Team:Marburg SYNMIKRO/Notebook

From 2012.igem.org

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<div class="conboxb">
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= Week 1 =
= Week 1 =
<html>
<html>
<div class="fixcon">
<div class="fixcon">
-
<div class="toggleall" onClick="javascript:toggledisplay('klapp1');javascript:toggledisplay('klapp2');javascript:toggledisplay('klapp3');javascript:toggledisplay('klapp4');javascript:toggledisplay('klapp5');javascript:toggledisplay('klapp6');javascript:toggledisplay('klapp7');javascript:toggledisplay('klapp8');javascript:toggledisplay('klapp9');javascript:toggledisplay('klapp10');" return false">[all]</div>
+
<div class="toggleall" onClick="javascript:toggledisplay('klapp1');javascript:toggledisplay('klapp2');javascript:toggledisplay('klapp3');javascript:toggledisplay('klapp4');javascript:toggledisplay('klapp5');javascript:toggledisplay('klapp6');javascript:toggledisplay('klapp7');javascript:toggledisplay('klapp8');javascript:toggledisplay('klapp9');javascript:toggledisplay('klapp10');javascript:toggledisplay('klapp11');" return false">[all]</div>
<div class="fixtoggle" onClick="javascript:toggledisplay('klapp1'); togglearrow(this);" return false"></div>
<div class="fixtoggle" onClick="javascript:toggledisplay('klapp1'); togglearrow(this);" return false"></div>
<div class="smalldate">09.07. - 15.07.2012</div>
<div class="smalldate">09.07. - 15.07.2012</div>
Line 81: Line 85:
==09.07.2012==
==09.07.2012==
; Inoculation
; Inoculation
-
: ''E. coli'' strain C600 Mucts 62' in DYT-medium
+
: ''E. coli'' strain C600 Mucts62 in dYT-medium
==10.07.2012==
==10.07.2012==
; Isolation of chromosomal DNA  
; Isolation of chromosomal DNA  
-
: ''E. coli'' strain C600 Mucts 62'''
+
: ''E. coli'' strain C600 Mucts62 (lysogenic for temperature sensitive phage Mu ''c''ts62)
; Inoculation
; Inoculation
-
: ''E. coli'' strain DH5a and TOP10 in DYT-medium
+
: ''E. coli'' strain DH5&alpha; and TOP10 in dYT-medium
==11.07.2012==
==11.07.2012==
Line 95: Line 99:
==12.07.2012==
==12.07.2012==
; Transformation
; Transformation
-
: GFP, GFP_fusion, CFP, mRFP, RBS, P108, P100, PLac with ''E.coli'' Top10
+
: GFP, GFP_fusion, CFP, mRFP, RBS, P108, P100, Plac with ''E.coli'' TOP10
{|class="wikitable"  
{|class="wikitable"  
Line 116: Line 120:
|PLac||[http://partsregistry.org/Part:BBa_I14032 BBa_I14032]||2||11P||pSB2K3||Kanamycin
|PLac||[http://partsregistry.org/Part:BBa_I14032 BBa_I14032]||2||11P||pSB2K3||Kanamycin
|}
|}
 +
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<div class="conboxb">
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Line 132: Line 143:
==16.07.2012==
==16.07.2012==
-
; PCR
+
; PCR  
-
: AmiC (primer: MS1 + MS2)
+
: AmiC (primer: [[Team:Marburg_SYNMIKRO/Primer#MS1|MS1]] + [[Team:Marburg_SYNMIKRO/Primer#MS2|MS2]])
-
: HU (primer: MS3 + MS4)
+
: HU (primer: [[Team:Marburg_SYNMIKRO/Primer#MS3|MS3]] + [[Team:Marburg_SYNMIKRO/Primer#MS4|MS4]])
-
: GroES (primer: MS5 + MS6)
+
: GroES (primer: [[Team:Marburg_SYNMIKRO/Primer#MS5|MS5]] + [[Team:Marburg_SYNMIKRO/Primer#MS6|MS6]])
-
: GixR (primer: MS7 + MS8)
+
: GixR (primer: [[Team:Marburg_SYNMIKRO/Primer#MS7|MS7]] + [[Team:Marburg_SYNMIKRO/Primer#MS8|MS8]])
-
: Enhancer (primer: MS9 + MS10)
+
: Enhancer (primer: [[Team:Marburg_SYNMIKRO/Primer#MS9|MS9]] + [[Team:Marburg_SYNMIKRO/Primer#MS10|MS10]])
-
: &rArr; template-DNA: chromosomal DNA, annealing: 30 sec; 60 °C, elongation: 30 sec; 72 °C, 35 cycles
+
: &rArr; template-DNA: chromosomal DNA, denaturation: 30 sec at 95°C; annealing: 30 sec at 60°C; elongation: 30 sec at 72°C; 35 cycles
; Inoculation
; Inoculation
Line 158: Line 169:
:RBS
:RBS
:P108
:P108
-
:<strike>LacIQ</strike>
+
:LacIq
:P100
:P100
==18.07.2012==
==18.07.2012==
-
; Agarose gel
+
; 1% Agarose gel
-
: pipet scheme
+
[[File:Syn_MR_Gel1.jpg|thumb|800px|center|M:1kb gene ruler plus, 1:enhancer, 2:GroES, 3:AmiC, 4:HU, 5:GixR, 6:GFP, 7:GFP-Fusion, 8:CFP, 9:mRFP, 10:RBS, 11:LacIQ, 12:P108, 13:P100, 14:λ-DNA (PCR 18.07.2012)]]
-
Marker|enhancer|GroES|AmiC|HU|GixR|GFP|GFP-Fusion|CFP|mRFP|RBS|LacIQ|J23108|J23100|λ-DNA
+
-
(bild 1)
+
; DNA Gel extraction
; DNA Gel extraction
Line 173: Line 182:
; GixR Hybridization
; GixR Hybridization
-
* 5 µl primer MS7 (100 µM) + 5 µl primer MS8 (100 µM)
+
* 5 µl primer [[Team:Marburg_SYNMIKRO/Primer#MS7|MS7]] (100 µM) + 5 µl primer [[Team:Marburg_SYNMIKRO/Primer#MS8|MS8]] (100 µM)
** 5 min 99°C
** 5 min 99°C
** over night, room temperature
** over night, room temperature
Line 186: Line 195:
; PCR
; PCR
-
: AmiC (primer: MS1 + MS2)
+
: AmiC (primer: [[Team:Marburg_SYNMIKRO/Primer#MS1|MS1]] + [[Team:Marburg_SYNMIKRO/Primer#MS2|MS2]])
: &rArr; template-DNA: chromosomal DNA, annealing: 30 sec; 85 °C, elongation: 36 sec; 72 °C, 35 cycles, 1) 2 µl DMSO; 1 µl chrom. DNA | 2) 2 µl DMSO; 5 µl chrom. DNA | 3) without DMSO; 1 µl chrom. DNA | 4) without DMSO; 5 µl chrom. DNA | 5) 2 µl DMSO; without chrom. DNA
: &rArr; template-DNA: chromosomal DNA, annealing: 30 sec; 85 °C, elongation: 36 sec; 72 °C, 35 cycles, 1) 2 µl DMSO; 1 µl chrom. DNA | 2) 2 µl DMSO; 5 µl chrom. DNA | 3) without DMSO; 1 µl chrom. DNA | 4) without DMSO; 5 µl chrom. DNA | 5) 2 µl DMSO; without chrom. DNA
-
; Test restriction
+
; Test restriction digest
: 7) CFP (EcoRI/PstI)
: 7) CFP (EcoRI/PstI)
: 8) GFP (EcoRI/PstI)
: 8) GFP (EcoRI/PstI)
Line 198: Line 207:
: &rArr; all negative
: &rArr; all negative
-
; Agarose gel
+
; 1% Agarose gel
-
: pipet scheme
+
[[File:Syn_MR_Gel2.jpg|thumb|800px|center|M:1kb gene ruler plus, 1-5:AmiC PCR conditions (see above), 6-9: control digest (E/P), 6:GFP, 7:GFP-Fusion, 8:CFP, 9:mRFP (PCR 19.07.2012)]]
-
1)|2)|3)|4)|5)|marker|7)|8)|9)|10)
+
 
-
(bild 2)
+
; Control digest
; Control digest
: CFP (EcoRI/PstI)
: CFP (EcoRI/PstI)
Line 210: Line 218:
==20.07.2012==
==20.07.2012==
-
; Agarose gel
+
; 1% Agarose gel from the control digest
-
: pipet scheme
+
[[File:Syn_MR_Gel3.jpg|thumb|400px|center|M:1kb gene ruler plus, 1:GFP, 2: GFP-Fusion, 3: CFP, 4: mRFP (control digest 18.07.2012)]]
-
: &rArr; marker|GFP|GFP-fusion|CFP|mRFP (all positive)
+
-
(bild 3)
+
-
 
+
<html>
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<div class="conboxb">
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= Week 3 =
= Week 3 =
Line 238: Line 249:
: Enhancer + pSB1K3
: Enhancer + pSB1K3
: cyclisation of pSB1C3, pSB1K3, pSB1A3, pSB1T3 (couldn’t work)
: cyclisation of pSB1C3, pSB1K3, pSB1A3, pSB1T3 (couldn’t work)
 +
 +
==27.07.2012==
 +
Our lab-dragon "Hugo" is born
 +
[[File:Syn_MR_Hugo.jpg|frameless|border|650px|center]]
Line 244: Line 259:
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+
<html>
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</div>
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</html>
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<html>
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<div class="conboxb">
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</html>
= Week 4 =
= Week 4 =
<html>
<html>
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; PCR
; PCR
-
: CFP (primer: MS16 + MS17) &rArr; template-DNA: BBa_E0010
+
: CFP (primer: [[Team:Marburg_SYNMIKRO/Primer#MS16|MS16]] + [[Team:Marburg_SYNMIKRO/Primer#MS17|MS17]]) &rArr; template-DNA: [http://partsregistry.org/Part:BBa_E0020 BBa_E0020]
: &rArr; positive
: &rArr; positive
-
: mRFP (primer: MS14 + MS15) &rArr; template-DNA: BBa_E1010  
+
: mRFP (primer: [[Team:Marburg_SYNMIKRO/Primer#MS14|MS14]] + [[Team:Marburg_SYNMIKRO/Primer#MS15|MS15]]) &rArr; template-DNA: [http://partsregistry.org/Part:BBa_E1010 BBa_E1010]
: &rArr; positive
: &rArr; positive
-
: Gin (primer: MS11 + MS13) &rArr; template-DNA: chromosomal DNA (that was wrong)  
+
: Gin (primer: [[Team:Marburg_SYNMIKRO/Primer#MS11|MS11]] + [[Team:Marburg_SYNMIKRO/Primer#MS13|MS13]]) &rArr; template-DNA: chromosomal DNA (that was wrong)  
-
: RBS-Gin (primer: MS13 + MS12) &rArr; template-DNA: chromosomal DNA (that was wrong)  
+
: RBS-Gin (primer: [[Team:Marburg_SYNMIKRO/Primer#MS13|MS13]] + [[Team:Marburg_SYNMIKRO/Primer#MS12|MS12]]) &rArr; template-DNA: chromosomal DNA (that was wrong)  
: &rArr; annealing: 30 sec; 60 °C, elongation: 40 sec; 72 °C, 35 cycles
: &rArr; annealing: 30 sec; 60 °C, elongation: 40 sec; 72 °C, 35 cycles
Line 280: Line 300:
-
; Agarose gel
+
; 1% Agarose gel
-
: pipet scheme
+
[[File:Syn_MR_Gel4.jpg|thumb|400px|center|M:Marker, 1: Gix, 2-6: AmiC (PCR 16.07.2012)]]
-
: &rArr; gix|marker|AmiC from 16.07.| AmiC from 16.07.| AmiC from 19.07.| AmiC from 19.07.
+
 
-
: &rArr; all negative
+
 
-
(bild 4,5)
+
; 1% Agarose gel
 +
[[File:Syn_MR_Gel5.jpg|thumb|300px|center|M:1kb gene ruler plus, 1: AmiC(PCR 16.07.2012)]]
 +
 
 +
 
 +
; 1% Agarose gel
 +
[[File:Syn_MR_Gel6.jpg|thumb|300px|center|M:1kb gene ruler plus, 1: RBS-Gin from 30.07, 2:AmiC from 16.07., 3:Gin from 30.07., 4:CFP from 30.07., 5:mRFP from 30.07., 6: control (PCR 30.07.2012)]]
-
; Agarose gel
 
-
: pipet scheme
 
-
: &rArr; marker|RBS-Gin from 30.07.|AmiC from 16.07.|Gin from 30.07.|CFP from 30.07.|mRFP from 30.07.|control
 
