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09.07.2012

Inoculation

E. coli strain C600 Mucts 62' in DYT-medium

10.07.2012

Isolation of chromosomal DNA

E. coli strain C600 Mucts 62

Inoculation

E. coli strain DH5a 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

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, annealing: 30 sec; 60 °C, elongation: 30 sec; 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

pipet scheme

Marker|enhancer|GroES|AmiC|HU|GixR|GFP|GFP-Fusion|CFP|mRFP|RBS|LacIQ|J23108|J23100|λ-DNA (bild 1)

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

7) CFP (EcoRI/PstI)

8) GFP (EcoRI/PstI)

9) GFP-fusion (EcoRI/PstI)

10) mRFP (EcoRI/PstI)

⇒ all negative

Agarose gel

pipet scheme

1)|2)|3)|4)|5)|marker|7)|8)|9)|10) (bild 2)

Control digest

CFP (EcoRI/PstI)

GFP (EcoRI/PstI)

GFP-fusion (EcoRI/PstI)

mRFP (EcoRI/PstI)

⇒ restriction over night

20.07.2012

Agarose gel

pipet scheme

⇒ marker|GFP|GFP-fusion|CFP|mRFP (all positive)

(bild 3)

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)

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

pipet scheme

⇒ gix|marker|AmiC from 16.07.| AmiC from 16.07.| AmiC from 19.07.| AmiC from 19.07.

⇒ all negative

(bild 4,5)

Agarose gel

pipet scheme

⇒ marker|RBS-Gin from 30.07.|AmiC from 16.07.|Gin from 30.07.|CFP from 30.07.|mRFP from 30.07.|control

⇒ all negative

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

pipet scheme

Marker|RBS-Gin1 +DMSO|RBS-Gin2 -DMSO|Gin1 +DMSO|Gin2 -DMSO|control

(bild 6)

Agarose gel

pipet scheme

Marker|AmiC1 +DMSO| AmiC2 –DMSO

(bild 7)

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

pipet scheme

Marker| pSB1A3 1,2,3| pSB1C3 1,2,3,4| pSB1K3 1,2,3,4

(bild 8)

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)

Agarose gel

pipet scheme

Marker|Terminator 1,2,3,4

(bild 9)

Transformation

pSB1T3 recycled

AmiC in T3

Gin-RBS in K3

Gin in K3

pSB3C5 linear (false)

03.08.2012

Plasmid preparation

Enhancer in pJET

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)

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)

⇒ positive: pSB3C5 c3a,4a,3b,4b,; Enhancer-pJET c1,2,3,4

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)

Agarose gel

pipet scheme

Marker| Enhancer|pSB1C3

(bild 11)

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)

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

(bild 13)

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

(bild 15)

Inoculation

GixR in C3

Gin in T3

Gin-RBS in T3

pSB1C3

Enhancer in C3

mRFP in A3

13.08.12

PCR

GroES (primer: MS5 + MS6 (b))

HU (primer: MS3 + MS4 (b))

⇒ with and without DMSO, template-DNA: chromosomal DNA, annealing: 30 sec; 60 °C, elongation: 40 sec; 72 °C, 35 cycles

Agarose gel

pipet scheme

Marker|HU +DMSO|HU -DMSO|GroES +DMSO|GroES –DMSO|control

(bild 16)

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 + MS6 (b))

HU (primer: MS3 + MS4 (b))

⇒ 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

Agarose gel

pipet scheme

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)

Resuspension of a biobrick

BBa_J04450 in pSB1A3

BBa_J04450 in pSB1T3

BBa_J04450 in pSB1C3

BBa_J04450 in pSB1K3

Agarose gel

pipet scheme

Marker|HU -DMSO|HU +DMSO|GroES -DMSO|GroES +DMSO

(bild 18)

⇒ 2-step PCR didn’t work, HU 1,2

DNA Gel extraction

HU 1,2

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 + MS6 (b))

⇒ with and without DMSO, template-DNA: chromosomal DNA, annealing: 30 sec; 55 °C, elongation: 30 sec; 72 °C , 35 cycles

Agarose gel

pipet scheme

Marker| GroES 1, 2 +DMSO

(bild 19)

Plasmid preparation

P-RBS

Test restriction

P-RBS (EcoRI/PstI)

Agarose gel

pipet scheme

control|P-RBS|marker

(bild 20)

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)

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)

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)

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

Agarose gel

pipet scheme

marker|: pSB1A3|pSB1T3|pSB1C3|pSB1K3

(bild 23)

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 --> MS24Prefix_rev + MS25Suffix_fwd

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 - ∞

miniprep

GroES, Enh 3/4 (+old), P-RBS (+old), GFP-T (+old)

Test restriction

of miniprep

over night, 37°C

Agarose gel (1%)

watch pic

Inoculation

E.coli BBa_I14032

over night, 37°C

22.08.2012

PCR

CFP

mRFP

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 - ∞

Agarose gel (1%)

watch picture

miniprep

of LacIQ

Test restriction

of LacIQ with EcoRI and PstI

watch picture

Agarose gel

test restriction of GroES and GFP-T (->21.08.12), gel 1%

watch picture

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

Svetti please check! -->lacIQ evtl ganz rausnehmen?! ;)

24.08.2012

Svetti please check!

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 ∞

colony-PCR

parameters: 98°C 5min, >> 98°C 1min, 72°C 1min << x30, 72°C 5min, 4°C ∞

Agarose gel of backbone PCR

no result

elution of the backbones of Friday the 24th

Agarose gel of colony PCR

watch picture

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 ~

watch picture

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

watch picture

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

bitte mal nachschlagen, die Notizen peil ich nicht ;)

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

Agarose gel

watch picture

cut out lines at 1.5kb

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

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

PCR

of colonies (CFP-T, mRFP-T)

parameters: 98°C 5', >>98°C 1', 72°C 1'<<x30, 72°C 5', 4°C

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)

result watch picture

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

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