Team:Marburg SYNMIKRO/Notebook

From 2012.igem.org

(Difference between revisions)
Line 163: Line 163:
==18.07.2012==
==18.07.2012==
; Agarose gel
; Agarose gel
-
[[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]]
+
[[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:?-DNA]]
; DNA Gel extraction
; DNA Gel extraction
Line 184: Line 184:
; PCR
; PCR
-
: AmiC (primer: MS1 + MS2)
+
: AmiC (primer: [[Team:Marburg_SYNMIKRO/Primer#MS1|MS1]] + [[Team:Marburg_SYNMIKRO/Primer#MS2|MS2]])
Line 252: Line 252:
; PCR
; PCR
-
: CFP (primer: MS16 + MS17) ⇒ template-DNA: BBa_E0010
+
: CFP (primer: [[Team:Marburg_SYNMIKRO/Primer#MS16|MS16]] + [[Team:Marburg_SYNMIKRO/Primer#MS17|MS17]]) ⇒ template-DNA: BBa_E0010
: ⇒ positive
: ⇒ positive
-
: mRFP (primer: MS14 + MS15) ⇒ template-DNA: BBa_E1010  
+
: mRFP (primer: [[Team:Marburg_SYNMIKRO/Primer#MS14|MS14]] + [[Team:Marburg_SYNMIKRO/Primer#MS15|MS15]]) ⇒ template-DNA: BBa_E1010  
: ⇒ positive
: ⇒ positive
-
: Gin (primer: MS11 + MS13) ⇒ template-DNA: chromosomal DNA (that was wrong)  
+
: Gin (primer: [[Team:Marburg_SYNMIKRO/Primer#MS11|MS11]] + [[Team:Marburg_SYNMIKRO/Primer#MS13|MS13]]) ⇒ template-DNA: chromosomal DNA (that was wrong)  
-
: RBS-Gin (primer: MS13 + MS12) ⇒ template-DNA: chromosomal DNA (that was wrong)  
+
: RBS-Gin (primer: [[Team:Marburg_SYNMIKRO/Primer#MS13|MS13]] + [[Team:Marburg_SYNMIKRO/Primer#MS12|MS12]]) ⇒ template-DNA: chromosomal DNA (that was wrong)  
: ⇒ annealing: 30 sec; 60 °C, elongation: 40 sec; 72 °C, 35 cycles
: ⇒ annealing: 30 sec; 60 °C, elongation: 40 sec; 72 °C, 35 cycles
Line 295: Line 295:
; 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]])
: ⇒ template-DNA: chromosomal DNA, annealing: 30 sec; 55 °C, elongation: 40 sec; 72 °C, 35 cycles
: ⇒ template-DNA: chromosomal DNA, annealing: 30 sec; 55 °C, elongation: 40 sec; 72 °C, 35 cycles
Line 615: Line 615:
; 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]])
: ⇒ with and without DMSO, template-DNA: chromosomal DNA, annealing: 30 sec; 60 °C, elongation: 40 sec; 72 °C, 35 cycles
: ⇒ with and without DMSO, template-DNA: chromosomal DNA, annealing: 30 sec; 60 °C, elongation: 40 sec; 72 °C, 35 cycles
Line 654: Line 654:
; 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]])
: ⇒ with and without DMSO, template-DNA: chromosomal DNA, annealing: 30 sec; 55 °C, elongation: 30 sec; 72 °C, 35 cycles
: ⇒ with and without DMSO, template-DNA: chromosomal DNA, annealing: 30 sec; 55 °C, elongation: 30 sec; 72 °C, 35 cycles
Line 688: Line 688:
; PCR
; PCR
-
: GroES (primer: MS5 + MS6 (b))
+
: GroES (primer: [[Team:Marburg_SYNMIKRO/Primer#MS5|MS5]] + [[Team:Marburg_SYNMIKRO/Primer#MS6b|MS6b]])
: ⇒ with and without DMSO, template-DNA: chromosomal DNA, annealing: 30 sec; 55 °C, elongation: 30 sec; 72 °C , 35 cycles
: ⇒ with and without DMSO, template-DNA: chromosomal DNA, annealing: 30 sec; 55 °C, elongation: 30 sec; 72 °C , 35 cycles
Line 781: Line 781:
; PCR
; PCR
-
: Backbone (primer MS24 + MS25)
+
: Backbone (primer [[Team:Marburg_SYNMIKRO/Primer#MS24|MS24]] + [[Team:Marburg_SYNMIKRO/Primer#MS25|MS25]])
: ⇒ template-DNA: pSB1A3, pSB1T3, pSB1C3 and pSB1K3, annealing: 30 sec; 55 °C, elongation: 30 sec; 72 °C , 35 cycles
: ⇒ template-DNA: pSB1A3, pSB1T3, pSB1C3 and pSB1K3, annealing: 30 sec; 55 °C, elongation: 30 sec; 72 °C , 35 cycles
Line 810: Line 810:
: 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 833: Line 833:
; 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
; 1% Agarose gel
; 1% Agarose gel
Line 871: Line 871:
: pSB1K3-BBa_J04450 (template DNA)
: pSB1K3-BBa_J04450 (template DNA)
: pSB1T3-BBa_J04450 (template DNA)
: pSB1T3-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
; 1% Agarose gel
; 1% Agarose gel
Line 898: Line 898:
: initial denaturation 98°C - 5min
: initial denaturation 98°C - 5min
: denaturation: 98°C - 10s; annealing: 60°C - 30s; elongation: 72°C - 40s -->repeat 30 times
: denaturation: 98°C - 10s; annealing: 60°C - 30s; elongation: 72°C - 40s -->repeat 30 times
-
: 72°C - 10min --> 4°C - ∞
+
: 72°C - 10min --> 4°C - 8
-
: primer: MS22, MS23  
+
: primer: [[Team:Marburg_SYNMIKRO/Primer#MS22|MS22]] + [[Team:Marburg_SYNMIKRO/Primer#MS23|MS23]]
; 1% Agarose gel
; 1% Agarose gel
Line 937: Line 937:
; 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  
