Team:UT-Tokyo/Counter/Project/Lab

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Contents 1 Favorite Parts 2 Other Pars 3 DNA construction 3.1 Transformation 3.2 Electrophoresis 3.3 Colony PCR 3.4 Miniprep 3.5 Digestion 3.6 Gel extraction 3.7 Ligation 4 Assay method 4.1 Influence on growth of E. coli 4.2 Sigma factor and its crresponding promoter 4.3 Reset function 4.4 Riboregulator 4.5 Positive feedback 5 7,14,2014 5.1 Lab member 5.2 Contents 5.2.1 Making Plate Culture 6 7,15,2014 6.1 Lab member 6.2 Contents 6.2.1 Making DW 100mL 6.2.2 TF 7 7,16,2014 7.1 Lab member 7.2 Contents 7.2.1 TF 7.2.2 preculture 8 7,17,2014 8.1 Lab member 8.2 Contents 8.2.1 Making glycerol stock/miniprep 8.2.2 making reagent 8.2.3 preculture 9 7,18,2014 9.1 Lab member 9.2 Contents 9.2.1 miniprep 9.2.2 Making competent cell 9.2.3 making reagent 10 7,22,2014 10.1 Lab member 10.2 Contents 10.2.1 Cut check 10.2.2 TF 10.2.3 TF 10.2.4 preculture 11 7,23,2014 11.1 Lab member 11.2 Contents 11.2.1 miniprep 11.2.2 Making plate 12 7,28,2014 12.1 Lab member 12.2 Contents 12.2.1 digestion,gel extraction 12.2.2 colony PCR 12.2.3 Measuring cfu 13 7,29,2014 13.1 Lab member 13.2 Contents 13.2.1 TF 13.2.2 Measuring cfu 13.2.3 Electrophoresis check 14 7,30,2014 14.1 Lab member 14.2 Contents 14.2.1 colony check 14.2.2 Cut check 14.2.3 colony PCR 14.2.4 preculture 15 7,31,2014 15.1 Lab member 15.2 Contents 15.2.1 miniprep 15.2.2 digestion,gel extraction 15.2.3 ligation 16 8,1,2014 16.1 Lab member 16.2 Contents 16.2.1 TF 16.2.2 ligation Check 17 8,4,2014 17.1 Lab member 17.2 Contents 17.2.1 Plate check 17.2.2 digestion 17.2.3 Electrophoresis 17.2.4 preculture 18 8,5,2014 18.1 Lab member 18.2 Contents 18.2.1 miniprep 18.2.2 Cut check 18.2.3 Elrctrophoresis 18.2.4 preculture 19 8,6,2014 19.1 Lab member 19.2 Contents 19.2.1 miniprep 19.2.2 digestion, gel extraction 19.2.3 making gel 19.2.4 ligation 19.2.5 TF 20 8，7,2014 20.1 Lab member 20.2 Contents 20.2.1 colony PCR 20.2.2 digestion, gel extraction 20.2.3 ligation 20.2.4 Cut check 21 8,8,2014 21.1 Lab member 21.2 Contents 21.2.1 Cut check sample Electrophoresis 21.2.2 digestion, gel extraction 21.2.3 ligation 21.2.4 TF 21.2.5 overlap extension PCR →PCR clean up 21.2.6 making gel 22 8,9,2014 22.1 Lab member 22.2 Contents 22.2.1 Plate and test tube check 23 8,11,2014 23.1 Lab member 23.2 Contents 23.2.1 colony PCR 23.2.2 overlap extension PCR →PCR clean up 23.2.3 Making Plate 23.2.4 preculture 24 8,12,2014 24.1 Lab member 24.2 Contents 24.2.1 miniprep 24.2.2 digestion →gel extraction 24.2.3 colony PCR 24.2.4 ligation →TF 24.2.5 Making　reagent 25 8,13,2014 25.1 Lab member 25.2 Contents 25.2.1 miniprep 25.2.2 digestion→ gel extrction 25.2.3 ligation→TF 25.2.4 colony PCR 25.2.5 preculture 26 8,14,2014 26.1 Lab member 26.2 Contents 26.2.1 miniprep 26.2.2 overlap extension PCR 26.2.3 digestion → gel extranction 26.2.4 gel making 26.2.5 colony PCR 26.2.6 ligation → TF 27 8,15,2014 27.1 Lab member 27.2 Contents 27.2.1 miniprep 27.2.2 digestion → gel extraction 27.2.3 colony PCR 27.2.4 ligation → TF 27.2.5 preculture 28 8,16,2014 28.1 Lab member 28.2 Contents 28.2.1 miniprep 29 8,18,2014 29.1 Lab member 29.2 Contents 29.2.1 digestion → gel extraction 29.2.2 sequence 29.2.3 colony PCR 29.2.4 ligation → TF 29.2.5 preculture 30 8,19,2014 30.1 Lab member 30.2 Contents 30.2.1 miniprep 30.2.2 digestion 30.2.3 making gel 30.2.4 colony PCR 30.2.5 sequence 31 8,20,2014 31.1 Lab member 31.2 Contents 31.2.1 PCR 31.2.2 TF 32 8,21,2014 32.1 Lab member 32.2 Contents 32.2.1 digestion → gel extraction 32.2.2 colony PCR 32.2.3 ligation → TF 32.2.4 preculture 33 8,22,2014 33.1 Lab member 33.2 Contents 33.2.1 miniprep 33.2.2 colony PCR 33.2.3 digestion → gel extraction 33.2.4 ligation → TF 33.2.5 preculture 34 8,23,2014 34.1 Lab member 34.2 Contents 34.2.1 miniprep 35 8,25,2014 35.1 Lab member 35.2 Contents 35.2.1 digestion → gel extraction 35.2.2 Plate　making 35.2.3 colony PCR 35.2.4 sequence 35.2.5 ligation → TF 35.2.6 preculture 36 8,26,2014 36.1 Lab member 36.2 Contents 36.2.1 miniprep 36.2.2 colony PCR 36.2.3 digestion → gel extraction 36.2.4 ligation → TF 36.2.5 preculture 36.2.6 sequence 36.2.7 sequence 37 8,27,2014 37.1 Lab member 37.2 Contents 37.2.1 miniprep 37.2.2 digestion → gel extraction 37.2.3 colony PCR 37.2.4 ligation → TF 37.2.5 preculture 37.2.6 Remarks 38 8,28,2014 38.1 Lab member 38.2 Contents 38.2.1 miniprep 38.2.2 digestion →gel extraction 38.2.3 overlap extension PCR 38.2.4 check & colony PCR 38.2.5 ligation → TF 39 8,29,2014 39.1 Lab member 39.2 Contents 39.2.1 miniprep 39.2.2 digestion →gel extraction 39.2.3 PCR 39.2.4 check & colony PCR 39.2.5 sequence order 39.2.6 ligation → TF 39.2.7 preculture 40 8,30,2014 40.1 Lab member 40.2 Contents 40.2.1 miniprep 40.2.2 PCR 41 9,1,2014 41.1 Lab member 41.2 Contents 41.2.1 PCR clean up 41.2.2 digestion → gel extraction 41.2.3 Making plate 41.2.4 colony PCR 41.2.5 Ligation 41.2.6 preculture 42 9,2,2014 42.1 Lab member 42.2 Contents 42.2.1 miniprep 42.2.2 digestion → gel extraction 42.2.3 sequence 42.2.4 PCR 42.2.5 check & colony PCR 42.2.6 ligation → TF 42.2.7 preculture 42.2.8 We got a result!! 43 9,3,2014 43.1 Lab member 43.2 Contents 43.2.1 sequence 43.2.2 digestion → gel extraction 43.2.3 colony PCR 43.2.4 PCR 43.2.5 ligation → TF 44 9,4,2014 44.1 Lab member 44.2 Contents 44.2.1 miniprep 44.2.2 digestion → gel extraction 44.2.3 PCR check & clean up 44.2.4 Making Plate 44.2.5 digestion 44.2.6 colony PCR 44.2.7 ligation → TF 44.2.8 miniprep 44.2.9 preculture 45 9,5,2014 45.1 Lab member 45.2 Contents 45.2.1 miniprep 45.2.2 digestion → gel extraction 45.2.3 ligation → TF 45.2.4 PCR 45.2.5 colony PCR 45.2.6 preculture 46 9,6,2014 46.1 Lab member 46.2 Contents 46.2.1 miniprep 46.2.2 PCR 46.2.3 check & colony PCR 47 9,8,2014 47.1 Lab member 47.2 Contents 47.2.1 digestion → gel extaction 47.2.2 colony PCR 47.2.3 PCR 47.2.4 ligation → TF 47.2.5 preculture 48 9,9,2014 48.1 Lab member 48.2 Contents 48.2.1 miniprep 48.2.2 digestion → gel extraction 48.2.3 colony PCR 48.2.4 sequence 48.2.5 ligation → TF 48.2.6 preculture 49 9,10,2014 49.1 Lab member 49.2 Contents 49.2.1 miniprep 49.2.2 digestion → gel extraction 49.2.3 colony PCR 49.2.4 ligation → TF 49.2.5 preculture 50 9,11,2014 50.1 Lab member 50.2 Contents 50.2.1 miniprep 50.2.2 digestion → gel extraction 50.2.3 sequence 50.2.4 Plate Making 50.2.5 colony PCR 50.2.6 ligation → TF 50.2.7 preculture 51 9,12,2014 51.1 Lab member 51.2 Contents 51.2.1 miniprep 51.2.2 digestion → gel extraction 51.2.3 colony PCR 51.2.4 sequence 51.2.5 ligation → TF 51.2.6 ligation 51.2.7 preculture 52 9,13,2014 52.1 Lab member 52.2 Contents 52.2.1 miniprep 53 9,15,2014 53.1 Lab member 53.2 Contents 53.2.1 digestion → gel extraction 53.2.2 colony PCR 53.2.3 ligation → TF 53.2.4 preculture 54 9,16,2014 54.1 Lab member 54.2 Contents 54.2.1 miniprep 54.2.2 colony PCR 54.2.3 digestion → gel extraction 54.2.4 ligation → TF 54.2.5 preculture 54.2.6 assay 55 9,17,2014 55.1 Lab member 55.2 Contents 55.2.1 miniprep 55.2.2 digestion → gel extraction 55.2.3 colony PCR 55.2.4 ligation → TF 55.2.5 assay 56 9,18,2014 56.1 Lab member 56.2 Contents 56.2.1 miniprep 56.2.2 digestion → gel extraction 56.2.3 colony PCR 56.2.4 ligation → TF 56.2.5 preculture 57 9,19,2014 57.1 Lab member 57.2 Contents 57.2.1 miniprep 57.2.2 digestion → gel extraction 57.2.3 colony PCR 57.2.4 ligation → TF 57.2.5 preculture 58 9,20,2014 58.1 Lab member 58.2 Contents 58.2.1 miniprep 59 9,22,2014 59.1 Lab member 59.2 Contents 59.2.1 digestion → gel extraction 59.2.2 colony PCR 59.2.3 ligation - TF 60 9,23,2014 60.1 Lab member 60.2 Contents 60.2.1 digestion, gel extraction 60.2.2 miniprep 60.2.3 colony PCR 60.2.4 ligation,TF 61 9,24,2014 61.1 Lab member 61.2 Contents 61.2.1 miniprep 61.2.2 digestion,gel extraction 61.2.3 ligation,TF 62 9,25,2014 62.1 Lab member 62.2 Contents 62.2.1 miniprep 62.2.2 digestion,gel extraction 62.2.3 ligation,TF 62.2.4 Making competent cell 62.2.5 preculture 62.2.6 competent cell(made at 0911) check 62.2.7 result of check 63 9,26,2014 63.1 Lab member 63.2 Contents 63.2.1 miniprep 63.2.2 digestion 63.2.3 colony PCR 63.2.4 ligation,TF 63.2.5 preculture 64 9,27,2014 64.1 Lab member 64.2 Contents 64.2.1 TF 64.2.2 miniprep 64.2.3 TF 65 9,29,2014 65.1 Lab member 65.2 Contents 65.2.1 digestion,gel extraction 65.2.2 Gel making 65.2.3 ligation 65.2.4 preculture 65.2.5 PCR 66 9,30,2014 66.1 Lab member 66.2 Contents 66.2.1 miniprep 66.2.2 digestion,gel extraction 66.2.3 colony PCR 66.2.4 Making M9 medium 66.2.5 digestion(cutcheck) 67 10,1,2014 67.1 Lab member 67.2 Contens 67.2.1 miniprep 67.2.2 sequence order 68 10,2,2014 68.1 Lab member 68.2 Contents 68.2.1 assay 68.2.2 culture 68.2.3 PCR 68.2.4 Making M9 medium 68.2.5 PCR check 68.2.6 PCR 68.2.7 preculture 69 10,3,2014 69.1 Lab member 69.2 Contents 69.2.1 PCR clean up and check 69.2.2 digestion,gel extraction 69.2.3 PCR 69.2.4 gel making 69.2.5 ligation,TF 69.2.6 PCR 70 10,4,2014 70.1 Lab member 70.1.1 assay1 70.1.2 colony PCR 70.1.3 preculture 71 10,5,2014 71.1 Lab member 71.2 Contents 71.2.1 miniprep 71.2.2 preculture 72 10,6,2014 72.1 Lab member 72.2 Contents 72.2.1 assay1,3 72.2.2 Making M9 medium 72.2.3 result of OD600 72.2.4 assay1 72.2.5 preculture 73 10,7,2014 73.1 Lab member 73.2 Contents 73.2.1 assay 73.2.2 OD600 before subculture 73.2.3 PCR 73.2.4 PCR 74 10,8,2014 74.1 Lab member 74.2 Contents 74.2.1 making gel 74.2.2 subculture 74.2.3 PCR clean up and check 74.2.4 sequence order 74.2.5 TF 75 10,9,2014 75.1 Lab member 75.2 Contents 75.2.1 digestion,gel extraction 75.2.2 making gel 75.2.3 making plate 75.2.4 ligation 75.2.5 preculture 76 10,10,2014 76.1 Lab member 76.1.1 assay1,3 76.1.2 making M9 medium