-
: &rArr; all negative
 
; Resuspension of a biobrick
; Resuspension of a biobrick
-
: BBa_B0010 (terminator)
+
: [http://partsregistry.org/Part:BBa_B0010 BBa_B0010](Terminator)
; DNA Gel extraction
; DNA Gel extraction
Line 299: Line 320:
; PCR
; PCR
-
: AmiC (primer: MS1 + MS2)
+
: AmiC (primer: [[Team:Marburg_SYNMIKRO/Primer#MS1|MS1]] + [[Team:Marburg_SYNMIKRO/Primer#MS2|MS2]])
-
: RBS-Gin (primer: MS12 + MS13)
+
: RBS-Gin (primer: [[Team:Marburg_SYNMIKRO/Primer#MS12|MS12]] + [[Team:Marburg_SYNMIKRO/Primer#MS13|MS13]])
-
: Gin (primer: MS11 + MS13)
+
: Gin (primer: [[Team:Marburg_SYNMIKRO/Primer#MS11|MS11]] + [[Team:Marburg_SYNMIKRO/Primer#MS13|MS13]])
: &rArr; template-DNA: chromosomal DNA, annealing: 30 sec; 55 °C, elongation: 40 sec; 72 °C, 35 cycles
: &rArr; template-DNA: chromosomal DNA, annealing: 30 sec; 55 °C, elongation: 40 sec; 72 °C, 35 cycles
Line 327: Line 348:
; GixR Hybridization
; GixR Hybridization
-
* 5 µl primer MS7 (100 µM) + 5 µl primer MS8 (100 µM)
+
* 5 µl primer [[Team:Marburg_SYNMIKRO/Primer#MS7|MS7]] (100 µM) + 5 µl primer [[Team:Marburg_SYNMIKRO/Primer#MS8|MS8]] (100 µM)
** 5 min 99°C
** 5 min 99°C
** over night, room temperature
** over night, room temperature
Line 357: Line 378:
: pSB1C3 (EcoRI/PstI)
: pSB1C3 (EcoRI/PstI)
-
; Agarose gel
+
; 1% Agarose gel
-
: pipet scheme
+
[[File:Syn_MR_Gel7.jpg|thumb|500px|center|M:1kb gene ruler plus, 1: RBS-Gin (+DMSO), 2:RBS-Gin (-DMSO), 3:Gin (+DMSO), 4:Gin (-DMSO), 5: control (PCR 31.07.2012)]]
-
: Marker|RBS-Gin1 +DMSO|RBS-Gin2 -DMSO|Gin1 +DMSO|Gin2 -DMSO|control
+
-
(bild 6)
+
-
; Agarose gel
+
; 1% Agarose gel
-
: pipet scheme
+
[[File:Syn_MR_Gel8.jpg|thumb|500px|center|M:1kb gene ruler plus, 1: AmiC (+DMSO), 2:AmiC (-DMSO), (PCR 31.07.2012)]]
-
: Marker|AmiC1 +DMSO| AmiC2 –DMSO
+
-
(bild 7)
+
; DNA Gel extraction
; DNA Gel extraction
Line 394: Line 411:
: GixR in pSB1C3
: GixR in pSB1C3
-
; Agarose gel
+
; 1% Agarose gel
-
: pipet scheme
+
[[File:Syn_MR_Gel9.jpg|thumb|500px|center|M:1kb gene ruler plus, 1-3: pSB1A3, 4-7:pSB1C3, 8-11: pSB1K3 (Control digest 01.08.2012)]]
-
: Marker| pSB1A3 1,2,3| pSB1C3 1,2,3,4| pSB1K3 1,2,3,4
+
 
-
(bild 8)
+
 
: pSB1A3 c2
: pSB1A3 c2
: pSB1C3 c4
: pSB1C3 c4
Line 430: Line 447:
: Terminator c1,2,3,4 (EcoRI/PstI)
: Terminator c1,2,3,4 (EcoRI/PstI)
-
; Agarose gel
+
 
-
: pipet scheme
+
; 2% Agarose gel
-
: Marker|Terminator 1,2,3,4
+
[[File:Syn_MR_Gel10.jpg|thumb|500px|center|M:1kb gene ruler plus, 1-4: Terminator, M2:Middle Range gene ruler (Control digest 01.08.2012)]]
-
(bild 9)
+
; Transformation
; Transformation
Line 447: Line 463:
; Plasmid preparation
; Plasmid preparation
: Enhancer in pJET
: Enhancer in pJET
-
 
-
 
Line 454: Line 468:
</div>
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</div>
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<div class="conboxb">
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= Week 5 =
= Week 5 =
<html>
<html>
Line 487: Line 506:
; GixR Hybridization
; GixR Hybridization
-
* 1(3) µl primer MS7 (100 µM) + 1(3) µl primer MS8 (100 µM) + T4-Ligasebuffer  
+
* 1(3) µl primer [[Team:Marburg_SYNMIKRO/Primer#MS7|MS7]]7 (100 µM) + 1(3) µl primer [[Team:Marburg_SYNMIKRO/Primer#MS8|MS8]] (100 µM) + T4-Ligasebuffer  
** 5 min 95°C
** 5 min 95°C
** over night, room temperature
** over night, room temperature
Line 527: Line 546:
: pJET (XbaI)
: pJET (XbaI)
-
; Agarose gel
 
-
: pipet scheme
 
-
: Marker|pSB3C5 c1a,2a,3a,4a|Marker|pSB3C5 c1b,2b,3b,4b|Marker|Enhancer-pJET c1,2,3,4|Marker
 
-
(bild 10)
 
-
: &rArr; positive: pSB3C5 c3a,4a,3b,4b,; Enhancer-pJET c1,2,3,4
 
 +
; 1% Agarose gel
 +
[[File:Syn_MR_Gel11.jpg|thumb|700px|center|M:1kb gene ruler plus, 1-4: pSB3C5, 5-8:pSB3C5, 9-12: Enhancer-pJET (Control digest 07.08.2012)]]
 +
 +
: &rArr; positive: pSB3C5 3,4,7,8,; Enhancer-pJET 9-12
==08.08.2012==
==08.08.2012==
Line 547: Line 565:
: pSB1C3 (EcoRI/PstI)
: pSB1C3 (EcoRI/PstI)
-
; Agarose gel
+
; 2% Agarose gel
-
: pipet scheme
+
[[File:Syn_MR_Gel12.jpg|thumb|500px|center|M:1kb gene ruler plus, 1: Enhancer, 2:pSB1C3, (Control digest 08.08.2012)]]
-
: Marker| Enhancer|pSB1C3
+
-
(bild 11)
+
; DNA Gel extraction
; DNA Gel extraction
Line 587: Line 603:
: CFP (EcoRI/PstI)
: CFP (EcoRI/PstI)
-
; Agarose gel
 
-
: pipet scheme
 
-
: Marker|GFP-fusion 1,2,3,4,5,6,7,8,9| Marker|10,11,12,13,14,15,16,17,18
 
-
(bild 12)
 
-
: Marker|J23108-RBS 1,2,3,4,5,6,7,8,9| Marker|10,11,12,13,14,15,16,17,18
+
; 1% Agarose gel
-
(bild 13)
+
[[File:Syn_MR_Gel13.jpg|thumb|500px|center|M:1kb gene ruler plus, 1-18: GFP-Fusion - Terminator, (Control digest 09.08.2012)]]
 +
 
 +
; 1% Agarose gel
 +
[[File:Syn_MR_Gel14.jpg|thumb|500px|center|M:1kb gene ruler plus, 1-18: P108-RBS, (Control digest 09.08.2012)]]
 +
 
 +
 
 +
; 1% Agarose gel
 +
[[File:Syn_MR_Gel15.jpg|thumb|500px|center|M:1kb gene ruler plus, 1-18: P100-RBS, (Control digest 09.08.2012)]]
-
: Marker|J23100-RBS 1,2,3,4,5,6,7,8,9| Marker|10,11,12,13,14,15,16,17,18
 
-
(bild 14)
 
-
: Marker|GFP-fusion 19,20| J23108-RBS 19,20| J23100-RBS 19,20| Marker |CFP 1,2,3,4,5
+
; 1% Agarose gel
-
(bild 15)
+
[[File:Syn_MR_Gel16.jpg|thumb|500px|center|M:1kb gene ruler plus, 1-2: GFP-Fusion - Terminator, 3-4: P108-RBS, 5-6: P100-RBS, 7-11: CFP (Control digest 09.08.2012)]]
Line 609: Line 626:
: Enhancer in C3
: Enhancer in C3
: mRFP in A3
: mRFP in A3
-
 
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<div class="conboxb">
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= Week 6 =
= Week 6 =
Line 626: Line 648:
; PCR
; PCR
-
: GroES (primer: MS5 + MS6 (b))
+
: GroES (primer: [[Team:Marburg_SYNMIKRO/Primer#MS5|MS5]] + [[Team:Marburg_SYNMIKRO/Primer#MS6b|MS6b]])
-
: HU (primer: MS3 + MS4 (b))
+
: HU (primer: [[Team:Marburg_SYNMIKRO/Primer#MS3|MS3]] + [[Team:Marburg_SYNMIKRO/Primer#MS4b|MS4b]])
: &rArr; with and without DMSO, template-DNA: chromosomal DNA, annealing: 30 sec; 60 °C, elongation: 40 sec; 72 °C, 35 cycles
: &rArr; with and without DMSO, template-DNA: chromosomal DNA, annealing: 30 sec; 60 °C, elongation: 40 sec; 72 °C, 35 cycles
-
; Agarose gel
+
 
-
: pipet scheme
+
; 1% Agarose gel
-
: Marker|HU +DMSO|HU -DMSO|GroES +DMSO|GroES –DMSO|control
+
[[File:Syn_MR_Gel17.jpg|thumb|500px|center|M:1kb gene ruler plus, 1: HU (+DMSO), 2: HU (-DMSO), 3: GroES (+DMSO), 4: GroES (-DMSO), (PCR 13.08.2012)]]
-
(bild 16)
+
; Transformation
; Transformation
Line 666: Line 687:
; PCR
; PCR
-
: GroES (primer: MS5 + MS6 (b))
+
: GroES (primer: [[Team:Marburg_SYNMIKRO/Primer#MS5|MS5]] + [[Team:Marburg_SYNMIKRO/Primer#MS6b|MS6b]])
-
: HU (primer: MS3 + MS4 (b))
+
: HU (primer: [[Team:Marburg_SYNMIKRO/Primer#MS3|MS3]] + [[Team:Marburg_SYNMIKRO/Primer#MS4b|MS4b]])
-
: &rArr; with and without DMSO, template-DNA: chromosomal DNA, annealing: 30 sec; 55 °C, elongation: 30 sec; 72 °C or annealing/elongation: 30 sec;  72 °C, 35 cycles
+
: &rArr; with and without DMSO, template-DNA: chromosomal DNA, annealing: 30 sec; 55 °C, elongation: 30 sec; 72 °C, 35 cycles
-
; Agarose gel
+
; 1% Agarose gel
-
: pipet scheme
+
[[File:Syn_MR_Gel18.jpg|thumb|500px|center|M:1kb gene ruler plus, 1: HU (-DMSO), 2: HU (+DMSO), 3: GroES (-DMSO), 4: GroES (+DMSO), (PCR 15.08.2012)]]
-
: Marker|HU +DMSO|HU -DMSO|GroES +DMSO|GroES –DMSO| HU +DMSO two-step PCR|HU -DMSO two-step PCR |GroES +DMSO two-step PCR |GroES –DMSO two-step PCR
+
-
(bild 17)
+
 +
; DNA Gel extraction
 +
: HU 1,2
; Resuspension of a biobrick
; Resuspension of a biobrick
Line 683: Line 704:
: BBa_J04450 in pSB1K3
: BBa_J04450 in pSB1K3
-
; Agarose gel
 
-
: pipet scheme
 
-
: Marker|HU -DMSO|HU +DMSO|GroES -DMSO|GroES +DMSO
 
-
(bild 18)
 
-
 
-
: &rArr; 2-step PCR didn’t work, HU 1,2
 
-
 
-
; DNA Gel extraction
 
-
: HU 1,2
 
; Transformation
; Transformation
Line 709: Line 721:
; PCR
; PCR
-
: GroES (primer: MS5 + MS6 (b))
+
: GroES (primer: [[Team:Marburg_SYNMIKRO/Primer#MS5|MS5]] + [[Team:Marburg_SYNMIKRO/Primer#MS6b|MS6b]])
: &rArr; with and without DMSO, template-DNA: chromosomal DNA, annealing: 30 sec; 55 °C, elongation: 30 sec; 72 °C , 35 cycles
: &rArr; with and without DMSO, template-DNA: chromosomal DNA, annealing: 30 sec; 55 °C, elongation: 30 sec; 72 °C , 35 cycles
-
; Agarose gel
+
 
-
: pipet scheme
+
; 1% Agarose gel
-
: Marker| GroES 1, 2 +DMSO
+
[[File:Syn_MR_Gel19.jpg|thumb|500px|center|M:1kb gene ruler plus, 1: GroES (-DMSO), 2: GroES (+DMSO), (PCR 16.08.2012)]]
-
(bild 19)
+
; Plasmid preparation
; Plasmid preparation
Line 723: Line 734:
: P-RBS (EcoRI/PstI)
: P-RBS (EcoRI/PstI)
-
; Agarose gel
+
; 1% Agarose gel
-
: pipet scheme
+
[[File:Syn_MR_Gel20.jpg|thumb|500px|center|M:1kb gene ruler plus, 1: control, 2: P108-RBS, (control digest 16.08.2012)]]
-
: control|P-RBS|marker
+
-
(bild 20)
+
-
 
+
; Restriction
; Restriction
Line 776: Line 784:
: BBa_J04450 in pSB1K3 (EcoRI/PstI)
: BBa_J04450 in pSB1K3 (EcoRI/PstI)
-
; Agarose gel
 
-
: pipet scheme
 
-
: marker|BBa_J04450 in pSB1A3 1,2,3,4|BBa_J04450 in pSB1K3 1,2,3,4| marker| BBa_J04450 in pSB1C3 1,2,3,4
 
-
(bild 21)
 
 +
; 1% Agarose gel
 +
[[File:Syn_MR_Gel21.jpg|thumb|500px|center|M:1kb gene ruler plus, 1-4: BBa_J04450 in pSB1A3, 5-8: BBa_J04450 in pSB1K3 P108-RBS, 9-12: BBa_J04450 in pSB1C3, (control digest 17.08.2012)]]
-
; Agarose gel
 
-
: pipet scheme
 
-
: marker|P-RBS 1,2,3,4| P-RBS 1,2,3,4| marker| BBa_J04450 in pSB1K3 1,2,3,4
 
-
 
-
(bild 22)
 
 +
; 1% Agarose gel
 +
[[File:Syn_MR_Gel22.jpg|thumb|500px|center|M:1kb gene ruler plus, 1-4: P-RBS (E/P), 5-8: P-RBS (RsaI), 9-12: BBa_J04450 in pSB1T3, (control digest 17.08.2012)]]
<html>
<html>
</div>
</div>
</html>
</html>
-
 
