Team:Marburg/Labbook/Curli
15/04/28
GXL-PCR
Used primers:
Promotor (plac): IC1 + IC2
csgA: IC3 + IC4
RBS + mCherry: IC5 + IC6
Backbone: IC7 + IC8
15/05/07
Resuspending of speedvaced gel extracted plac, mCherry, Curly
high values (100g/µl-250ng/µl) but weird curves
CPEC
15/05/08
Gel of CEPC reaction
No clear band visible, with a lot of imagination there is a band, which would be right in size -> transform anyway
Transformation of CEPC Curly
--> next day: colonies are visible
15/06/08
Overnightcultures
W3110 Δcsagα cells in 1 mL LB-Media
eCsgAα-Cells in 1 mL LB-Cm-Media
15/06/09
Overdayculture
W3110 Δcsagα + pC1 in 3 mL LB-Cm-Media (1% Glucose)
Platereader experiment
1. Add 30 µL overnight culture (probe B1 in the 96-well-plate)
2. After 2 h: induction with IPTG (probe B2 in the 96-well-plate)
3. After 2 h: fluorescence measurement with the plate reader
4. Non fluorescent cells (Control)
5. culture 4h after induction
6. culture 4h after induction 1:10 diluted
| 1 | 2 | 3 | 4 | 5 | 6 | blank | |
| OD | 0,7117 | 0,2091 | 0,2882 | 0,1249 | 0,4069 | 0,0909 | 0,0379 |
| fluorescence | 3850 | 229 | 303 | 203 | 1297 | 342 | 225 |
| F/OD | 5410 | 1095 | 1051 | 1625 | 3188 | 3762 |
Overday culture (W3110 DcsagA cells)
- 50 mL SOB-Medium
- Add 50 µL overnight culture
- Incubate 3 h at 30°C, then 1 h at 37°C
- OD = 0,46
- Preparation of electrocompetent cells
Electrocompetent cells
W3110 Δcsagα cells
Transformation
pC1 (constructed Curli-Plasmid)and iGEM (Curli, Lyon, 2014) plasmid (p70) into W3110 Δcsagα cells --> repetition because not many colonies (pC1) and not successful (p70)
15/06/10
Transformation
p70 in DH5α-Cells and pC1 in W3110-Cells, repetition of the streak out for the W3110 ΔcsgA cells (no colony picking possible)
15/06/11
Colony-PCR
Overnightcultures
W3110_pC1,W3110Δ_pC1,p70 in LB-Cm-Media
15/06/12
Platereader experiment
- Preparation of a day culture from overnight culture
- After 2h, prepared 96 well plate with different dillution of IPTG (10µL) (0mM, 0.01mM, 0.1mM, 0.5mM, 1mM, 10mM) to each strain (90µL) (DH5α, W3110 WT, W3110 ΔcsgA)
- Read out by fluorescence Photometer
15/06/24
Restriction
Template: pC1
Enzymes: NheI, BamHI
Temperature: 37°C
PCR-Purification
Ligation
Ligation of pC1 with SpyTag (DNA via primer-annealing)--> pC3Transformation
Trafo of pC3 into DH5α15/06/25
CV-staining
Preparation of SDS-probes
W3110, W3110 ΔcsgA, W3310 ΔcsgA pC1, W3310 ΔcsgA p70
15/06/26
SDS-Page
15/07/02
Colony-PCR
MiniPrep
Miniprep of pC315/07/03
CV-Staining
15/07/06
Transformation
pC3 into W3110Δ, W3110 RH, W3110 RHΔ --> not successful
Transformation
pC3 into W3110 --> not successful
preparation of Cryo-Stocks
plate W3110 and W3110Δ on plates
15/07/07
Transformation