Favorite Parts

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Name	Type	Description	Designer	Length
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461000" abp="49">BBa_K1461000</A>	RBS	crRBS	Takefumi Yoshikawa	52
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461001" abp="64">BBa_K1461001</A>	Regulatory	ecf20 promoter	Takefumi Yoshikawa	80
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461002" abp="79">BBa_K1461002</A>	Regulatory	ecf11 promoter	Takefumi Yoshikawa	80
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461003" abp="94">BBa_K1461003</A>	RNA	taRNA	Takefumi Yoshikawa	71
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461004" abp="109">BBa_K1461004</A>	Coding	ecf20	Takefumi Yoshikawa	582
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461005" abp="124">BBa_K1461005</A>	Coding	ecf11	Takefumi Yoshikawa	609
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461006" abp="139">BBa_K1461006</A>	Coding	anti ecf20	Takefumi Yoshikawa	432
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461007" abp="154">BBa_K1461007</A>	Coding	anti ecf11	Takefumi Yoshikawa	663

Other Pars

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Name	Type	Description	Designer	Length
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461008" abp="194">BBa_K1461008</A>	Coding	GFP (AAV tag +)	Takefumi Yoshikawa	742
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461009" abp="209">BBa_K1461009</A>	Coding	GFP (AAV tag +)	Takefumi Yoshikawa	742
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461200" abp="329">BBa_K1461200</A>	Composite	taRNA (+d.term)	Takefumi Yoshikawa	208
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461201" abp="344">BBa_K1461201</A>	Composite	ecf20 (+RBS, +d.term)	Takefumi Yoshikawa	737
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461202" abp="359">BBa_K1461202</A>	Composite	ecf11 (+RBS, +d.term)	Takefumi Yoshikawa	764
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461203" abp="374">BBa_K1461203</A>	Composite	cr-ecf20 (+RBS, +d.term)	Takefumi Yoshikawa	777
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461204" abp="389">BBa_K1461204</A>	Composite	cr-ecf11 (+RBS, +d.term)	Takefumi Yoshikawa	804
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461205" abp="404">BBa_K1461205</A>	Composite	cr-GFP (+RBS, +d.term)	Takefumi Yoshikawa	915
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461206" abp="419">BBa_K1461206</A>	Composite	anti ecf20 (+RBS, +d.term)	Takefumi Yoshikawa	587
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461207" abp="434">BBa_K1461207</A>	Composite	anti ecf11 (+RBS, +d.term)	Takefumi Yoshikawa	818
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461300" abp="959">BBa_K1461300</A>	Composite	RBS-GFP-4miR-142-3p bindng sites-d.term	Shunsuke Sumi	1007
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461301" abp="974">BBa_K1461301</A>	Composite	RBS-GFP-4 miR-142-5p bindng sites-d.term	Shunsuke Sumi	999
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461302" abp="989">BBa_K1461302</A>	Composite	RBS-GFP-miR 142 3p non-binding site-d.term	Shunsuke Sumi	1001
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461303" abp="1004">BBa_K1461303</A>	Composite	RBS-GFP-miR 142 5p non-binding site-d.term	Shunsuke Sumi	1007
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461304" abp="1019">BBa_K1461304</A>	Composite	RBS-GFP-2miR 142 3p bindng sites-2miR 142 5p bindng sites-d.term	Shunsuke Sumi	1003
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461305" abp="1034">BBa_K1461305</A>	Composite	RBS-GFP-2miR 142 3p non-bindng sites-2miR 142 5p non-bindng sites-d.term	Shunsuke Sumi	1003
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461208" abp="449">BBa_K1461208</A>	Generator	taRNA (arabinose-induced) (+d.term)	Takefumi Yoshikawa	346
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461209" abp="464">BBa_K1461209</A>	Generator	arabinose-induced ecf20 generator	Takefumi Yoshikawa	875
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461210" abp="479">BBa_K1461210</A>	Generator	constitutive ecf20 generator	Takefumi Yoshikawa	780
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461211" abp="494">BBa_K1461211</A>	Generator	arabinose-induced ecf11 generator	Takefumi Yoshikawa	902
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461212" abp="509">BBa_K1461212</A>	Generator	constitutive ecf11 generator	Takefumi Yoshikawa	807
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461213" abp="524">BBa_K1461213</A>	Generator	arabinose-induced cr-ecf20 generator	Takefumi Yoshikawa	915
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461214" abp="539">BBa_K1461214</A>	Generator	constitutive cr-ecf20 generator	Takefumi Yoshikawa	820
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461215" abp="554">BBa_K1461215</A>	Generator	arabinose-induced cr-ecf11 generator	Takefumi Yoshikawa	942
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461216" abp="569">BBa_K1461216</A>	Generator	constitutive cr-ecf11 generator	Takefumi Yoshikawa	847
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461217" abp="584">BBa_K1461217</A>	Generator	arabinose-induced cr-GFP generator	Takefumi Yoshikawa	1053
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461218" abp="599">BBa_K1461218</A>	Generator	constitutive cr-GFP generator	Takefumi Yoshikawa	958
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461219" abp="614">BBa_K1461219</A>	Generator	ecf20-induced GFP generator	Takefumi Yoshikawa	963
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461220" abp="629">BBa_K1461220</A>	Generator	ecf11-induced GFP generator	Takefumi Yoshikawa	963
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461221" abp="644">BBa_K1461221</A>	Generator	ecf20 generator (positive feedback)	Takefumi Yoshikawa	825
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461222" abp="659">BBa_K1461222</A>	Generator	ecf11 generator (positive feedback)	Takefumi Yoshikawa	852
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461223" abp="674">BBa_K1461223</A>	Generator	anti ecf20 generator (pLac)	Takefumi Yoshikawa	795
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461224" abp="689">BBa_K1461224</A>	Generator	anti ecf11 generator (pLac)	Takefumi Yoshikawa	1026
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461225" abp="704">BBa_K1461225</A>	Measurement	ecf20 promoter measurement system	Takefumi Yoshikawa	1846
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461226" abp="719">BBa_K1461226</A>	Measurement	ecf11 promoter measurement system	Takefumi Yoshikawa	1873
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461231" abp="794">BBa_K1461231</A>	Measurement	arabinose-induced GFP and taRNA generator	Takefumi Yoshikawa	1407
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461232" abp="809">BBa_K1461232</A>	Measurement	single counter	Takefumi Yoshikawa	1312
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461233" abp="824">BBa_K1461233</A>	Measurement	ecf20 promoter mesurement system (positive feedback)	Takefumi Yoshikawa	2679
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461234" abp="839">BBa_K1461234</A>	Measurement	ecf11 promoter mesurement system (positive feedback)	Takefumi Yoshikawa	2733
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461235" abp="854">BBa_K1461235</A>	Measurement	resettable counter device using ecf20 (w/o crRBS, taRNA)	Takefumi Yoshikawa	3482
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461236" abp="869">BBa_K1461236</A>	Measurement	resettable counter device using ecf11 (w/o crRBS, taRNA)	Takefumi Yoshikawa	3767
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461237" abp="884">BBa_K1461237</A>	Measurement	sigma factors cross-talk assay 1	Takefumi Yoshikawa	1873
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461238" abp="899">BBa_K1461238</A>	Measurement	sigma factors cross-talk assay 2	Takefumi Yoshikawa	1846
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461227" abp="734">BBa_K1461227</A>	Project	resettable single counter system using ecf20	Takefumi Yoshikawa	2671
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461228" abp="749">BBa_K1461228</A>	Project	reset function using ecf20	Takefumi Yoshikawa	2576
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461229" abp="764">BBa_K1461229</A>	Project	resettable single counter system using ecf11	Takefumi Yoshikawa	2834
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461230" abp="779">BBa_K1461230</A>	Project	reset function using ecf11	Takefumi Yoshikawa	2929
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461239" abp="914">BBa_K1461239</A>	Project	σ-Re Counter (double, using ecf20)	Takefumi Yoshikawa	3821
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461240" abp="929">BBa_K1461240</A>	Project	σ-Re Counter (double, using ecf11)	Takefumi Yoshikawa	4106
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461241" abp="944">BBa_K1461241</A>	Project	σ-Re Counter (triple)	Takefumi Yoshikawa	6615
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461104" abp="284">BBa_K1461104</A>	Regulatory	EpCAM promoter	Shunsuke Sumi	?
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461105" abp="299">BBa_K1461105</A>	Regulatory	EpCAM promoter(mutant)	Shunsuke Sumi	?
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461100" abp="224">BBa_K1461100</A>	RNA	miR-142-3p target binding site	ドリキャス?	25
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461101" abp="239">BBa_K1461101</A>	RNA	miR-142-5p target bindng site	Shunsuke Sumi	23
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461102" abp="254">BBa_K1461102</A>	RNA	miRNA-142 binding site control1	Shunsuke Sumi	23
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461103" abp="269">BBa_K1461103</A>	RNA	miRNA-142 binding site control2	Shunsuke Sumi	25
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461306" abp="1049">BBa_K1461306</A>	Translational_Unit	pCMV-RBS-GFP-4 miR 142 3p site-d.term	Shunsuke Sumi	1595
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461307" abp="1064">BBa_K1461307</A>	Translational_Unit	pCMV-RBS-GFP-4 miR 142 5p site-d.term	Shunsuke Sumi	1587
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461308" abp="1079">BBa_K1461308</A>	Translational_Unit	pCMV-RBS-GFP-4 miR 142 3p non-binding sites-d.term	Shunsuke Sumi	1589
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461309" abp="1094">BBa_K1461309</A>	Translational_Unit	pCMV-RBS-GFP-4 miR 142 5p non-bindgin sites-d.term	Shunsuke Sumi	1595
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461310" abp="1109">BBa_K1461310</A>	Translational_Unit	pCMV-RBS-GFP-2 miR 142 3p bindgin sites-2 miR 142 5p binding site-d.term	Shunsuke Sumi	1591
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461311" abp="1124">BBa_K1461311</A>	Translational_Unit	pCMV-RBS-GFP-2 miR 142 3p non-bindgin sites-2 miR 142 5p non-binding site-d.term	Shunsuke Sumi	1591
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461312" abp="1139">BBa_K1461312</A>	Translational_Unit	pEpCAM-RBS-GFP-4 miR 142 3p site-d.term	Shunsuke Sumi	738
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461313" abp="1154">BBa_K1461313</A>	Translational_Unit	pEpCAM-RBS-GFP-4 miR 142 5p site-d.term	Shunsuke Sumi	?
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461314" abp="1169">BBa_K1461314</A>	Translational_Unit	pEpCAM-RBS-GFP-4 miR 142 3p non-bindng site-d.term	Shunsuke Sumi	?
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461315" abp="1184">BBa_K1461315</A>	Translational_Unit	pEpCAM-RBS-GFP-4 miR 142 5p non-bindng site-d.term	Shunsuke Sumi	?
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461316" abp="1199">BBa_K1461316</A>	Translational_Unit	pEpCAM-RBS-GFP-2 miR 142 3p bindgin sites-2 miR 142 5p binding site-d.term	Shunsuke Sumi	?
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461317" abp="1214">BBa_K1461317</A>	Translational_Unit	pEpCAM-RBS-GFP-2 miR 142 3p non-bindgin sites-2 miR 142 5p non-binding site-d.term	Shunsuke Sumi	?
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461318" abp="1229">BBa_K1461318</A>	Translational_Unit	pEpCAM(mutant)-RBS-GFP-4 miR 142 3p site-d.term	Shunsuke Sumi	?
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461319" abp="1244">BBa_K1461319</A>	Translational_Unit	pEpCAM(mutant)-RBS-GFP-4 miR 142 5p site-d.term	Shunsuke Sumi	?
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461320" abp="1259">BBa_K1461320</A>	Translational_Unit	pEpCAM(mutant)-RBS-GFP-4 miR 142 3p non-bindng site-d.term	Shunsuke Sumi	?
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461321" abp="1274">BBa_K1461321</A>	Translational_Unit	pEpCAM(mutant)-RBS-GFP-4 miR 142 5p non-bindng site-d.term	Shunsuke Sumi	?
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461322" abp="1289">BBa_K1461322</A>	Translational_Unit	pEpCAM(mutant)-RBS-GFP-2 miR 142 3p bindgin sites-2 miR 142 5p binding site-d.term	Shunsuke Sumi	?
<A href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1461323" abp="1304">BBa_K1461323</A>	Translational_Unit	pEpCAM(mutant)-RBS-GFP-2 miR 142 3p non-bindgin sites-2 miR 142 5p non-binding site-d.term	Shunsuke Sumi	?