+
<html>
 +
</div>
 +
</html>
 +
<html>
 +
<div class="conboxb">
 +
</html>
= Week 7 =
= Week 7 =
<html>
<html>
Line 805: Line 813:
; PCR
; PCR
-
: Backbone (primer MS24 + MS25)
+
: Backbone (primer [[Team:Marburg_SYNMIKRO/Primer#MS24|MS24]] + [[Team:Marburg_SYNMIKRO/Primer#MS25|MS25]])
: &rArr; template-DNA: pSB1A3, pSB1T3, pSB1C3 and pSB1K3, annealing: 30 sec; 55 °C, elongation: 30 sec; 72 °C , 35 cycles
: &rArr; template-DNA: pSB1A3, pSB1T3, pSB1C3 and pSB1K3, annealing: 30 sec; 55 °C, elongation: 30 sec; 72 °C , 35 cycles
-
; Agarose gel
+
; 1% Agarose gel
-
: pipet scheme
+
[[File:Syn_MR_Gel23.jpg|thumb|500px|center|M:1kb gene ruler plus, 1: pSB1A3, 2: pSB1T3, 3: pSB1C3, 4: pSB1K3 (PCR 20.08.2012)]]
-
: marker|: pSB1A3|pSB1T3|pSB1C3|pSB1K3
+
-
(bild 23)
+
; Transformation
; Transformation
Line 836: Line 842:
: pSB1C3-BBa_J04450 (template DNA)
: pSB1C3-BBa_J04450 (template DNA)
: pSB1K3-BBa_J04450 (template DNA)
: pSB1K3-BBa_J04450 (template DNA)
-
: used primer --> MS24Prefix_rev + MS25Suffix_fwd
+
: used primer --> [[Team:Marburg_SYNMIKRO/Primer#MS24|MS24]] + [[Team:Marburg_SYNMIKRO/Primer#MS25|MS25]]
: initial denaturation 98°C - 5min
: initial denaturation 98°C - 5min
: denaturation: 98°C - 1min; annealing: 55°C - 30s; elongation: 72°C - 2.5min -->repeat 35 times
: denaturation: 98°C - 1min; annealing: 55°C - 30s; elongation: 72°C - 2.5min -->repeat 35 times
-
: 72°C - 5min --> 4°C - ∞
+
: 72°C - 5min --> 4°C - 8
; miniprep
; miniprep
Line 848: Line 854:
: over night, 37°C
: over night, 37°C
-
; Agarose gel (1%)
+
; 1% Agarose gel
-
: watch pic
+
: [[File:Syn_MR_Gel24.jpg|thumb|800px|center|M:1kb gene ruler plus, 1:pSB1A3, 2:pSB1K3, 3:pSB1C3, 4:pSB1T3, 5: negative control, (PCR 21.08.2012)]]
; Inoculation
; Inoculation
Line 859: Line 865:
; PCR
; PCR
-
: CFP
+
: CFP ([[Team:Marburg_SYNMIKRO/Primer#MS16|MS16]] + [[Team:Marburg_SYNMIKRO/Primer#MS17|MS17]])
-
: mRFP
+
: mRFP ([[Team:Marburg_SYNMIKRO/Primer#MS14|MS14]] + [[Team:Marburg_SYNMIKRO/Primer#MS15|MS15]])
: initial denaturation 98°C - 5min
: initial denaturation 98°C - 5min
: denaturation: 98°C - 10s; annealing: 60°C - 30s; elongation: 72°C - 40s -->repeat 35 times
: denaturation: 98°C - 10s; annealing: 60°C - 30s; elongation: 72°C - 40s -->repeat 35 times
-
: 72°C - 5min --> 4°C - ∞
+
: 72°C - 5min --> 4°C - 8
-
; Agarose gel (1%)
+
; 1% Agarose gel
-
: watch picture
+
: [[File:Syn_MR_Gel25.jpg|thumb|800px|center|M:1kb gene ruler plus, 1:mRFP, 2:mRFP, 3:CFP, 4:CFP (PCR 22.08.2012)]]
; miniprep
; miniprep
Line 873: Line 879:
; Test restriction
; Test restriction
: of LacIQ with EcoRI and PstI
: of LacIQ with EcoRI and PstI
-
: watch picture
 
-
; Agarose gel  
+
; 1% Agarose gel
-
: test restriction of GroES and GFP-T (->21.08.12), gel 1%
+
: [[File:Syn_MR_Gel26.jpg|thumb|800px|center|M:1kb gene ruler plus, 1-4:LacIQ, (control digest 22.08.2012)]]
-
: watch picture
+
 
 +
; 1% Agarose gel
 +
: [[File:Syn_MR_Gel27.jpg|thumb|800px|center|M:1kb gene ruler plus, 1-4:GroES, 5-12: GFT-Terminator, (control digest 21.08.2012)]]
 +
 
 +
; 1% Agarose gel
 +
: [[File:Syn_MR_Gel28.jpg|thumb|800px|center|M:1kb gene ruler plus, 1-12:Enhancer, (control digest 21.08.2012)]]
 +
 
 +
; 1% Agarose gel
 +
: [[File:Syn_MR_Gel29.jpg|thumb|800px|center|M:1kb gene ruler plus, 1-4:Enhancer, 5-12: P-RBS, (control digest 21.08.2012)]]
 +
 
: test restriction of Enhancer (->21.08.12), gel 2%
: test restriction of Enhancer (->21.08.12), gel 2%
: watch picture
: watch picture
Line 886: Line 900:
== 23.08.2012 ==
== 23.08.2012 ==
-
Svetti please check!
+
; PCR
-
-->lacIQ evtl ganz rausnehmen?! ;)
+
: pSB1K3-BBa_J04450 (template DNA)
 +
: pSB1T3-BBa_J04450 (template DNA)
 +
: used primer --> [[Team:Marburg_SYNMIKRO/Primer#MS24|MS24]] + [[Team:Marburg_SYNMIKRO/Primer#MS25|MS25]]
 +
: initial denaturation 98°C - 5min
 +
: denaturation: 98°C - 1min; annealing: 55°C - 30s; elongation: 72°C - 2.5min -->repeat 35 times
 +
: 72°C - 5min --> 4°C - 8
 +
 
 +
; 1% Agarose gel
 +
: [[File:Syn_MR_Gel31.jpg|thumb|800px|center|M:gene ruler mix ladder, 1-2:pSB1K3-BBa_J04450, 3-4: pSB1T3-BBa_J04450, (PCR 23.08.2012)]]
 +
 
 +
; Restriction
 +
: GixR (EcoRI/PstI)
 +
 
 +
; Ligation
 +
: GixR in pSB1C3
 +
 
 +
; Transformation
 +
: GixR in pSB1C3
 +
 
== 24.08.2012 ==
== 24.08.2012 ==
-
Svetti please check!
+
; PCR
 +
: pSB1A3-BBa_J04450 (template DNA)
 +
: pSB1T3-BBa_J04450 (template DNA)
 +
: pSB1C3-BBa_J04450 (template DNA)
 +
: pSB1K3-BBa_J04450 (template DNA)
 +
: initial denaturation 98°C - 5min
 +
: denaturation: 98°C - 10s; annealing: 60°C - 30s; elongation: 72°C - 40s -->repeat 30 times
 +
: 72°C - 10min --> 4°C - 8
 +
: primer: [[Team:Marburg_SYNMIKRO/Primer#MS22|MS22]] + [[Team:Marburg_SYNMIKRO/Primer#MS23|MS23]]
 +
; 1% Agarose gel
 +
: [[File:Syn_MR_Gel32.jpg|thumb|800px|center|M:gene ruler mix ladder, 1-2:pSB1A3-BBa_J04450, 3-4: pSB1K3-BBa_J04450, 5-6:pSB1C3-BBa_J04450, 7-8: pSB1T3-BBa_J04450, (PCR 24.08.2012)]]
 +
 +
; Restriction
 +
: RBS (XbaI/PstI)
 +
: CFP (EcoRI/PstI)
 +
: mRFP (EcoRI/PstI)
 +
 +
; Ligation
 +
: mRFP and CFP in pSB1C3
 +
 +
; Transformation
 +
: mRFP and CFP in pSB1C3
<html>
<html>
</div>
</div>
 +
</html>
 +
<html>
 +
</div>
 +
</html>
 +
<html>
 +
<div class="conboxb">
</html>
</html>
= Week 8 =
= Week 8 =
Line 915: Line 974:
; PCR  
; PCR  
-
: template: backbones (pSB1K3/T3/C3/A3
+
: template: backbones (pSB1K3/T3/C3/A3)
-
: parameters: 98°C 5min, >> 98°C 1min, 70°C 30s, 72°C 2.5min << x30, 72°C 5min, 4°C ∞
+
: parameters: 98°C 5min, >> 98°C 1min, 70°C 30s, 72°C 2.5min << x30, 72°C 5min, 4°C 8
 +
: primer: [[Team:Marburg_SYNMIKRO/Primer#MS22|MS22]] + [[Team:Marburg_SYNMIKRO/Primer#MS23|MS23]]
; colony-PCR
; colony-PCR
-
: parameters: 98°C 5min, >> 98°C 1min, 72°C 1min << x30, 72°C 5min, 4°C ∞
+
: parameters: 98°C 5min, >> 98°C 1min, 72°C 1min << x30, 72°C 5min, 4°C 8
 +
: primer: [[Team:Marburg_SYNMIKRO/Primer#MS18|MS18]] + [[Team:Marburg_SYNMIKRO/Primer#MS19|MS19]]
-
; Agarose gel of backbone PCR
+
; Agarose gel of backbone PCR  
: no result
: no result
; elution of the backbones of Friday the 24th
; elution of the backbones of Friday the 24th
-
; Agarose gel of colony PCR
+
 
-
: watch picture
+
; 1% Agarose gel
 +
: [[File:Syn_MR_Gel33.jpg|thumb|800px|center|M:1kb gene ruler plus, 1-2:gixR, 3-9: LacIQ-RBS-Gin, 10: CFP, (colony PCR 27.08.2012)]]
 +
 
 +
 
 +
; 1% Agarose gel
 +
: [[File:Syn_MR_Gel34.jpg|thumb|800px|center|M:1kb gene ruler plus, 1-4:CFP, 5-10: mRFP, (colony PCR 27.08.2012)]]
; Inoculation
; Inoculation
Line 943: Line 1,009:
: Enhancer, Terminator, Gix, P-RBS
: Enhancer, Terminator, Gix, P-RBS
: parameters: 98°C 5', 98°C 1', 66°C 30", 72°C 30", 72°C 5', 4°C ~
: parameters: 98°C 5', 98°C 1', 66°C 30", 72°C 30", 72°C 5', 4°C ~
-
: watch picture
+
: primer: [[Team:Marburg_SYNMIKRO/Primer#MS20|MS20]] + [[Team:Marburg_SYNMIKRO/Primer#MS21|MS21]]
 +
 
 +
; 1% Agarose gel
 +
: [[File:Syn_MR_Gel35.jpg|thumb|800px|center|M:1kb gene ruler plus, 1:Terminator, 2-3: Enhancer, 4-6: P-RBS, 7-8: GixR, 9-10: Enhancer, (PCR 28.08.2012)]]
; Transformation
; Transformation
-
: pBad, araC, pSacB
+
: [http://partsregistry.org/Part:BBa_I0500 pBad/araC] (plate:1, Well:N14)
 +
: pSacB
: over night, 37°C  
: over night, 37°C  
Line 964: Line 1,034:
: diluted templates: Terminator, P-RBS, Gix 1 --> 1:10
: diluted templates: Terminator, P-RBS, Gix 1 --> 1:10
: Enhancer, Gix 2 --> 1:20
: Enhancer, Gix 2 --> 1:20
-
: watch picture
+
: parameters: 98°C 5', 98°C 1', 66°C 30", 72°C 30", 72°C 5', 4°C ~
 +
: primer: [[Team:Marburg_SYNMIKRO/Primer#MS20|MS20]] + [[Team:Marburg_SYNMIKRO/Primer#MS21|MS21]]
 +
 
 +
 
 +
; 1% Agarose gel
 +
: [[File:Syn_MR_Gel36.jpg|thumb|800px|center|M:1kb gene ruler plus, 1:Terminator, 2-3: Enhancer, 4-6: P-RBS, 7-8: GixR, 9: Enhancer control, 10: P-RBS control, 11: GixR control, (PCR 29.08.2012)]]
; 3AA
; 3AA
Line 993: Line 1,068:
; Sequencing
; Sequencing
-
: bitte mal nachschlagen, die Notizen peil ich nicht ;)
+
: CFP_fwd
-
 
+
: CFP_rev
 +
: mRFP_fwd
 +
: mRFP_rev
 +
: P-RBS_fwd
 +
: GixR_fwd
 +
: GixR_fwd
 +
: Enhancer_fwd
== 30.08.2012 ==
== 30.08.2012 ==
Line 1,012: Line 1,093:
: Parameters No.1: 95°C 5', >>95°C 30", 60°C 30", 72°C 1'<< x30, 72°C 5', 4°C
: Parameters No.1: 95°C 5', >>95°C 30", 60°C 30", 72°C 1'<< x30, 72°C 5', 4°C
: Parameters No.2: 95°C 5', >>95°C 30", 72°C 1.5'<< x30, 72°C 5', 4°C
: Parameters No.2: 95°C 5', >>95°C 30", 72°C 1.5'<< x30, 72°C 5', 4°C
 +
: Primer [[Team:Marburg_SYNMIKRO/Primer#MS26|MS26]] + [[Team:Marburg_SYNMIKRO/Primer#MS27|MS27]]
-
; Agarose gel
+
 
-
: watch picture
+
; 1% Agarose gel
-
: cut out lines at 1.5kb
+
: [[File:Syn_MR_Gel37.jpg|thumb|800px|center|M:1kb gene ruler plus, 1,3,5:SacB, 4,5: none, (PCR 31.08.2012)]]
; Ligation
; Ligation
Line 1,046: Line 1,128:
: Tet --> hardly colonies
: Tet --> hardly colonies
: --> comp.TOP10 obviously all right, exclusion of Tet in future work
: --> comp.TOP10 obviously all right, exclusion of Tet in future work
-
 
<html>
<html>
</div>
</div>
 +
</html>
 +
<html>
 +
</div>
 +
</html>
 +
<html>
 +
<div class="conboxb">
</html>
</html>
= Week 9 =
= Week 9 =
Line 1,071: Line 1,158:
-
; PCR
+
; colony-PCR
: of colonies (CFP-T, mRFP-T)
: of colonies (CFP-T, mRFP-T)
: parameters: 98°C 5', >>98°C 1', 72°C 1'<<x30, 72°C 5', 4°C
: parameters: 98°C 5', >>98°C 1', 72°C 1'<<x30, 72°C 5', 4°C
 +
: primer [[Team:Marburg_SYNMIKRO/Primer#MS18|MS18]] + [[Team:Marburg_SYNMIKRO/Primer#MS19|MS19]]
; Agarose gel
; Agarose gel
Line 1,080: Line 1,168:
: pC3-CFP (14)
: pC3-CFP (14)
: pC3.mRFP (24)
: pC3.mRFP (24)
-
: result watch picture
 
