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
br>15/07/07
Transformation
promotor + RBS and GFP (iGEM Kit) into DH5α
TEXT
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