<img src="" class = "contTitle" />

DNA construction

Transformation

Method
1. Put the competent cell on ice and leave it until dissoloved.
2. Add DNA 10 μL to competent cell 100 μL on 2 ml tube.
3. Place on ice for 30 min.
4. Heatshock at 42℃ for 45 sec.
5. Place the tube on ice for 3 min.
6. Add 1000 μL of LB medium and place tube on 37℃ for 30 min.
7. Plate 1000 μL of the medium and spread well.
8. Incubate the plate on 37℃ over night.

Electrophoresis

Method
1. Add agarose to 50 ml or 25ml of 1xTAE buffer in Erlenmeyer flask per 1 gel plate. The concentration of gel is 1% for over 1kbp of DNA and use 2% to under 1kbp.
2. Wrap the top of Erlenmeyer Flask not to completely seal up.
3. Heat it by microwaves until the solution gets transparent.
4. Pour it into a gel maker and set a comb before gelling.
5. After gelling, remove the comb carefully not to break well.
6. Place the gel into tank for electrophoresis and fill 1xTAE up to over the level of gel.
7. Add 1 μl of 10xLoading buffer into 10 μl of DNA solution and vortex it.
8. Apply 11 μl of sample and 6 μl of ladder solution in each well.
9. Electrophoresis at 100 V for 20-30 min.
10. The band of bromophenol blue reaches 80% of the gel, stop electrophoresis and salvage the gel.
11. Check the band under blue light.

Colony PCR

Method
1. Mix DNA solution and reagent as follows.
(Add10xF-universal Primer and 10xR-Univarsal Primer at a final concentration of 5 μM. Then add 2xGoTaqMix and MillQ.)
2. Dispense it to each PCR tube for 10 μl on ice.
3. Pick up single colony on plate and dip to the tube.
4. Set to Thermal cycler. (95℃ 5 min, 95℃ 30 sec, 53℃ 30 sec, 72℃ 30-180 sec (step 2 - 4 repeated 30 times), 72℃ 3 min)
5．Check the length of DNA by Electrophoresis.

Miniprep

Material
WizardR Plus SV Minipreps DNA Purification System (promega)

Method
1. Pour 3 ml of LB broth to test tube.
2. Pick up a single colony and put it into the tube.
3. Incubate it at 37℃ for 12~16 hours with the tube shaking.
4. Transfer 1.5 ml of culture fluid to 1.5 ml tube.
5. Centrifuge at 15000 rpm for 1 min.
6. Throw away the supernatant
7. add the other 1.0 ml of culture.
8. Centrifuge at 15000 rpm for 1 min.
9. Throw away the supernatant
10. Add 250 μl of Cell-Resuspension Sol and Vortex it until precipitation dissolved.
11. Add 250 μl of Cell-Lysis Sol and invert it not to make bubble.
12. Add 10 μl of Alkaline Protease and invert it not to make bubble
13. Leave it for 3 min at room temperature.
14. Add 350 μl of Neutralization Sol and invert it not to make bubble.
15. Centrifuge at 15000 rpm 10 min.
14. Set a collection tube on column and apply the supernatant to it.
15. Centrifuge at15000 rpm for 1 min.
16. Add 750 μl of Column Wash Sol and Centrifuge for 1 min at 15000 rpm.
17. Remove the flow through.
18. Add 250 μl of Column Wash Sol and Centrifuge for 1 min at 15000 rpm.
19. Remove the flow through.
20. Centrifuge for 2 min at 15000 rpm to dry the column.
21. Transfer the column to another 1.5 ml tube.
22. Add 50 μl of nuclease-free water and leave it for 1 min at room temperature.
23. Centrifuge for 1 min at 15000 rpm.
24. Measure concentration of the flow though liquid.

Digestion

Material
The restriction enzymes (EcoRⅠ, XbaⅠ, SpeⅠ and PstⅠ) are purchased or provided as support for iGEM Japan from Promega.
Method
1. Mix DNA solution and reagent as follows.(unit μl)
2. Incubate for 2 hours at 37℃.
3. Add 2 μl of 10x Loading Buffer and Electrophoresis and Gel Extraction.

Gel extraction

Method
1. After electrophoresis, slice the band and put the gel slice into 1.5 ml tube.
2. Add 750 μl of Membrane Bind Sol and stand at 60℃ (Vortex every 2 min)
3. Apply to column-set collection tube and Centrifuge for 1 min at 15000 rpm.
4. Add 750 μl of Membrane Wash Sol to the column.
5. Centrifuge for 1 min and remove the flow through.
6. Add 500 μl of Membrane Wash Sol to the column.
7. Centrifuge for 1 min and remove the flow through.
8. Centrifuge for 2 min to dry the column.
9. Transfer the column to another 1.5 ml tube.
10. Add 20 μl of nuclease-free water and leave it for 1 min at room temperature.
11. Centrifuge for 1 min at 15000 rpm.
12. Measure concentration of the flow though liquid.

Ligation

Method
1. Calculate the mass ratio of Insert DNA to Vector DNA to equal
2. Mix the DNA solution and reagent as follows.
3. Stand it for 15 min at room temperature.

Assay method

Influence on growth of E. coli

Material
Strain: JM109
Medium: LB

Method
1. Culture O/N.
2. Take 30 uL of overnight culture into 3 mL LB medium (containing 50 ug/mL CP).
3. Incubate the culture at 37℃ and measure OD600 evry a hour.

Sigma factor and its crresponding promoter

Material
Strain: MG1655
Medium: M9

Method
1. Culture O/N.
2. Take 150 uL of overnight culture into 3 mL M9 medium (containing 50 ug/mL CP).
3. Incubate the culture until OD600 reaches the range of 0.45-0.95.
4. Add 3 uL arabinose (10%) to the culture.
5. Incubate the culture 3 hours.
6. Measure GFP fluorescence.

