Difference between revisions of "Team:Marburg/Labbook/Curli"
| Line 481: | Line 481: | ||
<br> | <br> | ||
<h1>15/06/24</h1> | <h1>15/06/24</h1> | ||
| − | <h2> | + | <h2><a href="https://2015.igem.org/Team:Marburg/Protocols/Protocol6">Restriction</a> </h2> |
| + | <p> Template: pC1<br> | ||
| + | Enzymes: NheI, BamHI<br> | ||
| + | Temperature: 37°C </p> | ||
| + | |||
| + | <h2><a href="https://2015.igem.org/Team:Marburg/Protocols/Protocol8">PCR-Purification</a> </h2> | ||
| + | |||
| + | <h2>Ligation</h2> | ||
| + | Ligation of pC1 with SpyTag (DNA via primer-annealing)--> pC3 | ||
| + | |||
| + | <h2><a href="https://2015.igem.org/Team:Marburg/Protocols/Protocol16">Transformation</a> </h2> | ||
| + | Trafo of pC3 into DH5α | ||
| + | |||
| + | |||
| + | <br> | ||
| + | <h1>15/06/25</h1> | ||
| + | <h2>CV-staining </h2> | ||
| + | |||
| + | <h2>preparation of SDS-probes</h2> | ||
| + | <p>W3110, W3110 ΔcsgA, W3310 ΔcsgA pC1, W3310 ΔcsgA p70</p> | ||
| + | |||
| + | <br> | ||
| + | <h1>15/06/26</h1> | ||
| + | <h2>SDS-Page</h2> | ||
Revision as of 20:49, 13 September 2015
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
TEXT
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