pSB4K5) 92.7ng/µL
torA was red for some reason. We do not know why.

Colony PCR

Quick TaqPrimer-rPrimer-fMilliQtotal
25112350

94℃, 2min
94℃, 30sec
55℃, 30sec
68℃, 6sec
→25cycles

Electrophoresis

Electrophoresis0316.JPG

The results were shown as photograph in the right.

Checking Transformation Efficiency
competent cells that were made on March 15.
DNA : 0.02ng → 668 colonies Transformation Efficiency : 3.3×10^7
DNA : 0.2ng → 1739 colonies Transformation Efficiency : 8.7×10^6

Restriction

pSB1C3EcoR1Spe1BSABufferMBufferHMilliQtotal
200.20.20.3306.330
50.200.20212.620

at 37℃, for 2 hours.
We did gel extraction for product with EcoR1, Spe1. We got 46.1ng/µL pSB1C3.

Kil(pSB4K5)EcoR1Pst1BSABufferHMilliQtotal
50.20.20.2212.420
50.200.2212.620

for overnight at 37℃.
We did this to confirm.

pspA (pSB1C3)EcoR1Pst1BSABufferHMilliQtotal
50.20.20.2212.420
50.200.2212.620

for overnight at 37℃.
We did this to confirm.

Ligation

torApSB1C3Ligation High Ver.2total
4239
MilliQpSB1C3Ligation High Ver.2total
4239

at 16℃, for overnight

  • torA→767fmol
  • pSB1C3→68fmol

March 17

Miniprep J23107-tatABCD 72.7ng/µL
pspA-DT 50.5ng/µL

Checking the Insert

J21037-tatABCDEcoR1Pst1BSABufferHMilliQtotal
50.20.20.2212.420
50.200.2212.620

Success.

pspA-DTEcoR1Pst1BSABufferHMilliQtotal
50.20.20.2212.420
50.200.2212.620

Failed.


March 19

Restriction

DTEcoR1Xba1BSABufferMMilliQtotal
100.20.20.3316.330

We did Gel extraction and got 17.2ng/µL of DT.

Ligation

KilDTLigation High Ver.2total
102618

We did this for an hour at 16℃.

Restriction

GFPEcoR1Spe1BSABufferMMilliQtotal
100.50.50.5315.530

We did this for 4 hours at 37℃

Ligation

pspADTLigation High Ver.2total
55515
pspApSB1C3Ligation High Ver.2total
4138

We did these for an hour at 16℃.

  • pspA (5µL)→377fmol
  • DT→39fmol
  • pspA (4µL)→339fmol
  • pSB1C3→34fmol

Transformation
pspA-DT, pspA (pSB1C3), torA (pSB1C3) and GFP-DT

March 20

Screaning PCR

Quick TaqPrimer-RPrimer-FMilliQtotal
25112350
  • pspA→○
  • pspA-DT→○
  • GFP-DT→○
  • torA→×
  • Kil-DT 6 of 8 sumples→○
Quick TaqVRVFMilliQtotal
25112350
  • pspA→○
  • pspA-DT→×

Restriction

torAEcoR1Spe1BSABufferMMilliQtotal
100.30.30.3316.130
DTEcoR1Xba1BSABufferMMilliQtotal
100.20.20.3316.330

We did this for overnight at 37℃. And we did purification.
torA 34.2ng/µL
DT 28.0ng/µL

March 21

Miniprep
GFP-DT-1 : 77.7ng/µL
GFP-DT-2 : 67.6ng/µL

Restriction

pSB1C3EcoR1Spe1BSABufferMMilliQtotal
100.20.20.3316.330
50.200.2212.620

We did this for 3 hours at 37℃, and then we did gel extraction. We got 25.1ng/µL pSB1C3

GFP-DTEcoR1Pst1Xba1BSABufferMMilliQtotal
50.20.200.2212.420
50.2000.2212.620
100.200.20.3316.330

We did this for 3 hours at 37℃, and then we did Purification. We got 30.7ng/µL GFP-DT.

Ligation

torApSB1C3pspADTGFP-DTLigation High Ver.2total
14100038
201700412
300530412
430005412

We did this for an hour at16℃.