Reset function

Material
Strain: MG1655
Medium: M9

Method
1. Culture O/N.
2. Take 150 uL of overnight culture into 3 mL M9 medium (containing 50 ug/mL CP).
3. Incubate the culture until OD600 reaches the range of 0.5-0.7.
4. Add 3 uL arabinose (10%) to the culture.
5. Incubate the culture 3 hours.
6. Measure GFP fluorescence.
7. Add 100mM IPTG 15 uL.
8. Incubate O/N.
9. Measure GFP fluorescence.

Riboregulator

Material
Strain: JM109
Medium: LB

Method
1. Culture O/N.
2. Take 150 uL of overnight culture into 3 mL M9 medium (containing 50 ug/mL CP).
3. Incubate the culture until OD600 reaches the range of 0.5-0.7.
4. Add 3 uL arabinose (10%) to the culture.
5. Incubate the culture 3 hours.
6. Measure GFP fluorescence.

Positive feedback

Material
Strain: MG1655
Medium: M9

Method
1. Culture O/N.
2. Take 150 uL of overnight culture into 3 mL M9 medium (containing 50 ug/mL CP).
3. Incubate the culture until OD600 reaches the range of 0.5-0.7.
4. Add 3 uL arabinose (10%) to the culture.
5. Culture 10 minute /1 hour.
6. Centrifuge at 3200 rpm for 5 min.
7. Throw away the supernatant.
9. Measure GFP fluorescence.

<img src="" class = "contTitle" />

<col width="9"> <col width="36" span="7"> <col width="40">

Jul.
Mon.	Tue.	Wed.	Thu.	Fri.	Sat.	Sun.
	1	2	3	4	5	6
7	8	9	10	11	12	13
<a href="Javascript:linkDate('0714')">14</a>	<a href="Javascript:linkDate('0715')">15</a>	<a href="Javascript:linkDate('0716')">16</a>	<a href="Javascript:linkDate('0717')">17</a>	<a href="Javascript:linkDate('0718')">18</a>	19	20
21	<a href="Javascript:linkDate('0722')">22</a>	<a href="Javascript:linkDate('0723')">23</a>	24	25	26	27
<a href="Javascript:linkDate('0728')">28</a>	<a href="Javascript:linkDate('0729')">29</a>	<a href="Javascript:linkDate('0730')">30</a>	<a href="Javascript:linkDate('0731')">31</a>

<col width="40"> <col width="36" span="7"> <col width="9"> <col width="64">

Aug.
Mon.	Tue.	Wed.	Thu.	Fri.	Sat.	Sun.
				<a href="Javascript:linkDate('0801')">1</a>	2	3
<a href="Javascript:linkDate('0804')">4</a>	<a href="Javascript:linkDate('0805')">5</a>	<a href="Javascript:linkDate('0806')">6</a>	<a href="Javascript:linkDate('0807')">7</a>	<a href="Javascript:linkDate('0808')">8</a>	<a href="Javascript:linkDate('0809')">9</a>	10
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Sep.
Mon.	Tue.	Wed.	Thu.	Fri.	Sat.	Sun.
<a href="Javascript:linkDate('0901')">1</a>	<a href="Javascript:linkDate('0902')">2</a>	<a href="Javascript:linkDate('0903')">3</a>	<a href="Javascript:linkDate('0904')">4</a>	<a href="Javascript:linkDate('0905')">5</a>	<a href="Javascript:linkDate('0906')">6</a>	7
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<a href="Javascript:linkDate('0929')">29</a>	<a href="Javascript:linkDate('0930')">30</a>

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Oct.
Mon.	Tue.	Wed.	Thu.	Fri.	Sat.	Sun.
		<a href="Javascript:linkDate('1001')">1</a>	<a href="Javascript:linkDate('1002')">2</a>	<a href="Javascript:linkDate('1003')">3</a>	<a href="Javascript:linkDate('1004')">4</a>	<a href="Javascript:linkDate('1005')">5</a>
<a href="Javascript:linkDate('1006')">6</a>	<a href="Javascript:linkDate('1007')">7</a>	<a href="Javascript:linkDate('1008')">8</a>	<a href="Javascript:linkDate('1009')">9</a>	<a href="Javascript:linkDate('1010')">10</a>	11	12
13	14	15	16	17	18	19
20	21	22	23	24	25	26
27	28	29	30	31

Lab member

Yoshikawa,Nakashima

Contents

Making Plate Culture

Ampicillin *10
Chloramphenicol*6

Lab member

Yoshikawa,Nakashima

Contents

Making DW 100mL

TF

4-17F(A-1), 4-4E(A-2), 3-19O(A-3), 3-4G(A-4), 4-1N(A-6), 3-3F(A-8), 4-13L(B-10)

Lab member

Yoshikawa,Nakashima

Contents

A-2:No colony

TF

A-2(recovery)

preculture

A-1, A-3, A-4, A-6, A-8, B-10,Escherichia coli JM109(for competent cell)

Lab member

Yoshikawa,Nakashima

Contents

A-2:There are something like colonies.

Making glycerol stock/miniprep

A-1, A-3, A-4, A-6, A-8
All samples:consentration is low.
B-10:disposed

making reagent

LB: 500mL
50mM CaCl2: 400mL
50mM CaCl2/ 20% glycerol: 200mL

preculture

A-1 #1
A-2 #1 #2
A-3 #1
A-4 #1
A-6 #1
A-8 #1
B-10 #1 #2
E. coli JM109（for competent cell）

1. colony number

Lab member

Yoshikawa,Nakashima

Contents

miniprep

A-1,A-3,A-6:from culture 2.5ml OK
A-4,A-8:from culture 1.5ml OK
A-2#1:made glycerol stock. Low consentration.
B-10#1:Low consentration.
A-2#2,B-10#2:did not grow

Making competent cell

100uL*120

making reagent

（2000×）Ampicillin 3ｍL　（100mg/mL）

Lab member

Yoshikawa,Nakashima

Contents

Cut check

A-4 (81ng/μL):ES Cut,XP cut.Checked in 1.5h, 2h, 2.5h, 3h:OK
Left:1kbp Ladder/A-4(Control)/ES 1.5h/ES 2h/ES 2.5h/ES 3h
Right:1kbp Ladder/A-4(Control)/XP 1.5h/XP 2h/XP 2.5h/XP 3h
<img src=""/>

TF

We measured cfu of competent cell we made.
We used Efficiency Kit （RFP Construct on pSB1C3）in distribution Kit.

TF

50pg, 20pg, 10pg, 5pg

competent cell:25uL

Sprinkling:100uL

preculture

B-10 #3, A-4

Lab member

Yoshikawa,Nakashima

Contents

plate check:Did not grow.

miniprep

A-4, B-10 #3(made glycerol stock):OK

Making plate

200ml,CP*10

Lab member

Nakashima

Contents

digestion,gel extraction

A-6 SP, B-10 XP：disposed
100bp Ladder, A-6 SP, B-10 XP, NC
<img src=""/>
(wrote at 0731
A-6
band near 2kbp:SP cut or single cut
band near 1.5kbp:Plasmid)

colony PCR

A-6 #1, B-10 #1
1kbp Ladder, A-6, B-10, NC
<img src=""/>
A-6:OK,B-10:wrong

Measuring cfu

competent cell:50uL

Lab member

Yoshikawa,Nakashima

Contents

Plate check:All Plates did not grow.

TF

B-10 by electroporation.

Measuring cfu

A-1:13,65,130pg

Electrophoresis check

1kbp Ladder, A-6(Plasmid), NC
<img src=""/>
There is band in 1500kbp,so 1500kbp in 0728 may be rest of cutting.

No contamination in Dye.

Lab member

Yoshikawa,Nakashima

Contents

colony check

cfu:Did not grow
B-10:One colony

Cut check

We checked whether A-6 band in 0728 was rest of cutting.

A-1 SP cut:checked on different times.

colony PCR

B-10(from Plate made in 0729)
1kbp Ladder, 1.5h, 2h, 2.5h, B-10(colony PCR), NC
<img src=""/>
band near 800bp:RFP between Spe1 site and Pst1 site of A-1.
band near 2kbp:Backbone cut by SP cut
band near 3kbp:rest of cutting

1.5h:incompletely cut
2h,2.5h:OK
We decided 2h for digestion.
B-10:OK

preculture

B-10

Lab member

Yoshikawa,Nakashima

Contents

miniprep

B-10 #1（from 140729 Plate）(made glycerol stock)

digestion,gel extraction

A-6 SP, B-10 XP

1kbp Ladder, A-6, B-10
<img src=""/>
B-10:incomplete cut
others:OK

ligation

C-7 (A-6 SP + B-10 XP)

Lab member

Yoshikawa,Nakashima

Contents

TF

C-7（Chemical & EP）

ligation Check

PCR reaction primed with Universal Primer, using ligation products as template.
<img src=""/>
OK!

Lab member

Yoshikawa,Nakashima

Contents

Plate check

No colony

digestion

A-6 SP, B-10 XP

Electrophoresis

1kbp Ladder, A-6, B-10
<img src=""/>
Incomplete cut.Disposed.

preculture

B-10

Lab member

Yoshikawa,Nakashima

Contents

miniprep

B-10　→Sample Lost!　Bye bye, GFP!

Cut check

SP(A-1), XP(A-1), EX(A-4), ES(A-4)
2h, 2.5h, 3h

Elrctrophoresis

1kbp Ladder, A-1, A-1 SP(2h, 2.5h, 3h), A-1 XP(2h,2.5h, 3h), A-4,

A-4 EX(2h, 2.5h 3h), A-4 ES(2h, 2.5h, 3h), None, 1kbp Ladder

<img src=""/>

Incomplete cut.

preculture

A-1, A-4, B-10

Lab member

Yoshikawa

Contents

miniprep

A-1, A-4, B-10

digestion, gel extraction

A-6 SP, B-10 XP

1kbp Ladder, A-6, B-10
<img src=""/>
A-6:OK
B-10:Incomplete cut
<img src=""/>

We chenged restriction enzyme.
B-10 XP
<img src=""/>
OK!
<img src=""/>

making gel

200ml

ligation

C-7（A-6 SP + B-10 XP）

TF

4-11L, C-7

Lab member

Yoshikawa,Nakashima,Tara,Itoh,Tsukada

Contents

colony PCR

C-7
<img src=""/>
<img src=""/>
All bands are self ligation of backbone.

digestion, gel extraction

A-6 SP, B-10 XP

1kbp Ladder, B-10, A-6
<img src=""/>

ligation

C-7 (A-6 SP + B-10 XP)

Cut check

(O/N)

（enzyme-buffer) S-B, S-H*, S-M*, P*-FD, E-FD

• Takara's product
  

Lab member

Nakashima,Nakamura,Yamanaka,Yoshikawa(Fresh),Yoshikawa

Contents

Cut check sample Electrophoresis

1kbp Ladder, S-B, S-H*, S-M*, NC, E-FD, P*-FD
<img src=""/>
FD buffer is appropriate for SP cut.

digestion, gel extraction

A-6 SP, B-10 XP
<img src=""/>
<img src=""/>
OK!

ligation

C-7 (A-6 SP + B-10 XP)

TF

C-7,4-11L(culture in test tube)

overlap extension PCR →PCR clean up

1+2
anealing 57℃,extension 10 sec, 30 cycle.

