Difference between revisions of "Team:Marburg/Labbook/Curli"

Line 638: Line 638:
 
<p>Trafo of pC4a, pC4b, pC5, pC6 into DH5α</p>
 
<p>Trafo of pC4a, pC4b, pC5, pC6 into DH5α</p>
  
 +
 +
<br>
 +
<h1>15/07/30</h1>
 +
<h2>Colony PCR</h2>
 +
<p> 3C5_1 + pC1, 3C5_1 + pC3, 4C5_2 pC1, 4C5_2 + pC3, pCam + pC1 and pCam + pC3</p>
 +
<figure style="text-align:center;">
 +
<img src="https://static.igem.org/mediawiki/2015/5/5a/MR_pic_Curli_LB_007.png" width="250" alt="Nutrinity" />
 +
<figcaption style="margin-top:5px;font-size:11pt;color:#606060;text-align:center;line-height:110%"><b>Figure 7:</b> positiv clones (marked with red arrows) for 4C5_2 + pc1 and 4C5_2 + pC3</figcaption>
 +
</figure>
 +
<figure style="text-align:center;">
 +
<img src="https://static.igem.org/mediawiki/2015/2/2b/MR_pic_Curli_LB_008.png" width="250" alt="Nutrinity" />
 +
<figcaption style="margin-top:5px;font-size:11pt;color:#606060;text-align:center;line-height:110%"><b>Figure 8:</b> no positiv clones for 4C5_2 pC1, 4C5_2 + pC3, pCam + pC1 and pCam + pC3</figcaption>
 +
</figure>
  
  

Revision as of 07:12, 16 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

Nutrinity
Figure 1: Nanodrop-Curve of plac, mCherry, Curly

high values (100g/µl-250ng/µl) but weird curves

CPEC


15/05/08

Gel of CEPC reaction

Nutrinity
Figure 2: CPEC of plac, mCherry, Curly

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

Nutrinity
Figure 3: lane 2-4: W3110_pC1; lane 5-7: W3110Δ_pC1; lane 8-10: p70

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)--> pC3

Transformation

Trafo of pC3 into DH5α

15/06/25

CV-staining

Preparation of SDS-probes

W3110, W3110 ΔcsgA, W3310 ΔcsgA pC1, W3310 ΔcsgA p70


15/06/26

SDS-Page

Nutrinity
Figure 4: SDS-Page of W3110, W3110 ΔcsgA, W3310 ΔcsgA pC1, W3310 ΔcsgA p70

15/07/02

Colony-PCR

Nutrinity
Figure 5: Colony-PCR of pC3 (lan 1-7)

MiniPrep

Miniprep of pC3

15/07/03

CV-Staining

Nutrinity
Figure 6: 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

Nutrinity
Figure 7: positiv clones (marked with red arrows) for 4C5_2 + pc1 and 4C5_2 + pC3
Nutrinity
Figure 8: no positiv clones for 4C5_2 pC1, 4C5_2 + pC3, pCam + pC1 and pCam + pC3














TEXT

Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet.

Duis autem vel eum iriure dolor in hendrerit in vulputate velit esse molestie consequat, vel illum dolore eu feugiat nulla facilisis at vero eros et accumsan et iusto odio dignissim qui blandit praesent luptatum zzril delenit augue duis dolore te feugait nulla facilisi. Lorem ipsum dolor sit amet, consectetuer adipiscing elit, sed diam nonummy nibh euismod tincidunt ut laoreet dolore magna aliquam erat volutpat.

Text

Ut wisi enim ad minim veniam, quis nostrud exerci tation ullamcorper suscipit lobortis nisl ut aliquip ex ea commodo consequat. Duis autem vel eum iriure dolor in hendrerit in vulputate velit esse molestie consequat, vel illum dolore eu feugiat nulla facilisis at vero eros et accumsan et iusto odio dignissim qui blandit praesent luptatum zzril delenit augue duis dolore te feugait nulla facilisi. Nam liber tempor cum soluta nobis eleifend option congue nihil imperdiet doming id quod mazim placerat facer possim assum. Lorem ipsum dolor sit amet, consectetuer adipiscing elit, sed diam nonummy nibh euismod tincidunt ut laoreet dolore magna aliquam erat volutpat. Ut wisi enim ad minim veniam, quis nostrud exerci tation ullamcorper suscipit lobortis nisl ut aliquip ex ea commodo consequat. Duis autem vel eum iriure dolor in hendrerit in vulputate velit esse molestie consequat, vel illum dolore eu feugiat nulla facilisis. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, At accusam aliquyam diam diam dolore dolores duo eirmod eos erat, et nonumy sed tempor et et invidunt justo labore Stet clita ea et gubergren, kasd magna no rebum. sanctus sea sed takimata ut vero voluptua. est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat. Consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Duis autem vel eum iriure dolor in hendrerit in vulputate velit esse molestie consequat, vel illum dolore eu feugiat nulla facilisis at vero eros et accumsan et iusto odio dignissim qui blandit praesent luptatum zzril delenit augue duis dolore te feugait nulla facilisi. Lorem ipsum dolor sit amet, consectetuer adipiscing elit, sed diam nonummy nibh euismod tincidunt ut laoreet dolore magna aliquam erat volutpat. Ut wisi enim ad minim veniam, quis nostrud exerci tation ullamcorper suscipit lobortis nisl ut aliquip ex ea commodo consequat. Duis autem vel eum iriure dolor in hendrerit in vulputate velit esse molestie consequat, vel illum dolore eu feugiat nulla facilisis at vero eros et accumsan et iusto odio dignissim qui blandit praesent luptatum zzril delenit augue duis dolore te feugait nulla facilisi. Nam liber tempor cum soluta nobis eleifend option congue nihil imperdiet doming id quod mazim placerat facer possim assum. Lorem ipsum dolor sit amet, consectetuer adipiscing elit, sed diam nonummy nibh euismod tincidunt ut laoreet dolore magna aliquam erat volutpat. Ut wisi enim ad minim veniam, quis nostrud exerci tation ullamcorper suscipit lobortis nisl ut aliquip ex ea commodo

iGEM Marburg - ZSM Karl-von-Frisch-Straße 16, D - 35043 Marburg