 +
; 1% Agarose gel
 +
: [[File:Syn_MR_Gel38.jpg|thumb|800px|center|M:1kb gene ruler plus, 1-6:CFP-Terminator, 7:CFP control, 8-13:mRFP-Terminator 14: mRFP control, 15-16: none, (colony-PCR 03.09.2012)]]
 +
== 04.09.2012 ==
 +
; Results
 +
: Transformation
 +
: CFP-T/mRFP-T in T3
 +
: 2 µl -> 5-6 colonies
 +
: 30 µl -> 1-2 colonies
 +
: Gix R in C3 (3.9) --> 4 colonies
 +
; colony-PCR
 +
: GixR
 +
: CFP-T
 +
: mRFP-T
 +
: parameters: 98°C 5min, >> 98°C 1min, 72°C 1min << x30, 72°C 5min, 4°C ∞
 +
: primer [[Team:Marburg_SYNMIKRO/Primer#MS20|MS20]] + [[Team:Marburg_SYNMIKRO/Primer#MS21|MS21]]
 +
; gel extraction
 +
: P108 (E/S)
 +
: RBS (X/P)
 +
; ligation
 +
: SacB1 + pSB1C3
 +
: P-RBS + pSB1C3
 +
; Transformation
 +
: ligation + TOP10 --> LB(cmp)
 +
 +
; 1% Agarose gel
 +
: [[File:Syn_MR_Gel40.jpg|thumb|800px|center|M:1kb gene ruler plus, 1-2:pBAD/araC, (mini-prep 31.08.2012)]]
 +
 +
 +
; Inoculation
 +
: pBad/araC
 +
: 5ml LB, over night, 37°C
 +
 +
; 1% Agarose gel
 +
: [[File:Syn_MR_Gel41.jpg|thumb|800px|center|M:1kb gene ruler plus, 1-4:GixR pSB1C3, 5-9:CFP-Terminator, 10:CFP control, (colony-PCR 04.09.2012)]]
 +
 +
 +
; 1% Agarose gel
 +
: [[File:Syn_MR_Gel42.jpg|thumb|800px|center|M:1kb gene ruler plus, 1-9:mRFP-Terminator, 10:mRFP control, (colony-PCR 04.09.2012)]]
 +
 +
 +
== 05.09.2012 ==
 +
; Results
 +
: Transformation
 +
: SacB in C3
 +
: P-RBS in C3
 +
 +
; colony PCR
 +
: SacB in C3
 +
: P-RBS in C3
 +
: parameters: 98°C 5min, >> 98°C 1min, 72°C 1min << x30, 72°C 5min, 4°C ∞
 +
: primer [[Team:Marburg_SYNMIKRO/Primer#MS20|MS20]] + [[Team:Marburg_SYNMIKRO/Primer#MS21|MS21]]
 +
 +
; miniprep
 +
: Gix in C3
 +
: pBad
 +
 +
; Results
 +
: Transformation
 +
: CFP-T (03.09) --> 10 colonies
 +
 +
; colony PCR
 +
: P108-RBS, SacB, GixR
 +
: parameters: 98°C 5min, >> 98°C 1min, 72°C 1min << x30, 72°C 5min, 4°C ∞
 +
: primer [[Team:Marburg_SYNMIKRO/Primer#MS19|MS19]] + [[Team:Marburg_SYNMIKRO/Primer#MS20|MS20]]
 +
 +
; 1% Agarose gel
 +
: [[File:Syn_MR_Gel43.jpg|thumb|800px|center|'''no marker''', 1:none, 2-7:SacB, 8-11:P108-RBS, (colony-PCR 05.09.2012)]]
 +
 +
; 1% Agarose gel
 +
: [[File:Syn_MR_Gel44.jpg|thumb|800px|center|'''no marker''', 1-5:P108-RBS, 6:none, 7-9:GixR, 10:Scar, 11-12: pBad, 13-15: pGixR,  (colony-PCR 05.09.2012)]]
 +
 +
 +
; restriction
 +
: pSB1A3 (E/P)
 +
: pSB1K3 (E/P)
 +
: pSB1C3 (E/P)
 +
: pSB1T3 (E/P)
 +
: GFP-T K3 (X/P)
 +
: GFP K3 (X/P)
 +
: CFP C3 (X/P)
 +
: mRFP C3 (X/P)
 +
: AmiC A (E/S)
 +
: GroES A (E/S)
 +
: Terminator (X/P)
 +
 +
 +
; Inoculation
 +
: pRBS
 +
: SacB
 +
: mRFP-Terminator
 +
: 5ml LB, over night, 37°C
 +
 +
== 06.09.2012 ==
 +
 +
; miniprep
 +
: pRBS
 +
: SacB
 +
: mRFP-Terminator
 +
 +
; ligation
 +
: AmiC (E/S) + GFP (X/P) + pSB1T3 (E/P)
 +
: AmiC (E/S) + RFP (X/P) + pSB1T3 (E/P)
 +
: AmiC (E/S) + GCFP (X/P) + pSB1T3 (E/P)
 +
: AmiC (E/S) + Terminator (X+P + pSB1T3 (E/P)
 +
: GroES (E/S) + GFP (X/P) + pSB1T3 (E/P)
 +
: GroES (E/S) + RFP (X/P) + pSB1T3 (E/P)
 +
: GroES (E/S) + CFP (X/P) + pSB1T3 (E/P)
 +
: GroES (E/S) + Terminator (X/P) + pSB1T3 (E/P)
 +
 +
; PCR of miniprep
 +
: SacB in pSB1C3
 +
: p-RBS in pSB1C3
 +
: terminator in pSB1T3
 +
: parameters: 98°C 5min, >> 98°C 30 sec, 72°C 1,5min <<  x30, 72°C 5min, 4°C ∞
 +
: primer [[Team:Marburg_SYNMIKRO/Primer#MS20|MS20]] + [[Team:Marburg_SYNMIKRO/Primer#MS21|MS21]]
 +
 +
; 1% Agarose gel
 +
: [[File:Syn_MR_Gel45.jpg|thumb|800px|center|M:1kb gene ruler plus, 1:Scar, 2-5:P-RBS, 6-9:SacB, 10-16: mRFP, 17: control, (PCR of miniprep (06.09.2012)]]
 +
 +
 +
; transformation
 +
: RBS-Gin T3
 +
: amiC A3
 +
: groES A3
 +
: groES-Terminator T3
 +
: groES-GFP T3
 +
: groES-CFP T3
 +
: groES-mRFP T3
 +
: amiC-Terminator T3
 +
: amiC-GFP T3
 +
: amiC-CFP T3
 +
: amiC-mRFP T3
 +
 +
== 07.09.2012 ==
 +
 +
; restriction (6.9.)
 +
 +
; ligation
 +
: AmiC in pSB1K3
 +
: GroES in pSB1K3
 +
: mRFP in pSB1K3
 +
: Terminator in pSB1K3
 +
: CFP in pSB1K3
 +
: GFP in pSB1K3
 +
: GFP+Terminator in C3
 +
: Enhancer in K3
 +
 +
; transformation
 +
: Ligation
 +
: AmiC
 +
: GroES
 +
: RBS-Gin
 +
 +
; miniprep
 +
: CFP-Terminator
 +
 +
; Sequencing
 +
: mRFP T3
 +
: P-RBS K12
 +
: P-RBS K15
 +
: SacB K6
 +
: SacB K6
<html>
<html>
</div>
</div>
 +
</html>
 +
<html>
 +
</div>
 +
</html>
 +
<html>
 +
<div class="conboxb">
</html>
</html>
= Week 10  =
= Week 10  =
Line 1,098: Line 1,353:
<div class="hiddenifjs" id="klapp10">
<div class="hiddenifjs" id="klapp10">
</html>
</html>
 +
 +
==10.09.2012==
 +
; Restriction
 +
: Gin-RBS (01.08)
 +
 +
; Ligation
 +
: Gin-RBS in pSB1C3
 +
 +
; Transformation
 +
: Gin-RBS in pSB1C3
 +
 +
; Hybridisation
 +
: GixR
 +
: 98 °C 20 min
 +
: minus 1 °C to 4 °C in 2 min
 +
 +
; Ligation and Transformation
 +
: GixR (X+S) + pSB1K3 (X+S)
 +
 +
; Inoculation (Tetracycline)
 +
: GroES-mRFP
 +
: GroES-GFP
 +
: GroES-CFP
 +
: amiC-mRFP
 +
: amiC-GFP
 +
: amiC-CFP
 +
: 5ml LB, over night, 37°C
 +
 +
; Inoculation (Kanamycine)
 +
: GroES-GFP
 +
: amiC-mRFP
 +
: amiC-GFP
 +
: GroES-Terminator
 +
: Enhancer
 +
: 5ml LB, over night, 37°C
 +
 +
; Inoculation (Chloramphenicol)
 +
: GFP-Terminator
 +
: 5ml LB, over night, 37°C
 +
 +
; Inoculation (Ampicillin)
 +
: Terminator
 +
: 5ml LB, over night, 37°C
== 11.09.2012 ==
== 11.09.2012 ==
Line 1,106: Line 1,404:
:AmiC-GFP &rArr;; no fluorescence
:AmiC-GFP &rArr;; no fluorescence
-
;colony-PCR
+
; colony-PCR
-
:GixR  
+
: GixR  
-
:RBS-Gin
+
: RBS-Gin
-
:Terminator
+
: Terminator
 +
: parameters: 98°C 5', >>98°C 1', 72°C 1'<<x30, 72°C 5', 4°C
 +
: primer [[Team:Marburg_SYNMIKRO/Primer#MS20|MS20]] + [[Team:Marburg_SYNMIKRO/Primer#MS21|MS21]]
;3A-Assembly (digestion, ligation, transformation)
;3A-Assembly (digestion, ligation, transformation)
Line 1,124: Line 1,424:
:GFP-Terminator
:GFP-Terminator
:Terminator
:Terminator
 +
 +
== 12.09.2012 ==
 +
; Results
 +
: Transformation
 +
: - P-RBS --> colonies
 +
: - pB10 --> no colonies
 +
: - RBS (B0030) --> colonies
 +
: - Gro-ES/ AmiC-Terminator --> no colonies
 +
 +
; colony-PCR
 +
: Gix (2. try)
 +
: Gin-RBS (2. try)
 +
: RBS (B0030)
 +
: P-RBS (108)
 +
: parameters: 98°C 5', >>98°C 1', 72°C 1'<<x30, 72°C 5', 4°C
 +
: primer [[Team:Marburg_SYNMIKRO/Primer#MS20|MS20]] + [[Team:Marburg_SYNMIKRO/Primer#MS21|MS21]]
 +
 +
; Agarose gel
 +
: Gin-RBS (5x)
 +
: Gin (1x)
 +
: Gix (5x)
 +
: RBS (6x)
 +
: P-RBS (6x)
 +
 +
; 1% Agarose gel
 +
: [[File:Syn_MR_Gel46.jpg|thumb|800px|center|M:1kb gene ruler plus, 1:Scar, 2-6:RBS-Gin, 7:Gin control 8-12: gixR, 13: control, (colony-PCR 12.09.2012)]]
 +
 +
 +
; 1% Agarose gel
 +
: [[File:Syn_MR_Gel47.jpg|thumb|800px|center|M:1kb gene ruler plus, 1:Scar, 2-6:RBS BBa_B0030, 7-12:P-RBS, 13: control, (colony-PCR 12.09.2012)]]
 +
 +
 +
; Restriction (miniprep 11.09.12)
 +
: gr-GFP
 +
: gr-CFP
 +
: gr-T K3
 +
: enhancer K3
 +
: am-RFP-K3
 +
: am-GFP-K3
 +
: GFP-T-C3
 +
: terminator A2
 +
 +
; 1% Agarose gel
 +
: [[File:Syn_MR_Gel48.jpg|thumb|800px|center|M:1kb gene ruler plus, 1-3:GroES-GFP, 4:GroES-GFP control, 5:GroES-CFP, 6:GroES-CFP control, 7-10: GroES-Terminator, 11: GroES-Terminator control, 12-15: Enhancer, 16: Enhancer control, (control digest 12.09.2012)]]
 +
 +
; 1% Agarose gel
 +
: [[File:Syn_MR_Gel49.jpg|thumb|800px|center|M:1kb gene ruler plus, 1-2:AmiC-mRFP, 3:AmiC-mRFP control, 4-5:AmiC-GFP, 6:AmiC-GFP control, 7-11: GFP-Terminator, 12: GFP-Terminator control, 13-15: Terminator, 16: Terminator control, (control digest 12.09.2012)]]
 +
 +
; Transformation
 +
: groES-T
 +
::2 Transformations:
 +
:::    - One with a 1:10 dilution of Terminator from ligation,
 +
:::    - another one with a 1:100 dilution of Terminator from ligation
 +
: amiC-T
 +
::2 Transformations:
 +
:::    - One with a 1:10 dilution of Terminator from ligation,
 +
:::    - another one with a 1:100 dilution of Terminator from ligation
 +
 +
; PCR
 +
: Gin-RBS
 +
::3 reactions:
 +
:::    -One using High Fidelity (HF) Reaction Buffer,
 +
:::    -one using GC-Buffer
 +
:::    -and another one using self-made reaction buffer
 +
: Primer used : [[Team:Marburg_SYNMIKRO/Primer#MS12|MS12]] + [[Team:Marburg_SYNMIKRO/Primer#MS13|MS13]]
 +
: Template DNA : ''E. coli'' C600 chromosomal DNA
 +
 +
== 13.09.2012 ==
 +
; Results
 +
: Transformation
 +
:: - pBAD (11.09.12) --> no colonies
 +
:: - AmiC-T (11.09.12) --> 2 x 2 colonies
 +
:: - GroES-T (11.12.09)--> 2 colonies
 +
 +
; colony-PCR
 +
: AmiC-T & GroES-T (transformation 11.09.12)
 +
: parameters: 98°C 5', >>98°C 1', 72°C 1'<<x30, 72°C 5', 4°C
 +
: primer [[Team:Marburg_SYNMIKRO/Primer#MS20|MS20]] + [[Team:Marburg_SYNMIKRO/Primer#MS21|MS21]]
 +
 +
; 1% Agarose gel
 +
: [[File:Syn_MR_Gel50.jpg|thumb|800px|center|M:1kb gene ruler plus, 1:RBS-Gin HF, 2:RBS-Gin GC, 3:RBS-Gin SM (PCR 13.09.2012)]]
 +
 +
 +
; Gel extraction
 +
: RBS-Gin (HF) --> 51 ng/µl
 +
: RBS-Gin (GC) --> 69 ng/µl
 +
: RBS-Gin (SM) --> 56 ng/µl
 +
 +
; Ligation
 +
: GroES-T & AmiC-T in pSB1K3
 +
 +
; Restriction (preparative digest)
 +
: GroES (A module)
 +
:: with SpeI
 +
: AmiC (A module)
 +
:: with SpeI
 +
: mRFP-C3 (B module)
 +
:: with EcoRI and XbaI
 +
: CFP-C3 (B module)
 +
:: with EcoRI and XbaI
 +
: Gin-RBS
 +
:: HF-PCR-Product, GC-PCR-product and SM-PCR-product
 +
::: with EcoRI and PstI
 +
 +
 +
; 1% Agarose gel
 +
: [[File:Syn_MR_Gel51.jpg|thumb|800px|center|M:1kb gene ruler plus, 1-4:AmiC-Terminator, 5-6:GroES-Terminator, 7:Scar, 8: control, (colony-PCR 13.09.2012)]]
 +
 +
 +
== 14.09.2012 ==
 +
 +
; Results
 +
: Transformation
 +
:: AmiC-T: no colonies
 +
:: GroES-T: no colonies
 +
 +
; Restriction
 +
: 2nd Digestion of A-modules (see digest from yesterday) additionally with EcoRI
 +
 +
; Ligation
 +
: Gin-RBS with pSB1C3
 +
:: 3 reactions:
 +
::: one with RBS-Gin HF-PCR-Product
 +
::: one with RBS-Gin GC-PCR-Product
 +
::: one with RBS-Gin SM-PCR-Product
 +
 +
; Inoculation
 +
: Gix-K3 (clone 3 and 4)
 +
: P-RBS-C3 (clone 1 and 3)
 +
: RBS (BBa_B0030) [clone 1 and 3]
 +
 +
; 1% Agarose gel
 +
: [[File:Syn_MR_Gel52.jpg|thumb|800px|center|M:1kb gene ruler plus, 1:AmiC, 2:GroES, 3:none, 4:GFP, 5:CFP, 6:mRFP,(digest 13.-14.09.2012)]]
 +
 +
; Gel extraction
 +
: GFP
 +
: mRFP
 +
 +
; 1% Agarose gel
 +
: [[File:Syn_MR_Gel53.jpg|thumb|800px|center|M:1kb gene ruler plus, 1:Terminator, (digest 14.09.2012)]]
 +
 +
; Gel extraction
 +
: Terminator clone 48
 +
 +
; Transformation
 +
: RBS-Gin(HF)-pSB1C3
 +
: RBS-Gin(GC)-pSB1C3
 +
: RBS-Gin(SM)-pSB1C3
 +
 +
<html>
 +
</div>
 +
</html>
 +
<html>
 +
</div>
 +
</html>
 +
<html>
 +
<div class="conboxb">
 +
</html>
 +
= Week 11  =
 +
<html>
 +
<div class="fixcon"><div class="fixtoggle" onClick="javascript:toggledisplay('klapp11'); togglearrow(this); return false"></div>
 +
<div class="smalldate"> 17.09. - 23.09.2012</div>
 +
</div>
 +
<div class="hiddenifjs" id="klapp11">
 +
</html>
 +
 +
== 17.09.2012 ==
 +
 +
; Restriction
 +
: AmiC
 +
: GroES
 +
: CFP
 +
: SacB
 +
 +
; 1% Agarose gel
 +
: [[File:Syn_MR_Gel54.jpg|thumb|800px|center|M:1kb gene ruler plus, 1:AmiC control, 2:AmiC, 3:GroES control, 4:GroES, 5:CFP control, 6:CFP, 7:SacB control, 8:SacB (digest 17.09.2012)]]
 +
 +
; Restriction
 +
: CFP-C3
 +
: SacB (2nd try)
 +
 +
; Resuspend
 +
: BBa_B001J
 +
 +
; Restreak
 +
: RBS-Gin (HF)
 +
: RBS-Gin (GC)
 +
: RBS-Gin (SM)
 +
 +
; DNA isolation
 +
: CFP-C3 (Miniprep28.08.12)--> 47,3 ng/µl
 +
 +
 +
; Miniprep
 +
: GixR pSB1K3 --> 50,5 ng/µl, 144,7 ng/µl
 +
: RBS(new)B0030 pSB1A2 --> 726,2 ng/µl, 242,6 ng/µl
 +
: P108-RBS pSB1C3 --> 200,5 ng/µl, 177,8 ng/µl
 +
 +
; 1% Agarose gel
 +
: [[File:Syn_MR_Gel55.jpg|thumb|800px|center|M:1kb gene ruler plus, 1-2:GixR, 3-4:RBS BBa_B0030, 3-4:P108-RBS, (mini prep 17.09.2012)]]
 +
 +
; Transformation
 +
: pSB2K3-pBAD & pSB1-A2-TT
 +
 +
; 1% Agarose gel
 +
: CFP-C3 (restriction 2nd try)
 +
: SacB-Cs (restriction 2nd try)
 +
: [[File:Syn_MR_Gel56.jpg|thumb|800px|center|M:1kb gene ruler plus, 1:CFP control, 2:CFP, 3:SacB, (digest 17.09.2012)]]
 +
 +
== 18.09.2012 ==
 +
 +
; Results
 +
; Transformation
 +
: 2x Terminator --> colonies
 +
: pBAD --> no colonies
 +
 +
; Colony PCR
 +
: RBS-Gin
 +
: 2x Terminator
 +
: parameters: 98°C 5', >>98°C 1', 72°C 1'<<x30, 72°C 5', 4°C
 +
: primer [[Team:Marburg_SYNMIKRO/Primer#MS20|MS20]] + [[Team:Marburg_SYNMIKRO/Primer#MS21|MS21]]
 +
 +
; 1% Agarose gel
 +
: [[File:Syn_MR_Gel57.jpg|thumb|800px|center|M:1kb gene ruler plus, 1-2:RBS-Gin HF, 3:RBS-Gin GC, 4:RBS-Gin SM, 5: Scar, 7-10:2xTerminator, 11:control, (colony-pcr 18.09.2012)]]
 +
 +
; Sequencing
 +
: SacB
 +
 +
; Gel extraction
 +
: CFP-C3 E+X
 +
 +
; Ligation
 +
: am-G: amiC E+S & GFP-A3 E+X
 +
: am-C: amiC E+S & CFP-C3 E+X
 +
: am-R: amiC E+S & mRFP-C3 E+X
 +
: gro-G: groES E+S & GFP-A3 E+X
 +
: gro-C: groES E+S & CFP-C3 E+X
 +
: gro-R: groES E+S & mRFP-C3 E+X
 +
 +
; Transformation
 +
: am-G
 +
: am-C
 +
: am-R
 +
: gr-G
 +
: gr-C
 +
: gr-R
 +
: pBAD
 +
 +
 +
== 19.09.2012 ==
 +
 +
; Results
 +
; Transformation
 +
: am-G --> colonies
 +
: am-C --> colonies
 +
: am-R --> colonies
 +
: gr-G --> colonies
 +
: gr-C --> colonies
 +
: gr-R --> colonies
 +
: pBAD --> no colonies
 +
 +
; Colony PCR
 +
: RBS-Gin (from 17.09.12)
 +
: 2x Terminator B0017 (from 17.09.12)
 +
: parameters: 98°C 5', >>98°C 1', 72°C 1'<<x30, 72°C 5', 4°C
 +
: primer [[Team:Marburg_SYNMIKRO/Primer#MS20|MS20]] + [[Team:Marburg_SYNMIKRO/Primer#MS21|MS21]]
 +
: --> Amplification failed
 +
 +
; Transformation
 +
: pBAD
 +
 +
; Microscopy-screening
 +
: AmiC-GFP --> fluorescence
 +
: GroES-GFP --> fluorescence
 +
: AmiC-CFP --> fluorescence
 +
: GroES-CFP --> fluorescence
 +
: AmiC-mRFP --> fluorescence
 +
: GroES-mRFP --> fluorescence
 +
 +
; Inoculation
 +
: RBS-Gin
 +
: TT (B0017)
 +
: AmiC-GFP
 +
: GroES-GFP
 +
: AmiC-CFP
 +
: GroES-CFP
 +
: AmiC-mRFP
 +
: GroES-mRFP
 +
 +
 +
== 20.09.2012 ==
 +
 +
; Miniprep
 +
: RBS-Gin
 +
: TT (B0017)
 +
: AmiC-GFP
 +
: GroES-GFP
 +
: AmiC-CFP
 +
: GroES-CFP
 +
: AmiC-mRFP
 +
: GroES-mRFP
 +
 +
; Restriction (E/P)
 +
: TT(B0017)
 +
: RBS-Gin
 +
 +
; 1% Agarose gel
 +
: [[File:Syn_MR_Gel58.jpg|thumb|800px|center|M:1kb gene ruler plus, 1,3,5,7:2xTerminator, 2,4,6,8:2xTerminator control, 9,11,13,15:RBS-Gin, 10,12,14,16:RBS-Gin control, (control digest 20.09.2012)]]
 +
 +
; Restriction
 +
: SacB ((XbaI + PstI) & (EcoRI + XbaI)
 +
: P-RBS (EcoRI + SpeI) & (SpeI + PstI)
 +
 +
; 1% Agarose gel
 +
: [[File:Syn_MR_Gel59.jpg|thumb|800px|center|M:1kb gene ruler plus, 1:P-RBS control, 2: P-RBS (E/S), 3: P-RBS (S/P), 4: SacB control, 5: SacB (X/P), 6:SacB (E/X), (digest 20.09.2012)]]
 +
 +
; Inoculation
 +
: GroES-GFP
 +
: GroES-CFP
 +
: GroES-mRFP
 +
 +
 +
== 21.09.2012 ==
 +
 +
 +
; Restriction
 +
: GFP-fusion (EcoRI + SpeI)
 +
: TT BBa_0017 (EcoRI + XbaI)
 +
 +
; 1% Agarose gel
 +
: [[File:Syn_MR_Gel60.jpg|thumb|800px|center|M:1kb gene ruler plus, 1:GFP-Fusion, 2:TT BBa_B0017, 3:SacB, (digest 21.09.2012)]]
 +
 +
; Gel extraction
 +
: GFP-Fusion
 +
: pSB1A2 (backbone GFP-Fusion)
 +
: TT
 +
: SacB
 +
 +
; Ligation
 +
: GFP bb Enhancer
 +
: GFP TT
 +
: GroES TT
 +
 +
; Transformation
 +
: GFP bb Enhancer
 +
: GFP TT
 +
: GroES TT
 +
: Promotor-RBS (17.09.12)
 +
 +
; Sequencing
 +
: RBS-Gin
 +
 +
; Microscopy-screening
 +
: AmiC-GFP --> Channel: DIC, Green, Red, Blue
 +
: GroES-GFP --> Channel: DIC, Green, Red, Blue
 +
: AmiC-CFP --> Channel: DIC, Green, Red, Blue
 +
: GroES-CFP --> Channel: DIC, Green, Red, Blue
 +
: AmiC-mRFP --> Channel: DIC, Red
 +
: GroES-mRFP --> Channel: DIC, Green, Red, Blue
 +
 +
== 22.09.2012 ==
 +
 +
; Inoculation
 +
: GroES-TT pSB1A2
 +
: GFP-TT pSB1A2
 +
: Enhancer
 +
: P108-RBS pSB1C3
 +
 +
<html>
 +
</div>
 +
</html>
<html>
<html>
Line 1,129: Line 1,800:
</script>
</script>
<script type="text/javascript">
<script type="text/javascript">
-
 