100bp Ladder, Pecf11, Pecf20, crRBS, taRNA
<img src=""/>
OK!(low concentration)

making gel

200ml

Lab member

Yoshikawa

Contents

Plate and test tube check

4-11L:OK!mixed with glycerol, and conserved in -80℃.
C-7:OK! conserved in refrigerator.

Lab member

Yoshikawa,Nakashima,Yamanaka,Nakamura

Contents

colony PCR

C-7 #1~11

1. 1~4, NC
   

<img src=""/>

1. 5~11,NC
   

<img src=""/>

1. 10 is OK!
   

overlap extension PCR →PCR clean up

Pecf11, Pecf20, crRBS, taRNA (1+2)+3,PCR product
1kbp Ladder, Pecf11(1+2+3), Pecf20(1+2+3), crRBS(1+2+3)

taRNA(1+2+3), Pecf11(all), Pecf20(all), crRBS(all), taRNA(all)

<img src=""/>

Making Plate

Ampicillin*20

preculture

A-6, B-10, C-7 #10

Lab member

Nakashima,Nakamura,Yamanaka,Yoshikawa(Fresh),Yoshikawa

Contents

miniprep

A-6, B-10, C-6 #10 (made glycerol stock)

digestion →gel extraction

A-8 EX, C-6 ES, pSB1A2(A-1) XP *2 sample, A-9 linear XP
A-10 linear XP, A-7 linear XP, B-1 linear XP

1kbp Ladder, A-1 XP, A-1 XP, A-8 EX, C-6 ES
<img src=""/>
C-6:wrong
A-1:OK!

colony PCR

4-11L
<img src=""/>
OK!

ligation →TF

A-9, A-10, A-7, B-1

Making　reagent

LB 800ml

Lab member

Yoshikawa,Nakashima,Tara,Nakamura,Takemura,Tsukada

Contents

miniprep

4-11L（made glycerol stock）

digestion→ gel extrction

A-8 EX, C-6 ES, A-6 SP, 4-11L XP

1kbp Ladder, A-6 SP, A-8 EX, C-6 ES, 4-11L XP
<img src=""/>
<img src=""/>
C-6:Incomplete cut
Backbone of 4-11L said to be pSB1A2, but ,actually,may be pSB1AK3.

ligation→TF

D-7 (4-11L XP + A-6 SP), D-7(C-6 ES + A-8 EX)

colony PCR

A-7 #1~2, A-9 #1~4, A-10 #1~4, B-1 #1~2 ,100bp Ladder
<img src=""/>
A-7 #1, A-10 #4：OK!

preculture

A-8, C-6, A-7 #1, A-10 #4

Lab member

Yoshikawa,Nakashima,Tara,Takemura,Nakamura

Contents

miniprep

A-7 #1（made glycerol stock）, A-10 #4（made glycerol stock）, A-8, C-6

overlap extension PCR

Pecf 11, Pecf 20, crRBS, taRNA
(35 cycles, annealing in 59℃)

digestion → gel extranction

A-7 SP, 4-11L XP, pSB1A2 (B-10), A-7 linear XP
A-9 linear XP, A-10 linear XP, B-1 linear XP

1kbp Ladder, A-7 SP, 4-11L XP
<img src=""/>
<img src=""/>
OK!
A-10 XP, 100bp Ladder
<img src=""/>
OK!(low concentration)

100bp Ladder, pSB1A2(B-10), pSB1A2(B-10), A-7, A-9, B-1
<img src=""/>
<img src=""/>
Incomplete cut of pSB1A2 is weigh on our mind.

gel making

2% 25mL
1% 200mL

colony PCR

D-7(Ampicillin), D-7(Chloramphenicol)

1kbp Ladder, D-7(Amp) #1~4, D-7(CP) #1~4
<img src=""/>

ligation → TF

D-6 (A-7 SP + 4-11L XP), A-7 (linear XP + pSB1A2 XP)
A-9, A-10, B-1 (linear XP + pSB1A2 XP)

Lab member

Yoshikawa,Nakashima,Tara,Takemura,Nakamura

Contents

miniprep

D-7 #3 (made glycerol stock)

digestion → gel extraction

A-10 SP, D-7 XP
<img src=""/>
<img src=""/>

colony PCR

A-7 #1~4, A-9 #1~4, A-10 #1~4, B-1 #1~4, D-6 #1~4
<img src=""/>
A-7 #3,4, A-10 #1,2,4, B-1 #1,2,4：OK!

D-6
<img src=""/>
OK!

ligation → TF

E-18 (A-10 SP + D-7 XP)

preculture

A-7 #3,4, A-10 #1,2,4, B-1 #1,2,4, D-6 #1

Lab member

Yoshikawa

Contents

miniprep

A-7 #3,4, A-10 #1,2,4, B-1 #1,2,4, D-6 #1
(made glycerol stock)

Lab member

Yoshikawa,Hiura

Contents

digestion → gel extraction

A-1 SP, A-2 SP, A-3 SP, D-6 XP *2, A-7 #3,4, B-10
<img src=""/>

sequence

A-7, A-7 #3,4, A-10, A-10 #1,2,4, B-1 #1,2,4
C-6 F, C-6 R, D-7 F, D-7 R, D-6 F, D-6 R

colony PCR

E-18 #1~3
<img src=""/>
OK!

ligation → TF

E-14 (A-3 SP + D-6 XP)
E-15 (A-1 SP + D-6 XP)
E-16 (A-2 SP + D-6 XP)

preculture

E-18 #1

Lab member

Yoshikawa,Nakamura,Yamanaka,Yoshikawa(Fresh)

Contents

miniprep

E-18 (made glycerol stock)

digestion

A-8 XP, B-1 #1,2,4 SP
→Today, sequences of these dna proved to be wrong, so we disposed them.

making gel

2% gel:25ml

colony PCR

C-5, E-14, E-15,E-16

sequence

A-7:RBS
A-7 #3:none
A-7 #4:none
A-10:OK
B-1: all none

Lab member

Yoshikawa,Nakamura,Tsukada,Yamanaka,Itoh

Contents

PCR

sigma11, sigma20, anti11, anti20

TF

1-21P

Lab member

Yoshikawa,Nakashima

Contents

digestion → gel extraction

pSB1C3(A-4), A-6, B-3(We did not extract.), A-6'(NEB buffer)
<img src=""/>

colony PCR

A-2, A-3, A-4, 1-21P, NC
<img src=""/>

ligation → TF

B-3, C-2, C-2'

preculture

1-21P

Lab member

Yoshikawa,Nakamura,Nakashima

Contents

miniprep

1-21P(made glycerol stock)

colony PCR

B-3, C-2, C-2
<img src=""/>

digestion → gel extraction

1-21P SP, D-7 XP, C-6 E, C-6 P, C-6(NC)
<img src=""/>
<img src=""/>

ligation → TF

K-1(1-21P SP + D-7 XP)

preculture

B-3 #1,4, D-7, C-2 #1,3, C-2' #3,4

Lab member

Yoshikawa

Contents

miniprep

D-7, B-3 #1,4, C-2 #1,3, C-2' #3,4

Lab member

Nakashima,Nakamura,Yamanaka,Hiura

Contents

digestion → gel extraction

A-8 EX *2, B-3 #1 ES, B-3 #4 ES, C-2 #1 ES, C-2 #3 ES
<img src=""/>
<img src=""/>

Plate　making

Ampicillin*10, Chloramphenicol*20

colony PCR

K-1
<img src=""/>

1. 1,4:OK!
   

sequence

B-3 #1,4, C-2 #1,3, C-2' #3,4, E-18, A-2, A-3

ligation → TF

D-3 1 (A-8 EX + C-2 #1 ES), D-3 2(A-8 EX + C-2 #3 ES)
C-10 1(A-8 EX + B-3 #1 ES), C-10 2(A-8 EX + B-3 #4 ES)

preculture

A-2, A-8, K-1 #1, C-2 #1,3, B-3 #1

Lab member

Yoshikawa,Nakashima,Yamanaka,Takemura,Tsukada

Contents

miniprep

K-1(made glycerol stock), A-2, A-8, B-3 #1, C-2 #1, C-2 #3

colony PCR

D-3 (1), D-3 (2), C-10 (1), C-10 (2)
<img src=""/>
OK!(C-10(1)#4 may be a little shorter.)

digestion → gel extraction

K-2 linear EP, K-3 linear EP, K-4 limear EP, K-5 linear EP, pSB1C3 (A-4) EP
<img src=""/>
We forgot to take the photo before we sliced the band.

ligation → TF

K-2, K-3, K-4, K-5

preculture

D-3 #1, C-10 #1

sequence

A-2:E-X-S-S-Pconst(weak)-S-P wrong

sequence

A-3:OK
B-3 #1:OK
B-3 #4:OK
C-2 #1:OK
C-2 #3:OK
C-2' #3:point mutation *2
C-2' #4:OK
E-18:OK

Lab member

Yoshikawa,Nakashima,Yoshikawa(fresh),Itoh,Tara,Nakamura

Contents

miniprep

D-3, C-10(made glycerol stock)

digestion → gel extraction

A-1 SP, A-3 SP, A-10 SP, D-3 XP *2
<img src=""/>
<img src=""/>
D-3 is a little strange.Contamination?

colony PCR

K-2~5 #1~4

• 100bp Ladder
  

<img src=""/>
500bp band:self ligation of backbone(A-4 200bop)
300bp band:If this band is blank vector, this band(EP cut) must be shorter than 300bp.

So, this band is OK!.

→K-2 #4, K-3 #1, K-4 #1,2, K-5 #2,3,4:OK!

ligation → TF

E-5(A-3 SP + D-3 XP), E-6(A-1 SP + D-3 XP), E-20(A-10 SP + D-3 XP)

preculture

E-15
D-3, C-10(Recovery)
K-2 #4, K-3 #1, K-5 #2

Remarks

<img src=""/>
We observed that pCMV expressed in Escherichia.coli.
Left:pCMV-GFP
Right:pConst(strong)-GFP
We may be able to carry out the characterization of pCMV and meet the gold medal requirement(parts implovement).