pC3 and pUC19 (as control) into W3110Δ, W3110 RH, W3110 RHΔ
Transformation
pC3 and pUC19 into W3110, DH5α
Overnight cultures
W3110, W3110Δ and DH5α + pC3 for cryo stock
15/07/08
Transformation did work (except for the DH5α strains, probably mistake while pipetting or something), prepared new plate with cells, because the growth was to compact
Cryo stocks
W3110, W3110Δ and DH5α + pC3 for cryo stock
15/07/09
Overnight cultures
W3110 + Spy,W3110 Δ + Spy, W3110 RH + Spy, W3110 Δ + Spy, W3110, W3110 Δ for CV-Staining and CR-staining
15/07/10
CR-Staining
preparation
CV-Staining
preparation
15/07/10
CR-Staining
incubation time: 1 day
15/07/13
CR-Staining
incubation time: 3 day
15/07/15
CR-Staining
incubation time: 5 day
15/07/17
CR-Staining
incubation time: 7 day
15/07/27
Transformation
promotor + RBS and GFP (iGEM Kit) into DH5α
15/07/28
Overnight cultures
streak out BBa_K147000 (AIDA) and BBa_K257018 (RFP + Catcher) (send from iGEM HQ)
15/07/29
Overnight cultures
W3110 pC3, W3110 dcsgA pC3
Congored-lb-agar-plates for W3110 and W3110 dcsgA
Restriction
PC4a:
Template a): promoter + RBS
Enzymes a): SpeI (after SpeI: dephosphorylation), PstI
Template b): GFP
Enzymes b): XBaI, SpeI
Template c): SpyCatcher
Enzymes c): Xba, PstI
PC4b:
Template a): promoter + RBS
Enzymes a): SpeI, PstI
Template b): GFP
Enzymes b): XBaI, SpeI
Template c): SpyCatcher
Enzymes c): Xba, PstI
PC5:
Template a): promoter + RBS
Enzymes a): SpeI, PstI
Template b): SpyCatcher
Enzymes b): Xba, SpeI
PC6:
Template a): pSB1C3
Enzymes a): EcoRI, PstI
Template b): SpyCatcher
Enzymes b): EcoRI, PstI
Ligation
Ligation of pC4a, pC4b, pC5, pC6
Transformation
Trafo of pC4a, pC4b, pC5, pC6 into DH5α
15/07/30
Colony PCR
3C5_1 + pC1, 3C5_1 + pC3, 4C5_2 pC1, 4C5_2 + pC3, pCam + pC1 and pCam + pC3
Miniprep
4C5_2 + pC1 and 4C5_2 + pC3 (send to sequenzing)
Colony PCR
DH5α + pC4a, DH5α + pC4b, DH5α + pC5 and DH5α + pC6
Cryo stocks
W3110 pC3, W3110 dcsgA pC3
15/07/31
Plasmidisolation
Miniprep of pC5 (clone 2 and 5) and pC6 (clone 1 and 3) --> send for sequencing (pC6 clone 3 in stock)15/08/03
Plasmidisolation
Miniprep of pC5 (clone 7 and 8) --> send for sequencing (pC5 clone 8 in stock)Streak out 4C5_pC6 (DH5a) on Agar-plates to prepare cryo-Stocks
Colony PCR of 3C5_C1, 3C5_C3, pCam_C1, pCam_C3
15/08/03
PCR
Phusion PCR of GGBP.
Restriction of pC11, Restriction of pC4, Restriction of pC12
Ligation and chemical transformation of pC12 into DH5a
15/08/05
Colony PCR
Colony PCR of pC4, pC11 and pC12
15/08/06
plasmid isolation, colony pcr
Cryo stocks eC28-eC34Elektrotransformation of pC7-pC10 in W3110 dscgA
Miniprep of pC4, pC11 and pC12
15/08/07
cryostocks
eC35-eC4115/08/10
overnight cultures
W3110, W3110 dcsgA; pC1, pC3, pC4, pC7-pC12 (in W3110 dcsgA)15/08/10
SDS-Page