March 22

PCR
We did PCR to amplify torA_signal that was product of PCR at March 5 with redesigned primers.

BufferdNTPsMgSO4Primer-FPrimer-RTemplateMilliQKOD plus neototal
5531.51.50.532.5150

94℃, 2min
98℃, 10sec
60℃, 30sec
68℃, 10sec
→30cycles
→Purification 110.7ng/µL

Restriction

torAEcoR1Spe1BSABufferMMilliQtotal
100.20.20.3316.330

Ligation

torApSSB1C3pspADTGFP-DTLigation hightotal
143000411
23000339
300550515
  • torA (4µL)→512fmol
  • pSB1C3→54fmol
  • torA (3µL)→384fmol
  • GFP-DT→36fmol
  • pspA→377fmol
  • DT→65fmol

March 23

Screening PCR
torA (pSB1C3), torA-GFP-DT and pspA-DT

Quick TaqVFVRMilliQtotal
25112350

E.coli in liquid culture that had pspA(pSB1C3) also expressed RFP.

Restriction

pspAEcoR1Spe1BufferMBSAMilliQtotal
50.30.330.321.130

And then we did ethanol precipitation

Ethanol precipitation
pspA 11.5ng/µL.

Miniprep
Lacp+pSB3C5-8 77.9µg/mL
Lacp+pSB3C5-10 69.0µg/mL
Kil+DT-4 58.0µg/mL
Kil+DT-7 15.2µg/mL

Restriction

Lacp+pSB3C5-8Spe1Pst1Buffer2BSAMilliQtotal
200.50.530.55.530
Kil+DT-4Xba1Pst1Buffer2BSAMilliQtotal
200.50.530.55.530

at 37℃ for 1.5 hours
And then we did Gel extraction.

Gel Extraction
Lacp+pSB3C5-8 40.4µg/mL
Kil+DT-4 26.8µg/mL

March 26

Miniprep
torA(pSB1C3) 18.5[ng/µL]
torA-GFP-DT 20.0[ng/µL]

Restriction

torA(pSB1C3)EcoR1Pst1BufferHBSAMilliQtotal
50.20.220.212.420
50.2020.212.620
torA-GFP-DTEcoR1Xba1Pst1BufferHBufferMBSAMilliQtotal
50.200.2200.212.420
50.200200.212.620
2000.20.2030.36.330

We did Gel extraction and then got ??? 28.7[ng/µL]

Ligation

Lacp (pSB3C5)torA-GFP-DTLigation High Ver.2total
1539
  • Lacp : 22fmol
  • torA-GFP-DT : 197fmol
pspApSB1C3Ligation High Ver.2total
101516
pspADTLigation High Ver.2total
101516
  • pspA : 180fmol
  • pSB1C3 : 18fmol
  • DT : 16fmol
LacP(pSB3C5)Kil-DTLigation High Ver.2total
1539

for 2 hours at 16℃

Transformation

Lacp-Kil-DTcompetent celltotal
11011

March 27

Miniprep by We retryed miniprep of torA(pSB1C3).
We got torA and its concentration was 39.3[ng/µL].

Transformation

NameWellSampleCompetent CellsTotalPlateColony
BBa_K117004 14J(2011 plate2)520???

We added 100[µL] of culture medium before we started culturing the E.coli.

Screening PCR

Quick TaqVF2VRMilliQTotal
25112350

Lacp-torA-GFP-DT 1, 3, 5, 6, 8, 9 was successful.
pspA-DT was failed.
E.coli that had pspA(pSB1C3) did not make any colony.
Lacp-Kil-DT 1,2,3,4,5,6,7,8 was failed.

Liquid culture
Lacp-torA-GFP-DT

Florigen

Team:Kyoto/Florigen/Note

Golden Gate Assembly

August 10

Golden Gate Assembly method This method helps us to constract some genes quickly and we can design the order of constractions. We are trying to construct a gene cycle composed of 6 genes( pSB1A3, lacI, GFP, RFP, GFP, DT). After this experiment is confirmed to work, we will so some experiment to check how the numbers of promorters influences the strength of transcription.

Retrieved from "http://2012.igem.org/Team:Kyoto/Notebook"