From 2012.igem.org

(Difference between revisions)

Revision as of 23:05, 26 September 2012

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

Name	Biobrick	Plate	Well	Backbone	Resistance
GFP	[http://partsregistry.org/Part:BBa_E0040 BBa_E0040]	1	14K	pSB1A2	Ampicillin
GFP_fusion	[http://partsregistry.org/Part:BBa_K125500 BBa_K125500]	3	2P	pSB1A2	Ampicillin
CFP	[http://partsregistry.org/Part:BBa_E0020 BBa_E0020]	1	6A	pSB1A2	Ampicillin
mRFP	[http://partsregistry.org/Part:BBa_E1010 BBa_E1010]	1	18F	pSB2K3	Kanamycin
RBS	[http://partsregistry.org/Part:BBa_J61100 BBa_J61100]	1	5J	pSB1A2	Ampicillin
P108	[http://partsregistry.org/Part:BBa_J23108 BBa_J23108]	2	2E	BBa_J61002	Ampicillin
P100	[http://partsregistry.org/Part:BBa_J23100 BBa_J23100]	1	18C	BBa_J61002	Ampicillin
PLac	[http://partsregistry.org/Part:BBa_I14032 BBa_I14032]	2	11P	pSB2K3	Kanamycin

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 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)

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 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: 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 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)

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 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)