Lab member

Yoshikawa,Nakashima,Itoh,Tara,Tsukada,Yamanaka

Contents

miniprep

K-2~5, E-23(made glycerol stock)
C-10, D-3

digestion →gel extraction

K-2 EX, K-3 EX, K-4 EX, K-5 EX, E-23 EP, C-6 ES *2, pSB1C3(A-4) EP, pSB1C3(A-4) XP
<img src=""/>
<img src=""/>
→pSB1C3 XP:keep in freezer

overlap extension PCR

A-7, A-9, B-1, B-2, D-8, D-9

check & colony PCR

check:E-5, E-6
colony PCR:(E-5 #1~4, E-6 #1~4, NC), A-7, A-9, D-8, B-1, B-2, D-9
<img src=""/>
A-7, A-9, B-1, D-8:OK!
B-2, D-9:Needs retry in higher concentration.
E-5 #2~4, E-6:OK!

ligation → TF

E-23'(E-23 EP + pSB1C3 EP), L-1~4(C-6 ES + K-2~5 EX)

Lab member

Yoshikawa,Nakashima,Nakamura,Tara,Tsukada,Yamanaka

Contents

miniprep

E-5(made glycerol stock)
A-4, C-6
E-6:No colony

digestion →gel extraction

A-7 linear XP, A-9 linear XP, A-10 SP, B-1 linear XP, D-3 XP, D-8 linear XP, E-5 SP, E-18 XP
<img src=""/>
<img src=""/>

PCR

B-2, D-9(retry)

check & colony PCR

E-23' #1~4, B-2, D-9
<img src=""/>
L-1~4
<img src=""/>
E-23 #1, L-1 #2,4, L-2 #4, L-3 #1~3, L-4 #2,4:OK!
PCR:failed

sequence order

E-23, K-1, K-2~5

ligation → TF

A-7, A-9, B-1, D-8( linear XP + pSB1C3 XP)
F-2 (E-18 XP + E-5 SP), E-20(A-10 SP + D-3 XP)

preculture

A-10, E-6, E-18, E-23', K-3, L-1~4

Lab member

Yoshikawa

Contents

miniprep

A-10, E-18, K-3
L-1~4, E-23', E-6, E-18(made glycerol stock)

PCR

B-2, D-9

Lab member

Nakashima,Itoh,Tara,Tsukada,Takemura

Contents

PCR clean up

B-2, D-9
<img src=""/>
failed

digestion → gel extraction

L-4 ES, E-6 ES, E-18 EX, L-1~3 ES, K-2~5 EX

<img src=""/>
<img src=""/>

Making plate

CP *30

colony PCR

A-7 #1~4, A-9 #1
<img src=""/>
OK!
D-8 #1~4, E-20 #1~4, F-2 #1~4 (100bp Ladder)
<img src=""/>
D-8 #1~4, E-20 #1~4,F-2#1,4:OK!
B-1 #1~4
<img src=""/>
OK!

Ligation

G-5 (E-6 ES + E-18 EX), M-1~4(K-2~5 EX + L-1~4 ES)

preculture

A-7 #1~4, A-9 #1, B-1 #1~4, D-8 #1~4, F-2, E-20

Lab member

Yoshikawa,Nakashima,Hiura,Tara,Tsukada,Takemura

Contents

miniprep

A-7 #1~4, A-9, B-1 #1~4, D-8 #1~4, F-2, E-2(made glycerol stock)

digestion → gel extraction

A-4 SP, A-7 SP #1, A-7 SP #2, A-9 SP
<img src=""/>
<img src=""/>
A-8XP
<img src=""/>
<img src=""/>

B-1 #1, B-1 #2, B-3 XP, B-10 XP, D-7 XP, D-8 XP, E-20 XP, F-2 SP
<img src=""/>
<img src=""/>
D-8:???

sequence

A-7 #1~4, B-1 #1~4
D-8 #1~4
A-9, A-4, E-5, E-6, E-20

PCR

B-2, D-9

check & colony PCR

M-1~3 #1~4
<img src=""/>
OK!
M-4 #1~4, B-2, D-9, PC(VF2→B-10←VR )
NC, G-5
<img src=""/>
M-4 #1~4,PC(VF2→B-10←VR ):OK!
(M-4 #2~4 is a little longer?)

ligation → TF

C-4(A-7 SP + B-3 XP), C-5(A-7 SP + B-10 XP), E-17(A-9 SP + D-7 XP)

D-1(B-1 SP + D-8 XP), E-21(A-4 SP + D-8 XP), J-4 (F-2 SP + E-20 XP)

preculture

M-1~4 #1, G-5 #1, E-20

We got a result!!

simpler version of assay 1.
Left:Pσ11→GFP generator
Right:Pconst(strong)→σ11 generator & Pσ11→GFP generator
<img src=""/>

Lab member

Yoshikawa,Nakashima,Hiura,Tara,Yamanaka,Takemura

Contents

M-1~4, G-5(made glycerol stock of M-3,4)

sequence

A-4: OK
A-7: all OK
A-9: OK
B-1:all OK
D-8#1: NG (2 skip)

    #2; OK

    #3: NG (1 mut.)

    #4; NG (2 mut. 2 skip)

E-5: OK
E-6: OK
E-20:OK

digestion → gel extraction

A-7 #1, 2 SP, C-9 XP, C-10 XP, K-2~4 EX, M-1~4 ES
<img src=""/>
<img src=""/>

colony PCR

C-4 a, C-5 a, D-1 a, E-17
<img src=""/>
C-4 a#2,3, C-5 a, D-1 a, E-17:OK!
E-21 b, J-4
<img src=""/>
J-4:wrong
E-21:a little shorter

PCR

B-2, D-9, B-7, B-2(Taq), D-9(Taq) Taq:positive control
anealing temperature:51℃～61℃(gradient)

ligation → TF

D-5 (A-7 SP + C-10 XP), D-6 (A-7 SP + C-9 XP), N-1~4 (M-1~4 ES + K-2~5 EX)

N-5 (M-1 ES K-3 EX), N-6 (M-3 ES + K-5 EX)

Lab member

Yoshikawa,Nakashima,Nakamura,Tara,Yamanaka,Tsukada

Contents

miniprep

E-20, D-1(made glycerol stock)

digestion → gel extraction

pSB1C3 (A-4) XS, pSB1C3 (A-4) XP, A-3 SP, D-1 XP
<img src=""/>
<img src=""/>

PCR check & clean up

D-9 #1,3,5,7,9
<img src=""/>
There are bands in #5,7,9
B-2 1,3,5,7,9,11, B-7.
<img src=""/>
There are bands in B-2 #5,7, B-7.
B-2 4,6, D-9 10,11,12
<img src=""/>
OK!
We decided to clean up B-2 #5, D-9 #11, B-7.

Making Plate

CP *30

digestion

B-2 linear SP, D-9 linear XP, B-7 linear XP

colony PCR

D-5, D-6, N-1, N-2 #1~4
<img src=""/>
D-5, D-6, N-1, N-2 #1,2:OK!
N-3~6 #1~4
<img src=""/>
N-3,N-4#1,3,4,N-5#1,4,N-6:OK!

ligation → TF

E-1 (A-3 SP + D-1 XP), B-2 (B-2 linear XP + pSB1C3 XP), D-9 (D-9 linear XP + pSB1C3 XP)

B-7 (B-7 linear XP + pSB1C3 XP), Emp. (pSB1C3 XS)

miniprep

M-1, M-2, C-4, C-5, E-17, E-21, J-4 (made glycerol stock)
K-2, K-5, C-9

preculture

F-2, D-5, D-6, N-1~6

Lab member

Yoshikawa,Nakashima,Nakamura,Takemura,Yamanaka

Contents

miniprep

F-2
N-1~6, D-5, D-6
→N-1, 2, 4, 5. 6 :failed

digestion → gel extraction

A-1 SP, A-3 SP, D-5 XP, D-6 SP, E-20 XP, K-4 EX, N-3 ES, F-2 SP
J-4 E(check)
<img src=""/>
<img src=""/>

ligation → TF

E-11 (A-3 SP + D-5 XP), E-12 (A-1 SP + D-5 XP), E-14 (A-3 SP + D-6 SP)

E-15 (A-1 SP + D-6 XP), O-3 (K-4 EX + N-3 ES)

PCR

B-2, D-9, B-7

colony PCR

B-2, B-7, D-9, E-1　#1~4
<img src=""/>
B-7 #2,3, D-9 #1, E-1 #3,4:OK!
Z-1 #2~4
<img src=""/>

preculture

B-7 #2,3, D-9 #1, E-1 #3, Z-1 #2, K-3, N-1,2,4~6

Lab member

<p>Yoshikawa,Nakashima

</p>

Contents

miniprep

B-7 #2,3, D-9 #1, E-1 #3, Z-1 #2 (made glycerol stock)
N-1,2,4~6, K-3

PCR

B-2, D-9, B-7

check & colony PCR

B-2, D-9, B-7, B-2#5~8, B-7 #2,3,5,6, D-9 #5,6.7,8
<img src=""/>

Lab member

Yoshikawa,Nakashima,Nakamura,Takemura,Yamanaka,Tara

Contents

digestion → gel extaction

B-2 linear XP (did not extracted)
N-1,2,4~6 ES
<img src=""/>
<img src=""/>
A-4 SP, pSB1C3 (A-4) XP, A-6 SP, B-7 #2 XP, B-7 #3 XP, D-9 #1 XP, K-2,3,5 EX
<img src=""/>
B-7 #2, D-9 #1:disposed(incomplete cut)
B-7 #3:disposed(a little longer)(This band proved to be correct.9/9 wrote)
<img src=""/>

colony PCR

E-11,12,14,15 #1~4
<img src=""/>
OK!
O-3 #1,2
<img src=""/>

1. 2:OK!
   