function ReplaceContentInContainer(matchClass)
function ReplaceContentInContainer(matchClass)
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Latest revision as of 01:44, 27 September 2012

Main Page

Contents

Week 1

[all]
09.07. - 15.07.2012


09.07.2012

Inoculation
E. coli strain C600 Mucts62 in dYT-medium

10.07.2012

Isolation of chromosomal DNA
E. coli strain C600 Mucts62 (lysogenic for temperature sensitive phage Mu cts62)
Inoculation
E. coli strain DH5α and TOP10 in dYT-medium

11.07.2012

Preparation of chemically competent E. coli cells
TOP10

12.07.2012

Transformation
GFP, GFP_fusion, CFP, mRFP, RBS, P108, P100, Plac with E.coli TOP10
NameBiobrickPlateWellBackboneResistance
GFP[http://partsregistry.org/Part:BBa_E0040 BBa_E0040]114KpSB1A2Ampicillin
GFP_fusion[http://partsregistry.org/Part:BBa_K125500 BBa_K125500]32PpSB1A2Ampicillin
CFP[http://partsregistry.org/Part:BBa_E0020 BBa_E0020]16ApSB1A2Ampicillin
mRFP[http://partsregistry.org/Part:BBa_E1010 BBa_E1010]118FpSB2K3Kanamycin
RBS[http://partsregistry.org/Part:BBa_J61100 BBa_J61100]15JpSB1A2Ampicillin
P108[http://partsregistry.org/Part:BBa_J23108 BBa_J23108]22EBBa_J61002Ampicillin
P100[http://partsregistry.org/Part:BBa_J23100 BBa_J23100]118CBBa_J61002Ampicillin
PLac[http://partsregistry.org/Part:BBa_I14032 BBa_I14032]211PpSB2K3Kanamycin



Week 2

16.07. - 22.07.2012

16.07.2012

PCR
AmiC (primer: MS1 + MS2)
HU (primer: MS3 + MS4)
GroES (primer: MS5 + MS6)
GixR (primer: MS7 + MS8)
Enhancer (primer: MS9 + MS10)
⇒ template-DNA: chromosomal DNA, denaturation: 30 sec at 95°C; annealing: 30 sec at 60°C; elongation: 30 sec at 72°C; 35 cycles
Inoculation
GFP
GFP_fusion
CFP
mRFP
RBS
P108
LacIQ
P100