Line 970: Line 972:
: 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 ~
 +
: primer: [[Team:Marburg_SYNMIKRO/Primer#MS20|MS20]] + [[Team:Marburg_SYNMIKRO/Primer#MS21|MS21]]
; 1% Agarose gele
; 1% Agarose gele
Line 993: Line 996:
: 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
 +
: 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 gele
; 1% Agarose gele
Line 1,049: Line 1,055:
: 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]]
 +
; 1% Agarose gele
; 1% Agarose gele
Line 1,110: Line 1,118:
: 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 1,145: Line 1,154:
: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 1,177: Line 1,188:
: RBS (B0030)
: RBS (B0030)
: P-RBS (108)
: 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
; Agarose gel
Line 1,225: Line 1,238:
:::    -one using GC-Buffer  
:::    -one using GC-Buffer  
:::    -and another one using self-made reaction buffer
:::    -and another one using self-made reaction buffer
-
: Primer used : MS12 & MS13
+
: Primer used : [[Team:Marburg_SYNMIKRO/Primer#MS12|MS12]] + [[Team:Marburg_SYNMIKRO/Primer#MS13|MS13]]
: Template DNA : ''E. coli'' C600 chromosomal DNA
: Template DNA : ''E. coli'' C600 chromosomal DNA
Line 1,237: Line 1,250:
; colony-PCR
; colony-PCR
: AmiC-T & GroES-T (transformation 11.09.12)  
: 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 gele
; 1% Agarose gele
Line 1,374: Line 1,388:
: RBS-Gin
: RBS-Gin
: 2x Terminator
: 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 gele
; 1% Agarose gele
Line 1,417: Line 1,433:
: RBS-Gin (from 17.09.12)
: RBS-Gin (from 17.09.12)
: 2x Terminator B0017 (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
: --> Amplification failed

Revision as of 17:30, 26 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

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:?-DNA
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
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
Control digest
CFP (EcoRI/PstI)
GFP (EcoRI/PstI)
GFP-fusion (EcoRI/PstI)
mRFP (EcoRI/PstI)
⇒ restriction over night

20.07.2012

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


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)


Week 4

30.07. - 05.08.2012


30.07.2012

PCR
CFP (primer: MS16 + MS17) ⇒ template-DNA: BBa_E0010
⇒ positive
mRFP (primer: MS14 + MS15) ⇒ template-DNA: 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


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


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


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
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)
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)
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
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 MS7 (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)


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
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 und CFP in pSB1C3
Transformation
mRFP und 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 gele
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
pBad, araC, 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 gele
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 gele
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 gele
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)




Week 10

10.09. - 16.09.2012


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 gele
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 gele
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 gele
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 gele
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 gele
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 gele
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 gele
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 gele
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 gele
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 gele
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 ???????? kann ich nicht lesen
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 gele
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 gele
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"