PCR

B-2
<img src=""/>
B-7,D-9
<img src=""/>
The band of the longest DNA is OK!

ligation → TF

O-1 (N-1 ES + K-2 EX), O-2 (N-2 ES + K-3 EX), O-4 (N-4 ES + K-5 EX), O-5 (N-5 ES + K-3 EX)
O-6 (N-6 ES + K-5 EX), B-2 (B-2 linear XP (9/8 PCR) + pSB1C3 XP)
B-2' (B-2 linear XP + pSB1C3 XP), D-9, B-7

preculture

D-5, D-6, N-3, E-21, A-3, K-4
E-11 #1, E-12 #1, E-14 #1, E-15 #1, O-3 #2, B-7 #6, D-9 #5,6

Lab member

Yoshikawa,Nakashima,Nakamura,Takemura,Yamanaka

Contents

miniprep

E-11,12,14,15, O-3, B-7 #6, D-9 #5,6 (made glycerol stock)
D-5, N-3, K-4, A-3
D-6:did not grow
D-5:diposed(doubt of cross contamination)

digestion → gel extraction

B-2 linear XP (did not extracted)
pSB1C3 (A-4) XP, A-6 SP, A-8 EX, B-7 #6 XP, D-9 linear XP, B-7 linear , E-1 EX, E-14 ES, E-15 ES, O-3 ES
<img src=""/>
<img src=""/>

colony PCR

B-2 #1~3, B-2' #1~4, B-7 #1~3, D-9 #1,2, O-1 #1~4
<img src=""/>
B-2' #1, B-7 #1,2, O-1:OK!
O-2,4,5,6 #1~4
<img src=""/>
O-2,3,4,6,O-5#2,3,4

sequence

B-7 #2: NG
B-7 #3: OK
D-9 #1: NG
E-1: OK
C-4: OK
C-5: OK
D-5: OK
D-6: OK
E-17: OK
E-21: OK
F-2: ?
J-4: OK
N-1: OK
N-2: M-2
N-3: OK
N-4: M-4
N-5: OK
N-6: OK

ligation → TF

D-9 (D-9 linear XP + pSB1C3 XP) *2, B-2 (B-2 linear XP + pSB1C3 XP)*2

P-3 (O-3 ES + A-8 EX), I-1 (E-14 ES + E-1 EX), I-2 (E-15 ES + E-1 EX)

preculture

F-2, D-5,6, D-9 #5,6, E-12, K-4
O-1,2,4,6 #1, O-5 #2, B-2' #1

Lab member

Nakashima,Hiura,Takemura,Yamanaka,Yoshikawa(Fresh)

Contents

miniprep

O-1,5,6, N-2,4, F-2, B-2' #1 (made glycerol stock)
E-12, K-4, D-5
D-6:did not grow

digestion → gel extraction

A-6 SP, A-7 SP, A-8 EX, B-2' XP, B-7 XP
<img src=""/>
<img src=""/>
K-3 EX, K-5 EX, O-1 ES, N-2 ES, N-4 ES
<img src=""/>
<img src=""/>
O-5 ES, O-6 ES
<img src=""/>
<img src=""/>

colony PCR

I-1 #1~4
<img src=""/>
B-2(140909) #1~8, D-9 #1~8
<img src=""/>
B-2 #2, D-9 all, I-1 #2~4, I-2 #1~4:OK!

ligation → TF

O-2 (N-2 ES + K-3 EX), O-4 (N-4 ES + K-5 EX), P-1 (O-1 ES + A-8 EX), P-5 (O-5 ES + A-8 EX)

P-6 (O-6 ES + A-8 EX), C-1' (B-2' XP + A-6 SP), C-3' (B-2 ' XP + A-7 SP), C-8 (B-7 XP + A-6 SP)

preculture

D-9 #5,6, D-9(140909) #1~4, B-2 (140909) #2, I-1 #2, I-2 #1, D-6 #1

Lab member

Yoshikawa,Nakashima,Nakamura,Hiura,Itoh,Tsukada

Contents

miniprep

D-9 #1~4, B-2 #2, I-1, I-2, D-6 (made glycerol stock)

digestion → gel extraction

A-1 EP, pSB1C3(A-3) EP *2, A-4 SP, A-6 SP, A-7 SP, A-8 EX, A-10 EX, B-2 #2 XP, E-6 EP, E-12 EP
<img src=""/>
<img src=""/>
E-15 EP, E-18 EP, E-20 EP, D-9 #1 XP, O-3 ES
<img src=""/>
<img src=""/>

sequence

B-2' #1, B-2 #2, F-2, E-11, E-12, E-14, E-15, O-1, N-2, O-3, N-4, O-5, O-6, D-9 #1~4

Plate Making

CP *30

colony PCR

C-1', C-3', C-8, O-2, O-4 #1~3
<img src=""/>
C-1', C-3', C-8, O-2, O-4 #1,2:OK!
P-1, P-5, P-6 #1~3
<img src=""/>
→P-1 #1~3, P-5 #2, P-6 #1~3:OK!

ligation → TF

C-1 (A-6 SP + B-2 #2 XP), C-3 (A-7 SP + B-2 #2 XP)

E-22a (A-4 SP + D-9 #1 XP), E-22b (A-4 SP + D-9 #2 XP), P-3 (O-3 ES + A-8 EX)

A-10 CP, A-1 CP, E-6 CP, E-12 CP, E-20 CP, E-15 CP (replace backbone to pSB1C3)

preculture

C-1' #1, C-3' #1, C-8 #1, O-2 #1, O-4 #1, P-1 #1, P-5 #2, P-6 #1, E-12,14,15,18, A-1, O-1,3,5

Lab member

Yoshikawa,Nakashima,Hiura,Itoh,Tsukada,Tara

Contents

miniprep

C-1', C-3', O-2, O-4, P-1, P-5, P-6 (made glycerol stock)
E-12, E-14, E-15, O-1, O-3, O-5, E-18, A-1

digestion → gel extraction

A-6 SP, A-8 EX *2, B-2 XP, C-1' ES, C-3' ES, C-8 ES, K-6 SP, O-2 ES, O-3 ES, O-4 ES
<img src=""/>
<img src=""/>
C-1,3:did not extracted
P-1 XP, P-5 XP, P-6 XP
<img src=""/>
<img src=""/>

colony PCR

A-1 CP, A-10 CP, C-3, E-6 CP #1~3
<img src=""/>
E-12 CP, E-15 CP, E-20 CP, E-22a, E-22b
<img src=""/>
except E-20#1,2:OK!

sequence

B-2' #1: NG
B-2 #2: OK
D-9 #1: NG

   #2: NG

   #3: NG

   #4: OK

O-1: OK
O-3: OK
O-5: OK
O-6: OK
N-2: OK
N-4: OK
E-11: Ok
E-12: OK
E-14: OK
E-15: OK
F-2: OK

ligation → TF

C-1 (A-6 SP + B-2 XP), P-2~4 (O-2~4 ES + A-8 EX), Q-1,5,6 (P-1,5,6 XP + K-6 SP)

ligation

P-3 (O-3 ES + A-8 EX)

preculture

A-4, A-6, A-8, C-3 #1, A-1 CP #1, E-12 CP #1, E-15 CP #1, E-20 CP #3, E-6 CP #1

Lab member

Yoshikawa

Contents

miniprep

A-4, A-6, A-8 C-3, E-6 CP, E-12 CP, E-15 CP, E-20 CP, A-1 CP(made glycerol stock)

Lab member

Yoshikawa,Nakashima,Takemura,Nakamura,Yamanaka,Tara

Contents

digestion → gel extraction

A-8 EX, C-3 ES
<img src=""/>
<img src=""/>

colony PCR

C-1 (σ20F, VR)
<img src=""/>
OK!

ligation → TF

D-4 (C-3 ES + A-8 EX) *2 (new and old competent cell)

preculture

C-1 #1

Lab member

Yoshikawa,Nakashima,Hiura,Itoh,Nakamura,Yamanaka

Contents

miniprep

C-1　(made glycerol stock)

colony PCR

D-4, P-2~4
<img src=""/>
A-10
<img src=""/>

Q-1,5,6:confirmed by fluorescence

digestion → gel extraction

A-4 SP, A-8 EX, C-1 ES, D-9 XP, E-18 EP, pSB1C3 (A-4) EP
<img src=""/>

ligation → TF

D-2 (A-8 EX + C-1 ES), E-18 CP (E-18 EP + pSB1C3 EP) *2, E-22 (A-4 SP + D-9 XP)

preculture

D-4, P-2~4, A-10 CP, Q-1,5,6

assay

<img src=""/>
PC (E-23': Pconst (strong)-GFP-d.term)
Experiment (K-1: pCMV-GFP-d.term)
NC (Z-1: pSB1C3)
absorbance:600nm and 395nm Absorbance of 395nm proved not to be able to measure.
We need fluorospectro-photometer.

Lab member

Yoshikawa,Nakashima,Hiura,Takemura,Yoshikawa(Fresh),Yamanaka

Contents

miniprep

D-4, P-2~4, Q-1,5,6, A-10 CP　(made glycerol stock)

digestion → gel extraction

A-1 CP SP, A-3 SP, D-4 XP, K-6 SP, P-2 XP, P-3 XP, P-4 XP
<img src=""/>
<img src=""/>

colony PCR

D-2, E-18 CP, E-22
<img src=""/>

ligation → TF

E-8 *2 (A-3 SP + D-4 XP), E-9 (A-1 CP SP + D-4 XP), Q-2~4 (K-6 SP + P-2~4 XP)

assay

Photo of plate(n=4)
<img src=""/>
<img src=""/>
<img src=""/>
<img src=""/>
<img src=""/>
<img src=""/>
<img src=""/>
<img src=""/>

Lab member

Yoshikawa,Nakashima,Hiura,Tsukada,Nakamura

Contents

miniprep

D-2, E-18 CP, E-22
(made glycerol stock)

digestion → gel extraction

A-1 CP SP, A-3 SP, A-9 SP, D-2 XP, E-22 SP, G-5 XP
<img src=""/>
<img src=""/>

colony PCR

E-8, E-9　#1~4
<img src=""/>
Q-2~4 :confirmed by fluorescent

ligation → TF

E-2 (A-3 SP + D-2 XP)
E-3 (A-1CP SP + D-2 XP)
E-19 (A-9 SP + D-2 XP)
H-5 (E-22 SP + G-5 XP)

preculture

E-8,9, Q-2~4

Lab member

Yoshikawa,Nakashima,Hiura,Tara,Yamanaka,Nakamura

Contents

miniprep

E-8,9, Q-2~4

digestion → gel extraction

A-9 SP, D-2 XP, E-22 XP, F-2 SP, G-5 SP
<img src=""/>
<img src=""/>

colony PCR

E-2, E-19 #1~4
E-3, H-5 :No colony
<img src=""/>

ligation → TF

H-5 (G-5 SP + E-22 XP)
H-4 (F-2 SP + E-22 XP)

preculture

E-2 E#1, E-19 #1

Lab member

Yoshikawa

Contents

miniprep

E-2, E-19

Lab member

Yoshikawa,Nakashima,Takemura,Nakamura,Tara,Yamanaka

Contents

digestion → gel extraction

A-1 SP, D-2 XP, E-2 ES, E-17 EX, E-22 XP, G-5 SP, J-4 SP
<img src=""/>
<img src=""/>

colony PCR

H-4
<img src=""/>
H-4#1,2,3:OK!

ligation - TF

H-5 (G-5 SP + E-22 XP)
J-6 (J-4 SP + E-22 XP)
E-3 (A-1 SP + D-2 XP)
F-1 (E-2 ES + E-17 EX)

Lab member

Yoshikawa,Nakashima,Nakamura,Tara,Yamanaka

Contents

digestion, gel extraction

A-1CP SP, D-2 XP, E-22 XP, G-5 SP
J-4 SP(stopped)
<img src=""/>
<img src=""/>

miniprep

H-4(made glycerol stock)

colony PCR

J-6
<img src=""/>
OK!