17.07.2012

Plasmid preparation
GFP
GFP_fusion
CFP
mRFP
RBS
P108
LacIq
P100

18.07.2012

1% Agarose gel
M:1kb gene ruler plus, 1:enhancer, 2:GroES, 3:AmiC, 4:HU, 5:GixR, 6:GFP, 7:GFP-Fusion, 8:CFP, 9:mRFP, 10:RBS, 11:LacIQ, 12:P108, 13:P100, 14:λ-DNA (PCR 18.07.2012)
DNA Gel extraction
Enhancer
GroES
HU
GixR Hybridization
  • 5 µl primer MS7 (100 µM) + 5 µl primer MS8 (100 µM)
    • 5 min 99°C
    • over night, room temperature
    • -80°C

19.07.2012

Fill-in reaction (primer extension)
Enhancer
Restriction
Enhancer (EcoRI/PstI)
PCR
AmiC (primer: MS1 + MS2)


⇒ template-DNA: chromosomal DNA, annealing: 30 sec; 85 °C, elongation: 36 sec; 72 °C, 35 cycles, 1) 2 µl DMSO; 1 µl chrom. DNA | 2) 2 µl DMSO; 5 µl chrom. DNA | 3) without DMSO; 1 µl chrom. DNA | 4) without DMSO; 5 µl chrom. DNA | 5) 2 µl DMSO; without chrom. DNA
Test restriction digest
7) CFP (EcoRI/PstI)
8) GFP (EcoRI/PstI)
9) GFP-fusion (EcoRI/PstI)
10) mRFP (EcoRI/PstI)
⇒ all negative
1% Agarose gel
M:1kb gene ruler plus, 1-5:AmiC PCR conditions (see above), 6-9: control digest (E/P), 6:GFP, 7:GFP-Fusion, 8:CFP, 9:mRFP (PCR 19.07.2012)
Control digest
CFP (EcoRI/PstI)
GFP (EcoRI/PstI)
GFP-fusion (EcoRI/PstI)
mRFP (EcoRI/PstI)
⇒ restriction over night

20.07.2012

1% Agarose gel from the control digest
M:1kb gene ruler plus, 1:GFP, 2: GFP-Fusion, 3: CFP, 4: mRFP (control digest 18.07.2012)

Week 3

23.07. - 29.07.2012

26.07.2012

Restriction
pSB1T3 (XbaI/SpeI and DpnI)
pSB1A3 (XbaI/SpeI and DpnI)
pSB1K3 (XbaI/SpeI and DpnI)
pSB1C3 (XbaI/SpeI and DpnI)
Ligation
GixR + pSB1A3
Enhancer + pSB1K3
cyclisation of pSB1C3, pSB1K3, pSB1A3, pSB1T3 (couldn’t work)

27.07.2012

Our lab-dragon "Hugo" is born

Syn MR Hugo.jpg


Week 4

30.07. - 05.08.2012


30.07.2012

PCR
CFP (primer: MS16 + MS17) ⇒ template-DNA: [http://partsregistry.org/Part:BBa_E0020 BBa_E0020]
⇒ positive
mRFP (primer: MS14 + MS15) ⇒ template-DNA: [http://partsregistry.org/Part:BBa_E1010 BBa_E1010]
⇒ positive
Gin (primer: MS11 + MS13) ⇒ template-DNA: chromosomal DNA (that was wrong)
RBS-Gin (primer: MS13 + MS12) ⇒ template-DNA: chromosomal DNA (that was wrong)
⇒ annealing: 30 sec; 60 °C, elongation: 40 sec; 72 °C, 35 cycles
Transformation
all vector-backbone-ligations
Enhancer (K3)
GixR (A3)

31.07.2012

Result
transformation
pSB1A3: 3 colonies
pSB1K3: 60 colonies
pSB1C3: 9 colonies
pSB1T3: 0 colonies
Enhancer (K3): 0 colonies
GixR (A3): 0 colonies


1% Agarose gel
M:Marker, 1: Gix, 2-6: AmiC (PCR 16.07.2012)


1% Agarose gel
M:1kb gene ruler plus, 1: AmiC(PCR 16.07.2012)


1% Agarose gel
M:1kb gene ruler plus, 1: RBS-Gin from 30.07, 2:AmiC from 16.07., 3:Gin from 30.07., 4:CFP from 30.07., 5:mRFP from 30.07., 6: control (PCR 30.07.2012)


Resuspension of a biobrick
[http://partsregistry.org/Part:BBa_B0010 BBa_B0010](Terminator)
DNA Gel extraction
CFP
mRFP
PCR
AmiC (primer: MS1 + MS2)
RBS-Gin (primer: MS12 + MS13)
Gin (primer: MS11 + MS13)
⇒ template-DNA: chromosomal DNA, annealing: 30 sec; 55 °C, elongation: 40 sec; 72 °C, 35 cycles
Restriction
pSB1A3 (EcoRI/PstI)
CFP (EcoRI/PstI)
mRFP (EcoRI/PstI)
Transformation
pSB1A3
pSB1C3
pSB1T3
Terminator
GixR
Enhancer
Inoculation
pSB1A3
pSB1C3
pSB1K3
Ligation
pSB1A3 + CFP
pSB1A3 + mRFP
GixR Hybridization
  • 5 µl primer MS7 (100 µM) + 5 µl primer MS8 (100 µM)
    • 5 min 99°C
    • over night, room temperature
    • -80°C
DNA Gel extraction
AmiC
Ligation
AmiC + pSV2 (suicide vector)


01.08.2012

Result
transformation
Only BBa_B0010 colonies present
Plasmid preparation
pSB1A3 ⇒ only clone 1, 3 Positive
pSB1C3 ⇒ all negative
pSB1K3 ⇒ all negative
Ligation
GixR hyb. + pJET2_1
⇒ over blunt ends
Restriction
GixR hyb. (EcoRI/PstI)
pSB1C3 (EcoRI/PstI)
1% Agarose gel
M:1kb gene ruler plus, 1: RBS-Gin (+DMSO), 2:RBS-Gin (-DMSO), 3:Gin (+DMSO), 4:Gin (-DMSO), 5: control (PCR 31.07.2012)
1% Agarose gel
M:1kb gene ruler plus, 1: AmiC (+DMSO), 2:AmiC (-DMSO), (PCR 31.07.2012)
DNA Gel extraction
RBS-Gin
Gin
AmiC
Test restriction
pSB1A3 (EcoRI)
Restriction
RBS-Gin (EcoRI/PstI)
Gin (EcoRI/PstI)
AmiC (EcoRI/PstI)
pSB1C3 (EcoRI/PstI)
pSB1K3 (EcoRI/PstI)
pSB1T3 (EcoRI/PstI)
Ligation
cyclisation of pSB1T3
Fill-in reaction (primer extension)
Enhancer
Ligation
Enhancer and GixR in pSV2
Enhancer in pSB1K3
GixR in pSB1C3
1% Agarose gel
M:1kb gene ruler plus, 1-3: pSB1A3, 4-7:pSB1C3, 8-11: pSB1K3 (Control digest 01.08.2012)


pSB1A3 c2
pSB1C3 c4
pSB1K3 c3
Transformation
pSB1A3 religation
CFP in A3
mRFP in A3
AmiC in pJET
GixR in pJET
Enhancer in pJET
GixR in C3
Enhancer in K3
Ligation
AmiC + pSB1T3
RBS-Gin + pSB1K3
Gin + pSB1K3


02.08.2012

Resuspension of a biobrick
pSB3C5
⇒ for ccdB
Plasmid preparation
BBa_B0010 (terminator)
Test restriction
Terminator c1,2,3,4 (EcoRI/PstI)


2% Agarose gel
M:1kb gene ruler plus, 1-4: Terminator, M2:Middle Range gene ruler (Control digest 01.08.2012)
Transformation
pSB1T3 recycled
AmiC in T3
Gin-RBS in K3
Gin in K3
pSB3C5 linear (false)


03.08.2012

Plasmid preparation
Enhancer in pJET


Week 5

06.08. - 12.08.2012


06.08.2012

Resuspension and transformation of a biobrick
BBa_J04450 in pSB1A3
BBa_J04450 in pSB1C3
BBa_J04450 in pSB1K3
BBa_J04450 in pSB1T3
Restriction
GFP-fusion (EcoRI/SpeI)
Terminator (XbaI/PstI)
Promoter BBa_J23100 (EcoRI/SpeI)
Promoter BBa_J23108 (EcoRI/SpeI)
RBS (XbaI/PstI)
Ligation
RBS-Gin in T3
Gin in T3
GFP-fusion (EcoRI/SpeI) + Terminator (XbaI/PstI) in pSB1K3
Promoter BBa_J23108 (EcoRI/SpeI) + RBS (XbaI/PstI) in pSB1K3
Promoter BBa_J23100 (EcoRI/SpeI) + RBS (XbaI/PstI) in pSB1K3
GixR Hybridization
  • 1(3) µl primer MS77 (100 µM) + 1(3) µl primer MS8 (100 µM) + T4-Ligasebuffer
    • 5 min 95°C
    • over night, room temperature
    • -80°C
Transformation
GixR in C3
Gin in T3
RBS-Gin in T3
mRFP in A3
CFP in A3

07.08.2012

Transformation
GFP-fusion
CFP
J23108-RBS
J23100-RBS
Plasmid preparation
GixR 1,2,3
Restriction
GixR hyb. 1,2,3 (EcoRI/PstI)
pSB1C3 (EcoRI/PstI)
Ligation
GixR hyb. 1,2,3 in pJET
GixR hyb. 1,2,3 in C3


Transformation
GixR hyb. 1,2,3 in pJET
GixR hyb. 1,2,3 in C3
Test restriction
pSB3C5 (XbaI)
pJET (XbaI)


1% Agarose gel
M:1kb gene ruler plus, 1-4: pSB3C5, 5-8:pSB3C5, 9-12: Enhancer-pJET (Control digest 07.08.2012)
⇒ positive: pSB3C5 3,4,7,8,; Enhancer-pJET 9-12

08.08.2012

Result transformation
GFP-fusion (20 clones)
J23108-RBS (20 clones)
J23100-RBS (20 clones)
GixR hyb. 1,2,3 in pJET (10 clones)
GixR hyb. 1,2,3 in C3 (10 clones)
CFP (5 clones)
Restriktion
Enhancer in pJET (EcoRI/PstI)
pSB1C3 (EcoRI/PstI)
2% Agarose gel
M:1kb gene ruler plus, 1: Enhancer, 2:pSB1C3, (Control digest 08.08.2012)
DNA Gel extraction
Enhancer
pSB1C3
Ligation
Enhancer 1,2 in C3
Transformation
Enhancer 1,2 in C3
C3
mRFP

09.08.2012

Result transformation
GFP-fusion ⇒ positive
J23108-RBS ⇒ positive
J23100-RBS ⇒ positive
CFP ⇒ positive
GixR in C3 c1 ⇒ negative
GixR in C3 c2 ⇒ negative
GixR in C3 c3 ⇒ negative
Plasmid preparation
GFP-fusion
J23108-RBS
J23100-RBS
CFP
Test restriction
GFP-fusion (EcoRI/PstI)
J23108-RBS (EcoRI)
J23100-RBS (EcoRI)
CFP (EcoRI/PstI)


1% Agarose gel
M:1kb gene ruler plus, 1-18: GFP-Fusion - Terminator, (Control digest 09.08.2012)
1% Agarose gel
M:1kb gene ruler plus, 1-18: P108-RBS, (Control digest 09.08.2012)


1% Agarose gel
M:1kb gene ruler plus, 1-18: P100-RBS, (Control digest 09.08.2012)


1% Agarose gel
M:1kb gene ruler plus, 1-2: GFP-Fusion - Terminator, 3-4: P108-RBS, 5-6: P100-RBS, 7-11: CFP (Control digest 09.08.2012)


Inoculation
GixR in C3
Gin in T3
Gin-RBS in T3
pSB1C3
Enhancer in C3
mRFP in A3

Week 6

13.08. - 19.08.2012


13.08.12

PCR
GroES (primer: MS5 + MS6b)
HU (primer: MS3 + MS4b)
⇒ with and without DMSO, template-DNA: chromosomal DNA, annealing: 30 sec; 60 °C, elongation: 40 sec; 72 °C, 35 cycles


1% Agarose gel
M:1kb gene ruler plus, 1: HU (+DMSO), 2: HU (-DMSO), 3: GroES (+DMSO), 4: GroES (-DMSO), (PCR 13.08.2012)
Transformation
LacIQ (BBa_I14032)


14.08.12

Restriction
pSB1K3 (EcoRI/PstI)
Enhancer 3,4 (EcoRI/ PstI)
GFP-fusion (EcoRI/SpeI)
Terminator (XbaI/PstI)
Test restriction
RBS-Gin in T3 (EcoRI/ PstI)
Ligation
GFP-fusion (EcoRI/SpeI) + Terminator (XbaI/PstI) in pSB1K3 (EcoRI/PstI)
Promotor J23108 (EcoRI/SpeI) + RBS (XbaI/PstI) in pSB1K3 (EcoRI/PstI)
Enhancer 3,4 (EcoRI/ PstI) + pSB1K3 (EcoRI/PstI)
Transformation
GFP-fusion-Terminator in pSB1K3
Promotor-RBS in pSB1K3
Enhancer 3 in pSB1K3
Enhancer 4 in pSB1K3
GixR in pJET


15.08.2012

PCR
GroES (primer: MS5 + MS6b)
HU (primer: MS3 + MS4b)
⇒ with and without DMSO, template-DNA: chromosomal DNA, annealing: 30 sec; 55 °C, elongation: 30 sec; 72 °C, 35 cycles


1% Agarose gel
M:1kb gene ruler plus, 1: HU (-DMSO), 2: HU (+DMSO), 3: GroES (-DMSO), 4: GroES (+DMSO), (PCR 15.08.2012)
DNA Gel extraction
HU 1,2
Resuspension of a biobrick
BBa_J04450 in pSB1A3
BBa_J04450 in pSB1T3
BBa_J04450 in pSB1C3
BBa_J04450 in pSB1K3


Transformation
BBa_J04450 in pSB1A3
BBa_J04450 in pSB1T3
BBa_J04450 in pSB1C3
BBa_J04450 in pSB1K3
Test restriction
pSB1T3 (EcoRI/PstI and DpnI)
pSB1K3 (EcoRI/PstI and DpnI)
pSB1C3 (EcoRI/PstI and DpnI)
pSB1A3 (EcoRI/PstI and DpnI)


16.08.2012

PCR
GroES (primer: MS5 + MS6b)
⇒ with and without DMSO, template-DNA: chromosomal DNA, annealing: 30 sec; 55 °C, elongation: 30 sec; 72 °C , 35 cycles


1% Agarose gel
M:1kb gene ruler plus, 1: GroES (-DMSO), 2: GroES (+DMSO), (PCR 16.08.2012)
Plasmid preparation
P-RBS
Test restriction
P-RBS (EcoRI/PstI)
1% Agarose gel
M:1kb gene ruler plus, 1: control, 2: P108-RBS, (control digest 16.08.2012)
Restriction
Enhancer 3,4 (EcoRI/ PstI)
HU (EcoRI/ PstI)
AmiC (EcoRI/ PstI)
Promoter, GFP (EcoRI/SpeI)
RBS; Terminator (XbaI/PstI)
Ligation
HU, AmiC in pSB1A3
Enhancer 3,4 in pSB1K3
P-RBS in pSB1K3
GFP fusion-Terminator in pSB1K3
DNA Gel extraction
GroES 1, P-RBS
Transformation
AmiC
HU
Enhancer 3,4, 3 alt,4 alt
Inoculation
BBa_J04450 in pSB1A3
BBa_J04450 in pSB1T3
BBa_J04450 in pSB1C3
BBa_J04450 in pSB1K3
AmiC
P-RBS

17.08.2012

Restriction
GroES (EcoRI/SpeI)
Ligation
GroES in pSB1C3
Transformation
GroES in pSB1C3
Test restriction
P-RBS (EcoRI/PstI)
BBa_J04450 in pSB1A3 (EcoRI/PstI)
BBa_J04450 in pSB1T3 (EcoRI/PstI)
BBa_J04450 in pSB1C3 (EcoRI/PstI)
BBa_J04450 in pSB1K3 (EcoRI/PstI)


1% Agarose gel
M:1kb gene ruler plus, 1-4: BBa_J04450 in pSB1A3, 5-8: BBa_J04450 in pSB1K3 P108-RBS, 9-12: BBa_J04450 in pSB1C3, (control digest 17.08.2012)


1% Agarose gel
M:1kb gene ruler plus, 1-4: P-RBS (E/P), 5-8: P-RBS (RsaI), 9-12: BBa_J04450 in pSB1T3, (control digest 17.08.2012)

Week 7

20.08. - 26.08.2012


20.08.2012

PCR
Backbone (primer MS24 + MS25)
⇒ template-DNA: pSB1A3, pSB1T3, pSB1C3 and pSB1K3, annealing: 30 sec; 55 °C, elongation: 30 sec; 72 °C , 35 cycles
1% Agarose gel
M:1kb gene ruler plus, 1: pSB1A3, 2: pSB1T3, 3: pSB1C3, 4: pSB1K3 (PCR 20.08.2012)
Transformation
BBa_JH023
Inoculation
GroES
P-RBS
GFP-T
Enhancer 3,4
AmiC
P-RBS alt
GFP-T alt

21.08.2012

Results
colonies of BBa_JH023
inoculations except AmiC and P-RBS alt successful
PCR
pSB1A3-BBa_J04450 (template DNA)
pSB1T3-BBa_J04450 (template DNA)
pSB1C3-BBa_J04450 (template DNA)
pSB1K3-BBa_J04450 (template DNA)
used primer --> MS24 + MS25
initial denaturation 98°C - 5min
denaturation: 98°C - 1min; annealing: 55°C - 30s; elongation: 72°C - 2.5min -->repeat 35 times
72°C - 5min --> 4°C - 8
miniprep
GroES, Enh 3/4 (+old), P-RBS (+old), GFP-T (+old)
Test restriction
of miniprep
over night, 37°C
1% Agarose gel
M:1kb gene ruler plus, 1:pSB1A3, 2:pSB1K3, 3:pSB1C3, 4:pSB1T3, 5: negative control, (PCR 21.08.2012)
Inoculation
E.coli BBa_I14032
over night, 37°C


22.08.2012

PCR
CFP (MS16 + MS17)
mRFP (MS14 + MS15)
initial denaturation 98°C - 5min
denaturation: 98°C - 10s; annealing: 60°C - 30s; elongation: 72°C - 40s -->repeat 35 times
72°C - 5min --> 4°C - 8
1% Agarose gel
M:1kb gene ruler plus, 1:mRFP, 2:mRFP, 3:CFP, 4:CFP (PCR 22.08.2012)
miniprep
of LacIQ
Test restriction
of LacIQ with EcoRI and PstI
1% Agarose gel
M:1kb gene ruler plus, 1-4:LacIQ, (control digest 22.08.2012)
1% Agarose gel
M:1kb gene ruler plus, 1-4:GroES, 5-12: GFT-Terminator, (control digest 21.08.2012)
1% Agarose gel
M:1kb gene ruler plus, 1-12:Enhancer, (control digest 21.08.2012)
1% Agarose gel
M:1kb gene ruler plus, 1-4:Enhancer, 5-12: P-RBS, (control digest 21.08.2012)
test restriction of Enhancer (->21.08.12), gel 2%
watch picture
test restriction of Enhancer and P-RBS (->21.08.12), gel 2%
watch picture


23.08.2012

PCR
pSB1K3-BBa_J04450 (template DNA)
pSB1T3-BBa_J04450 (template DNA)
used primer --> MS24 + MS25
initial denaturation 98°C - 5min
denaturation: 98°C - 1min; annealing: 55°C - 30s; elongation: 72°C - 2.5min -->repeat 35 times
72°C - 5min --> 4°C - 8
1% Agarose gel
M:gene ruler mix ladder, 1-2:pSB1K3-BBa_J04450, 3-4: pSB1T3-BBa_J04450, (PCR 23.08.2012)
Restriction
GixR (EcoRI/PstI)
Ligation
GixR in pSB1C3
Transformation
GixR in pSB1C3


24.08.2012

PCR
pSB1A3-BBa_J04450 (template DNA)
pSB1T3-BBa_J04450 (template DNA)
pSB1C3-BBa_J04450 (template DNA)
pSB1K3-BBa_J04450 (template DNA)
initial denaturation 98°C - 5min
denaturation: 98°C - 10s; annealing: 60°C - 30s; elongation: 72°C - 40s -->repeat 30 times
72°C - 10min --> 4°C - 8
primer: MS22 + MS23
1% Agarose gel
M:gene ruler mix ladder, 1-2:pSB1A3-BBa_J04450, 3-4: pSB1K3-BBa_J04450, 5-6:pSB1C3-BBa_J04450, 7-8: pSB1T3-BBa_J04450, (PCR 24.08.2012)
Restriction
RBS (XbaI/PstI)
CFP (EcoRI/PstI)
mRFP (EcoRI/PstI)
Ligation
mRFP and CFP in pSB1C3
Transformation
mRFP and CFP in pSB1C3

Week 8

27.08. - 02.09.2012


27.08.2012

Results
CFP: 1 colony
CFP (neu): Colonies
mRFP: colonies
mRFP (neu): no colonies
PCR
template: backbones (pSB1K3/T3/C3/A3)
parameters: 98°C 5min, >> 98°C 1min, 70°C 30s, 72°C 2.5min << x30, 72°C 5min, 4°C 8
primer: MS22 + MS23
colony-PCR
parameters: 98°C 5min, >> 98°C 1min, 72°C 1min << x30, 72°C 5min, 4°C 8
primer: MS18 + MS19
Agarose gel of backbone PCR
no result
elution of the backbones of Friday the 24th


1% Agarose gel
M:1kb gene ruler plus, 1-2:gixR, 3-9: LacIQ-RBS-Gin, 10: CFP, (colony PCR 27.08.2012)


1% Agarose gel
M:1kb gene ruler plus, 1-4:CFP, 5-10: mRFP, (colony PCR 27.08.2012)
Inoculation
pSB1C3_...
mRFP clone 20,24
CFP clone 14,15,17,18
gix clone 1,2
5ml LB, over night, 37°C

28.08.2012

production of competent TOP10
PCR
Enhancer, Terminator, Gix, P-RBS
parameters: 98°C 5', 98°C 1', 66°C 30", 72°C 30", 72°C 5', 4°C ~
primer: MS20 + MS21
1% Agarose gel
M:1kb gene ruler plus, 1:Terminator, 2-3: Enhancer, 4-6: P-RBS, 7-8: GixR, 9-10: Enhancer, (PCR 28.08.2012)
Transformation
[http://partsregistry.org/Part:BBa_I0500 pBad/araC] (plate:1, Well:N14)
pSacB
over night, 37°C
miniprep
GixR (1,2)
CFP (14,15,17,18)
mRFP (20,24)
alle in pSB1C3

29.08.2012

Results
Transformation: no colonies
PCR
Terminator, Gix, Enhancer, P-RBS
diluted templates: Terminator, P-RBS, Gix 1 --> 1:10
Enhancer, Gix 2 --> 1:20
parameters: 98°C 5', 98°C 1', 66°C 30", 72°C 30", 72°C 5', 4°C ~
primer: MS20 + MS21


1% Agarose gel
M:1kb gene ruler plus, 1:Terminator, 2-3: Enhancer, 4-6: P-RBS, 7-8: GixR, 9: Enhancer control, 10: P-RBS control, 11: GixR control, (PCR 29.08.2012)
3AA
CFP, mRFP with Terminator
2µl DNA
1µl SpeI
2µl Tango
15µl dH2O
--> 1h at 37°C
+1µl EcoRI
+2,5µl Tange
--> 1h at 37°C
--> inactivation for 20min at 80°C
Ligation
3AA products in pSB1T3
18.5µl H2O
2.5 µl Buffer
1µl Insert CFP or mRFP (EcoRI + SpeI)
1µl Insert Terminator (BBa_B0010) (XbaI + PstI)
1µl pSB1T3 (EcoRI + PstI)
1µl Ligase
--> 1h at RT
Transformation
pBad --> TOP10 + pSB2K3
CFP-T, mRFP-T --> TOP10 + pSB1T3
Sequencing
CFP_fwd
CFP_rev
mRFP_fwd
mRFP_rev
P-RBS_fwd
GixR_fwd
GixR_fwd
Enhancer_fwd

30.08.2012

Results
Transformation: no colonies --> testing Trafo with BBa_J04450 A/K/T/C
2 colonies at pBad-Trafo from 28.08.2012 --> liquid culture for miniprep
1 colony at pSacB-Trafo from 28.08.2012 --> tested on succrose-sensitivity


31.08.2012

PCR
SacB
Parameters No.1: 95°C 5', >>95°C 30", 60°C 30", 72°C 1'<< x30, 72°C 5', 4°C
Parameters No.2: 95°C 5', >>95°C 30", 72°C 1.5'<< x30, 72°C 5', 4°C
Primer MS26 + MS27


1% Agarose gel
M:1kb gene ruler plus, 1,3,5:SacB, 4,5: none, (PCR 31.08.2012)
Ligation
3AA (CFP-T, mRFP-T)
watch 29.08.
used vector: pSB1K3/T3
Transformation
of ligation in TOP10
miniprep
pBad/araC K.1+2
restriction
GixR rehybridization
cut with EcoRI and PstI
ligation
1µl GixRhyb E+P
1µl pSB1C3 E+P
1µl Th-Pol
2µl Buffer
15µl dH2O
Transformation
ligation + TOP10 --> LB(cam)
Results of test-trafo
Amp, Km, Cm OK --> Km with fewer colonies
Tet --> hardly colonies
--> comp.TOP10 obviously all right, exclusion of Tet in future work

Week 9

03.09. - 09.09.2012


03.09.2012

Results
Transformation
GixR in C3 --> no colonies
CFP-T/mRFP-T in C3(31.8.) --> no colonies
CFP-T/mRFP-T in T3(31.8.) --> many but small colonies
CFP-T/mRFP-T in T3(29.8.) --> less colonies


colony-PCR
of colonies (CFP-T, mRFP-T)
parameters: 98°C 5', >>98°C 1', 72°C 1'<<x30, 72°C 5', 4°C
primer MS18 + MS19
Agarose gel
6x colony pT3-CFP-T from 29./31.8.
6x colony pT3-mRFP-T from 29./31.8.
pC3-CFP (14)
pC3.mRFP (24)
1% Agarose gel
M:1kb gene ruler plus, 1-6:CFP-Terminator, 7:CFP control, 8-13:mRFP-Terminator 14: mRFP control, 15-16: none, (colony-PCR 03.09.2012)

04.09.2012

Results
Transformation
CFP-T/mRFP-T in T3
2 µl -> 5-6 colonies
30 µl -> 1-2 colonies
Gix R in C3 (3.9) --> 4 colonies
colony-PCR
GixR
CFP-T
mRFP-T
parameters: 98°C 5min, >> 98°C 1min, 72°C 1min << x30, 72°C 5min, 4°C ∞
primer MS20 + MS21
gel extraction
P108 (E/S)
RBS (X/P)
ligation
SacB1 + pSB1C3
P-RBS + pSB1C3
Transformation
ligation + TOP10 --> LB(cmp)
1% Agarose gel
M:1kb gene ruler plus, 1-2:pBAD/araC, (mini-prep 31.08.2012)


Inoculation
pBad/araC
5ml LB, over night, 37°C
1% Agarose gel
M:1kb gene ruler plus, 1-4:GixR pSB1C3, 5-9:CFP-Terminator, 10:CFP control, (colony-PCR 04.09.2012)


1% Agarose gel
M:1kb gene ruler plus, 1-9:mRFP-Terminator, 10:mRFP control, (colony-PCR 04.09.2012)


05.09.2012

Results
Transformation
SacB in C3
P-RBS in C3
colony PCR
SacB in C3
P-RBS in C3
parameters: 98°C 5min, >> 98°C 1min, 72°C 1min << x30, 72°C 5min, 4°C ∞
primer MS20 + MS21
miniprep
Gix in C3
pBad
Results
Transformation
CFP-T (03.09) --> 10 colonies
colony PCR
P108-RBS, SacB, GixR
parameters: 98°C 5min, >> 98°C 1min, 72°C 1min << x30, 72°C 5min, 4°C ∞
primer MS19 + MS20
1% Agarose gel
no marker, 1:none, 2-7:SacB, 8-11:P108-RBS, (colony-PCR 05.09.2012)
1% Agarose gel
no marker, 1-5:P108-RBS, 6:none, 7-9:GixR, 10:Scar, 11-12: pBad, 13-15: pGixR, (colony-PCR 05.09.2012)


restriction
pSB1A3 (E/P)
pSB1K3 (E/P)
pSB1C3 (E/P)
pSB1T3 (E/P)
GFP-T K3 (X/P)
GFP K3 (X/P)
CFP C3 (X/P)
mRFP C3 (X/P)
AmiC A (E/S)
GroES A (E/S)
Terminator (X/P)