ligation,TF

H-5(G-5 SP+E22 XP)
E-3(A-1 SP+D-2 XP)

Lab member

Nakashima,Yamanaka,Takemura

Contents

miniprep

F-1,J-6(made glycerol stock)
E-22,D-6
G-6 did not grow.
We disposed J-6.(dropped on the floor)

digestion,gel extraction

A-1 SP,E-2 ES,E-5 ES,E-17 EX,E-18CP EX,E-21 XP,G-5 SP,D-2 XP,F-1 SP,E-19 XP,E-22 XP
<img src=""/>
<img src=""/>

ligation,TF

J-2(F-1 SP+E-1 XP)
H-1(F-1 SP+E-21 XP)
F-3(E-5 ES+E-17 EX)
F-4(E-2 ES+E-18 EX)
E-3(A-1 SP+D-2 XP)
H-5(G-5 SP+E-22 XP)

Lab member

Nakashima,Nakamura,Tara,Tsukada,Yamanaka

Contents

miniprep

A-1CP, J-6

digestion,gel extraction

A-1 SP,D-2 XP,E-5 ES,E-17 EX,E-19 XP,E-21 XP,E-22 XP,F-1 SP,G-5 SP
<img src=""/>
<img src=""/>

ligation,TF

J-2(F-1 SP+E-19 XP)
H-1(F-1 SP+E-21 XP)
F-3(E-5 ES+E-17 EX)
E-3(A-1 SP+D-2 XP)
H-5(G-5 SP+E-22 XP)

Making competent cell

preculture

G-5,E-5,E-17,E. coli JM109

competent cell(made at 0911) check

Ampicillin,Chloramphenicol,non-antibiotics
culture in LB 3ml
LB midium 3ml, as N.C.
culture for 2h.
no TF

result of check

Ampicilln,non-antibiotics:become clouded
Chroramphenicol,LB:did not grow
So,we confirmed ampicillin resistant plasmid exists in competent cell and we disposed it.

Lab member

Yoshikawa,Nakashima,Tara,Yoshikawa(Fresh),Tsukada

Contents

miniprep

G-5,E-5,E-17

digestion

A-1CP SP,D-2 XP,G-5 EP(strange band appeared,disposed),pSB1C3(A-4) EP,A-1 SP
<img src=""/>
<img src=""/>

colony PCR

F-3,F-4,H-1,H-5,J-2
<img src=""/>
<img src=""/>
OK!

ligation,TF

E-3(D-2 XP+A-1SP)
E-3CP(D-2 XP+A-1CP SP)

preculture

F-3,F-4,H-1,H-5,J-2#1,G-5

Lab member

Yoshikawa,Nakashima

Contents

TF

E-3(chemical)

miniprep

F-3,F-4,H-1,H-5,J-2(made glycerol stock)
G-5

TF

E-3CP(electroporation)

Lab member

Yoshikawa,Nakashima,Nakamura,Tara,Takemura

Contents

digestion,gel extraction

A-1CP SP,pSB1C3(A-4) EP,D-2 XP,E-21 XP,G-5 EP,H-5 EP,J-2 SP
<img src=""/>
<img src=""/>

Gel making

ligation

G-5CP(G-5 EP+pSB1C3 EP)
H-5CP(H-5 EP+pSB1C3 EP)
J-5(J-2 SP+E-21 XP)
E-3(A-1CP SP+P-2 XP)

preculture

E-5,E-11,E-22
K-1,E-23',Z-1(for assay)

PCR

VF2-(ligation product of E-3)-VR E-3 lig:1uL
VF2:1uL
VR:1uL
Taq:5uL
MilliQ:2uL
Total:10uL
extention:1m12sec
<img src=""/>
We observed the band which had expected length.
The ligtion must be OK!

Lab member

Yoshikawa.Nakamura,Tsukada,Tara,Yamanaka

Contents

miniprep

E-2,E-5,E-11

digestion,gel extraction

We could not find D-2 sample(probably accidentally disposed).
So we stopped it.

colony PCR

G-5,H-5,J-5
<img src=""/>
H-5,J-5#1,2,3,4:OK!

Making M9 medium

1M MgSO4 50ml
1M CaCl2 10ml
20% glucose 50ml

digestion(cutcheck)

G-5 E,G-5 P,G-5 non-cut
<img src=""/>
Strange bands appeared.
We decided to read a sequence of this part.

Lab member

Nakashima,Tara,Nakamura,Takemura

Contens

miniprep

H-5CP,G-5CP,J-5(made glycerol stock)

sequence order

E-2,E-8,E-9,E-19,F-1,F-3,F-4,G-5,H-1,H-4
H-5(VF-VR,Psigma2F-anti2R),J-2,J-4,J-5,J-6

Lab member

Nakashima,Nakamura,Tara

Contents

assay

completely failed

In M9 medium, Escherichia.coli JM109 did not grow well.(doubling time:1h)
The glycerol stock needed to be put a lot.

culture

F-2,F-3,F-4(for M9 check)

PCR

G-1(F,R),G-4(F,R),G-5(F,R),H-1(F,R),H-4(F,R),H-5(F,R)
Template:1ng/uL,1uL

Making M9 medium

5*M9 24mL
20% Glu 1.2mL
MgSO4 240uL
CaCl2 12uL
Amino acid 10mL
mess up to 1L

PCR check

<img src=""/>

OK!(except G-5)

PCR

G-1,G-4,H-1,H-4
extension time:6m30sec
EpCAM nested
95C 3min-(-95C 30sec-48C 30sec-72C 3min-)*30-72C 5min-4C

preculture

E-17,E-18,F-1,F-2,F-3,F-4,Z-1(M9)
E-23'(LB)

Lab member

Nakashima,Tara,Nakamura

Contents

PCR clean up and check

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G-1,G-4,H-1,H-4:Band in correct position and unexpected position.
EpCAM:No band

digestion,gel extraction

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G-1deg linear EP,G-4deglinear EP,H-1deg linear EP,H-4deg linear EP(A-4)
(deg:degradation tag)

PCR

EpCAM nested
94C 3min-(-94C 30sec-48C 30sec-72C 3min-)*30-72C 5min-4C <img src=""/>
No band

gel making

ligation,TF

H-1'(H-1'linear EP+pSB1C3 EP)
H-4'(H-4'linear EP+pSB1C3 EP)
G-1'(G-1'linear EP+pSB1C3 EP)
G-4'(G-4'linear EP+pSB1C3 EP)

PCR

EpCAM nested
95C 3min-(-95C 30sec-46C 30sec-72C 3min-)*30-72C 5min-4C <img src=""/>
No band

Lab member

Yoshikawa

assay1

F-2 in 20mL culture
When OD600=0.516,we put 10% arabinose 20uL.
F-1 in 20mL culture
When OD600=0.514,we put 10% arabinose 20uL.
Z-1 in 20mL culture
When OD600=0.544,we put 10% arabinose 20uL.
O/N culture

Culture in 3mL:disposed(OD600 value did not agrees with each other.)

colony PCR

H-1deg,H-4deg,G-1eg,G-4deg
We confirmed by fluorescence.

preculture

H-1deg,H-4deg#1,G-1eg,G-4deg

Lab member

Yoshikawa

Contents

miniprep

H-1deg,H-4deg#1,G-1eg,G-4deg

preculture

(M9)

E-15CP,E-17,E-18CP,I-2,F-1,2,3,4,Z-1

Lab member

Yoshikawa,Nakashima,Tara

Contents

assay1,3

E-15,I-2,F-1,F-2,F-3,F-4,F-17,E-18,Z-1
Measured the OD 600 value.
Except I-2,F-2,the value is more than 1.0.
F-1,F-3,E-17,Z-1(20 fold dilution):Culture in 3mL. The composition of M9 proved to be wrong,so disposed.

Making M9 medium

5*M9 200mL
20% Glu 10mL
amino acids 10mL
1M MgSO4 2mL
1M CaCl2 100uL
MilliQ 778mL
Total 1000mL

result of OD600

F-3(non-Chloramphenicol)
1h:0.094
2h:0.086
F-3(Chloramphenicol) 1h:0.074
2h:0.073

assay1

(tentative)

We measured the fluorescence of F-1,F-2,Z-1(culture in 20mL).
Ex:501nm
F-2:We observed a peak in 511nm.
F-1,Z-1:No peak

preculture

F-1,F-2,F-3,F-4,E-15CP,I-2,Z-1,E-17,E-18CP(M9 medium)
E-23'(LB medium)

Lab member

Yoshikawa,Nakashima,Tara,Nakamura

Contents

assay

F-1,F-13,E-17,Z-1,E-15,I-2
culture in M9
except E-15,OD600 is more than 1.0.
E-15:over 0.85
F-2:only 0.3
E-23':over 2.0,culture in LB medium
All samples was cultured in 3 mL.(n=5)
F-1,Z-1:cultured in 20mL(flask)(n=1)

OD600 before subculture

E-15:1.113
E-11:0.933
E-18:1.190
E-23':forgot to measure
F-1:0.927
F-2:0.374
F-3:0.878
F-4:0.992
I-2:0.888
Z-1:1.084

PCR

J-5(F,R),J-6(F,R)
J-5:OK!
extension time
F:1800+200=2000bp,4min
R:1850+200=2050bp,4min

PCR

J-6(F,R)
J-5R
extension time:1min

Lab member

Yoshikawa,Nakashima,Tara

Contents

making gel

subculture

Escherichia.coliMG1655
100 fold dilution
20mL,flask

PCR clean up and check

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J-6F:low concentration
J-6R:shorter band is OK!
We decided that we made J-5,J-6
as H5deg-positive feedback circuit, and H6deg-positive feedback circuit.

sequence order

G-1deg,G-4deg,H-1deg,H-4deg

TF

(electroporation)

F-1,F-2,F-3,F-4,E-17,E-18CP,Z-1,I-2,E-15
G-1deg,G-4deg,H-1deg,h-4deg,J-4,J-4
also,2mL culture as recovery.

Lab member

Yoshiakwa,Nakashima,Tara

Contents

digestion,gel extraction

E-19 XP,E-20 XP,H-1deg SP,H-4deg SP

making gel

making plate

Chloramphenicol*10

ligation

J-5deg(E-19XP+H-1degSP)
J-6deg(E-20XP+H-4degSP)
We did not have competent cell of E.coli MG1655, so put it in freezer.

preculture

F-1,E-17,E-15,Z-1,I-2:both in LB medium and M9 medium.
MG1655:LB medium

Lab member

Yoshikawa,Nakashima,Tara,Yamanaka

assay1,3

I-2,E-15,Z-1,F-1,E-17(made glycerol stock,subculture in 20 fold dilution)
MG1655 did not grow, because we accidentally put antibiotics.
OD600(O/N culture)
E-15:1.795
E-17:1.781
F-1:1.937
I-2:1.886
Z-1:1.780

making M9 medium