Inoculation
pRBS
SacB
mRFP-Terminator
5ml LB, over night, 37°C

06.09.2012

miniprep
pRBS
SacB
mRFP-Terminator
ligation
AmiC (E/S) + GFP (X/P) + pSB1T3 (E/P)
AmiC (E/S) + RFP (X/P) + pSB1T3 (E/P)
AmiC (E/S) + GCFP (X/P) + pSB1T3 (E/P)
AmiC (E/S) + Terminator (X+P + pSB1T3 (E/P)
GroES (E/S) + GFP (X/P) + pSB1T3 (E/P)
GroES (E/S) + RFP (X/P) + pSB1T3 (E/P)
GroES (E/S) + CFP (X/P) + pSB1T3 (E/P)
GroES (E/S) + Terminator (X/P) + pSB1T3 (E/P)
PCR of miniprep
SacB in pSB1C3
p-RBS in pSB1C3
terminator in pSB1T3
parameters: 98°C 5min, >> 98°C 30 sec, 72°C 1,5min << x30, 72°C 5min, 4°C ∞
primer MS20 + MS21
1% Agarose gel
M:1kb gene ruler plus, 1:Scar, 2-5:P-RBS, 6-9:SacB, 10-16: mRFP, 17: control, (PCR of miniprep (06.09.2012)


transformation
RBS-Gin T3
amiC A3
groES A3
groES-Terminator T3
groES-GFP T3
groES-CFP T3
groES-mRFP T3
amiC-Terminator T3
amiC-GFP T3
amiC-CFP T3
amiC-mRFP T3

07.09.2012

restriction (6.9.)
ligation
AmiC in pSB1K3
GroES in pSB1K3
mRFP in pSB1K3
Terminator in pSB1K3
CFP in pSB1K3
GFP in pSB1K3
GFP+Terminator in C3
Enhancer in K3
transformation
Ligation
AmiC
GroES
RBS-Gin
miniprep
CFP-Terminator
Sequencing
mRFP T3
P-RBS K12
P-RBS K15
SacB K6
SacB K6

Week 10

10.09. - 16.09.2012

10.09.2012

Restriction
Gin-RBS (01.08)
Ligation
Gin-RBS in pSB1C3
Transformation
Gin-RBS in pSB1C3
Hybridisation
GixR
98 °C 20 min
minus 1 °C to 4 °C in 2 min
Ligation and Transformation
GixR (X+S) + pSB1K3 (X+S)
Inoculation (Tetracycline)
GroES-mRFP
GroES-GFP
GroES-CFP
amiC-mRFP
amiC-GFP
amiC-CFP
5ml LB, over night, 37°C
Inoculation (Kanamycine)
GroES-GFP
amiC-mRFP
amiC-GFP
GroES-Terminator
Enhancer
5ml LB, over night, 37°C
Inoculation (Chloramphenicol)
GFP-Terminator
5ml LB, over night, 37°C
Inoculation (Ampicillin)
Terminator
5ml LB, over night, 37°C

11.09.2012

microscopy-screening
GroES-GFP ⇒; no fluorescence
GroES-CFP ⇒; no fluorescence
AmiC-mRFP ⇒; no fluorescence
AmiC-GFP ⇒; no fluorescence
colony-PCR
GixR
RBS-Gin
Terminator
parameters: 98°C 5', >>98°C 1', 72°C 1'<<x30, 72°C 5', 4°C
primer MS20 + MS21
3A-Assembly (digestion, ligation, transformation)
GroES (E/S) + Terminator (X/P) + pSB1K3 (P/E)
AmiC (E/S) + Terminator (X/P) + pSB1K3 (P/E)
plasmid-prep and control-digestion (E/P)
GroES-GFP
GroES-CFP
AmiC-mRFP
AmiC-GFP
GroES-Terminator
Enhancer
GFP-Terminator
Terminator

12.09.2012

Results
Transformation
- P-RBS --> colonies
- pB10 --> no colonies
- RBS (B0030) --> colonies
- Gro-ES/ AmiC-Terminator --> no colonies
colony-PCR
Gix (2. try)
Gin-RBS (2. try)
RBS (B0030)
P-RBS (108)
parameters: 98°C 5', >>98°C 1', 72°C 1'<<x30, 72°C 5', 4°C
primer MS20 + MS21
Agarose gel
Gin-RBS (5x)
Gin (1x)
Gix (5x)
RBS (6x)
P-RBS (6x)
1% Agarose gel
M:1kb gene ruler plus, 1:Scar, 2-6:RBS-Gin, 7:Gin control 8-12: gixR, 13: control, (colony-PCR 12.09.2012)


1% Agarose gel
M:1kb gene ruler plus, 1:Scar, 2-6:RBS BBa_B0030, 7-12:P-RBS, 13: control, (colony-PCR 12.09.2012)


Restriction (miniprep 11.09.12)
gr-GFP
gr-CFP
gr-T K3
enhancer K3
am-RFP-K3
am-GFP-K3
GFP-T-C3
terminator A2
1% Agarose gel
M:1kb gene ruler plus, 1-3:GroES-GFP, 4:GroES-GFP control, 5:GroES-CFP, 6:GroES-CFP control, 7-10: GroES-Terminator, 11: GroES-Terminator control, 12-15: Enhancer, 16: Enhancer control, (control digest 12.09.2012)
1% Agarose gel
M:1kb gene ruler plus, 1-2:AmiC-mRFP, 3:AmiC-mRFP control, 4-5:AmiC-GFP, 6:AmiC-GFP control, 7-11: GFP-Terminator, 12: GFP-Terminator control, 13-15: Terminator, 16: Terminator control, (control digest 12.09.2012)
Transformation
groES-T
2 Transformations:
- One with a 1:10 dilution of Terminator from ligation,
- another one with a 1:100 dilution of Terminator from ligation
amiC-T
2 Transformations:
- One with a 1:10 dilution of Terminator from ligation,
- another one with a 1:100 dilution of Terminator from ligation
PCR
Gin-RBS
3 reactions:
-One using High Fidelity (HF) Reaction Buffer,
-one using GC-Buffer
-and another one using self-made reaction buffer
Primer used : MS12 + MS13
Template DNA : E. coli C600 chromosomal DNA

13.09.2012

Results
Transformation
- pBAD (11.09.12) --> no colonies
- AmiC-T (11.09.12) --> 2 x 2 colonies
- GroES-T (11.12.09)--> 2 colonies
colony-PCR
AmiC-T & GroES-T (transformation 11.09.12)
parameters: 98°C 5', >>98°C 1', 72°C 1'<<x30, 72°C 5', 4°C
primer MS20 + MS21
1% Agarose gel
M:1kb gene ruler plus, 1:RBS-Gin HF, 2:RBS-Gin GC, 3:RBS-Gin SM (PCR 13.09.2012)


Gel extraction
RBS-Gin (HF) --> 51 ng/µl
RBS-Gin (GC) --> 69 ng/µl
RBS-Gin (SM) --> 56 ng/µl
Ligation
GroES-T & AmiC-T in pSB1K3
Restriction (preparative digest)
GroES (A module)
with SpeI
AmiC (A module)
with SpeI
mRFP-C3 (B module)
with EcoRI and XbaI
CFP-C3 (B module)
with EcoRI and XbaI
Gin-RBS
HF-PCR-Product, GC-PCR-product and SM-PCR-product
with EcoRI and PstI


1% Agarose gel
M:1kb gene ruler plus, 1-4:AmiC-Terminator, 5-6:GroES-Terminator, 7:Scar, 8: control, (colony-PCR 13.09.2012)


14.09.2012

Results
Transformation
AmiC-T: no colonies
GroES-T: no colonies
Restriction
2nd Digestion of A-modules (see digest from yesterday) additionally with EcoRI
Ligation
Gin-RBS with pSB1C3
3 reactions:
one with RBS-Gin HF-PCR-Product
one with RBS-Gin GC-PCR-Product
one with RBS-Gin SM-PCR-Product
Inoculation
Gix-K3 (clone 3 and 4)
P-RBS-C3 (clone 1 and 3)
RBS (BBa_B0030) [clone 1 and 3]
1% Agarose gel
M:1kb gene ruler plus, 1:AmiC, 2:GroES, 3:none, 4:GFP, 5:CFP, 6:mRFP,(digest 13.-14.09.2012)
Gel extraction
GFP
mRFP
1% Agarose gel
M:1kb gene ruler plus, 1:Terminator, (digest 14.09.2012)
Gel extraction
Terminator clone 48
Transformation
RBS-Gin(HF)-pSB1C3
RBS-Gin(GC)-pSB1C3
RBS-Gin(SM)-pSB1C3

Week 11

17.09. - 23.09.2012

17.09.2012

Restriction
AmiC
GroES
CFP
SacB
1% Agarose gel
M:1kb gene ruler plus, 1:AmiC control, 2:AmiC, 3:GroES control, 4:GroES, 5:CFP control, 6:CFP, 7:SacB control, 8:SacB (digest 17.09.2012)
Restriction
CFP-C3
SacB (2nd try)
Resuspend
BBa_B001J
Restreak
RBS-Gin (HF)
RBS-Gin (GC)
RBS-Gin (SM)
DNA isolation
CFP-C3 (Miniprep28.08.12)--> 47,3 ng/µl


Miniprep
GixR pSB1K3 --> 50,5 ng/µl, 144,7 ng/µl
RBS(new)B0030 pSB1A2 --> 726,2 ng/µl, 242,6 ng/µl
P108-RBS pSB1C3 --> 200,5 ng/µl, 177,8 ng/µl
1% Agarose gel
M:1kb gene ruler plus, 1-2:GixR, 3-4:RBS BBa_B0030, 3-4:P108-RBS, (mini prep 17.09.2012)
Transformation
pSB2K3-pBAD & pSB1-A2-TT
1% Agarose gel
CFP-C3 (restriction 2nd try)
SacB-Cs (restriction 2nd try)
M:1kb gene ruler plus, 1:CFP control, 2:CFP, 3:SacB, (digest 17.09.2012)

18.09.2012

Results
Transformation
2x Terminator --> colonies
pBAD --> no colonies
Colony PCR
RBS-Gin
2x Terminator
parameters: 98°C 5', >>98°C 1', 72°C 1'<<x30, 72°C 5', 4°C
primer MS20 + MS21
1% Agarose gel
M:1kb gene ruler plus, 1-2:RBS-Gin HF, 3:RBS-Gin GC, 4:RBS-Gin SM, 5: Scar, 7-10:2xTerminator, 11:control, (colony-pcr 18.09.2012)
Sequencing
SacB
Gel extraction
CFP-C3 E+X
Ligation
am-G: amiC E+S & GFP-A3 E+X
am-C: amiC E+S & CFP-C3 E+X
am-R: amiC E+S & mRFP-C3 E+X
gro-G: groES E+S & GFP-A3 E+X
gro-C: groES E+S & CFP-C3 E+X
gro-R: groES E+S & mRFP-C3 E+X
Transformation
am-G
am-C
am-R
gr-G
gr-C
gr-R
pBAD


19.09.2012

Results
Transformation
am-G --> colonies
am-C --> colonies
am-R --> colonies
gr-G --> colonies
gr-C --> colonies
gr-R --> colonies
pBAD --> no colonies
Colony PCR
RBS-Gin (from 17.09.12)
2x Terminator B0017 (from 17.09.12)
parameters: 98°C 5', >>98°C 1', 72°C 1'<<x30, 72°C 5', 4°C
primer MS20 + MS21
--> Amplification failed
Transformation
pBAD
Microscopy-screening
AmiC-GFP --> fluorescence
GroES-GFP --> fluorescence
AmiC-CFP --> fluorescence
GroES-CFP --> fluorescence
AmiC-mRFP --> fluorescence
GroES-mRFP --> fluorescence
Inoculation
RBS-Gin
TT (B0017)
AmiC-GFP
GroES-GFP
AmiC-CFP
GroES-CFP
AmiC-mRFP
GroES-mRFP


20.09.2012

Miniprep
RBS-Gin
TT (B0017)
AmiC-GFP
GroES-GFP
AmiC-CFP
GroES-CFP
AmiC-mRFP
GroES-mRFP
Restriction (E/P)
TT(B0017)
RBS-Gin
1% Agarose gel
M:1kb gene ruler plus, 1,3,5,7:2xTerminator, 2,4,6,8:2xTerminator control, 9,11,13,15:RBS-Gin, 10,12,14,16:RBS-Gin control, (control digest 20.09.2012)
Restriction
SacB ((XbaI + PstI) & (EcoRI + XbaI)
P-RBS (EcoRI + SpeI) & (SpeI + PstI)
1% Agarose gel
M:1kb gene ruler plus, 1:P-RBS control, 2: P-RBS (E/S), 3: P-RBS (S/P), 4: SacB control, 5: SacB (X/P), 6:SacB (E/X), (digest 20.09.2012)
Inoculation
GroES-GFP
GroES-CFP
GroES-mRFP


21.09.2012

Restriction
GFP-fusion (EcoRI + SpeI)
TT BBa_0017 (EcoRI + XbaI)
1% Agarose gel
M:1kb gene ruler plus, 1:GFP-Fusion, 2:TT BBa_B0017, 3:SacB, (digest 21.09.2012)
Gel extraction
GFP-Fusion
pSB1A2 (backbone GFP-Fusion)
TT
SacB
Ligation
GFP bb Enhancer
GFP TT
GroES TT
Transformation
GFP bb Enhancer
GFP TT
GroES TT
Promotor-RBS (17.09.12)
Sequencing
RBS-Gin
Microscopy-screening
AmiC-GFP --> Channel: DIC, Green, Red, Blue
GroES-GFP --> Channel: DIC, Green, Red, Blue
AmiC-CFP --> Channel: DIC, Green, Red, Blue
GroES-CFP --> Channel: DIC, Green, Red, Blue
AmiC-mRFP --> Channel: DIC, Red
GroES-mRFP --> Channel: DIC, Green, Red, Blue

22.09.2012

Inoculation
GroES-TT pSB1A2
GFP-TT pSB1A2
Enhancer
P108-RBS pSB1C3

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