Team:Marburg/Labbook/Curli

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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

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

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

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

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

15/07/02

Colony-PCR

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

MiniPrep

Miniprep of pC3

15/07/03

CV-Staining

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

Figure 7: positiv clones (marked with red arrows) for 4C5_2 + pc1 and 4C5_2 + pC3

Figure 8: no positiv clones for 4C5_2 pC1, 4C5_2 + pC3, pCam + pC1 and pCam + pC3

--> new colonies of 3C5_1 + pC1, 3C5_1 + pC3, pCam + pC1 and pCam + pC3 (16 each)

Miniprep

4C5_2 + pC1 and 4C5_2 + pC3 (send to sequenzing)

Colony PCR

DH5α + pC4a, DH5α + pC4b, DH5α + pC5 and DH5α + pC6

Figure 9: pC4a (lane 2-16)

Figure 10: pC4b (lane 2-16)

Figure 11: pC5 (lane 2-9, positive: c2, c5, c7, c8) + pC6 (lane 10-16, positive: c1, c3, c4, c5, c6, c7)

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

Figure 12: 3C5_C1 (lane 2-9), 3C5_C3 (lane 10-16)

Figure 13:3C5_C1 (lane 2-9), 3C5_C3 (lane 10-16)

15/08/03

PCR

Phusion PCR of GGBP.

Figure 14:GGBP(lane 2-9)

PCR-Purification (GeneJet Purification Kit)
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

Figure 15: Colony-PCR of pC4

Figure 16: Colony-PCR of pC11

Figure 17: Colony-PCR of pC12

15/08/06

plasmid isolation, colony pcr

Cryo stocks eC28-eC34
Elektrotransformation of pC7-pC10 in W3110 dscgA
Miniprep of pC4, pC11 and pC12

15/08/07

cryostocks

eC35-eC41

15/08/10

overnight cultures

W3110, W3110 dcsgA; pC1, pC3, pC4, pC7-pC12 (in W3110 dcsgA)

15/08/11

SDS-Page

Figure 18: SDS-Page, SpyCatcher-GFP (41 kDa) (lane 2), SpyCatcher-GGBP (51) (lane 3), W3110_dcsgA (lane 6)

15/08/12

Crystal Red Staining

Figure 19: CR-Staining

15/08/25

primer design for Spy-LacZ and Spy-ADH

15/08/25

primer design for Spy-GFP-His-Tag

15/08/27

CR-Staining

Preparation (30°C, TB)

PCR

Figure 20: PCR of LacZ

Figure 21: PCR of ADHII with chromosomal DNA of yeast as template; Lane 2: 1 µl template, lane 3: 5 µl template

Restriction of LacZ

Ligation

ligation of LacZ into pC5 and pC6

15/08/28

Isolation of Spy-GFP: cell lysis via sonication (+/- lysosome)
Primer design for removing the pstI-site in csgA

Colony_PCR

no bands for LacZ visible --> not successful

CV-Staining

15/09/01

PCR

Figure 22: Lane 2: W3110, Phusion-PCR; Lane 3: W3110, GXL-PCR, Lane 4: DH5a, Phusion-PCR; Lane 5: DH5a, GXL-PCR

GFP template: pGFP, primer: 160/125
GGBP template: pC11, primer: OS_mglB_XbaI_f/OS_mglB_SpeI_r
PRBS template: pPRBS, primer: 160/125
GFP-HisTag primer: 125/OS_His_gfp_sp_r

Restriction

GFP, GGBP, ADH, PRBS, LacZ

Ligation

ligation of SpyCatcher with GFP, GGBP, ADH and PRBS
ligation of PRBS with ADH, GFP-His and LacZ

15/09/02

Splitting of Curli-fibres into monomers

--> see Ngyuen et al.

Colony-PCR

+ sequencing of GFP-SpyC,GGBP-SpyC,GFP-His

15/09/03

Restriction, Ligation and Transformation

csgA - pstI

15/09/07

Test-Digest

csgA - pstI digested with pstI; two bands are expected

Figure 23: test digest with pstI

15/09/08

Colony-PCR

Figure 24: Lane 1-8: lacZ-pC6, lane 10-17: GGBP-Sc-pC6 (clone 3,5,8 for seq)

Figure 25: Lane 2-9: PRBS-ADH-SC-pC5 (clone 5 for seq), lane 11-18:ADH-Sc-pC6 (clone 1,3 for seq)

Figure 26: Lane 2-9: PRBS-csgA, lane 11-18: GFP-Sc-pC6 (clone 1,3 for seq)

SDS-Page

WT dcsgA, 1 GFP-His, 2 GFP-His, 3 GFP-His, GFP-His-csgA new, 1 PRBS_csgA-Tag_mCherry, 2 PRBS_csgA-Tag_mCherry, 3 PRBS_csgA-Tag_mCherry, pellet GFP-His-csgA new

15/09/09

SDS-Page

repetition

Colony PCR

Figure 27: Lane 2-15: pC6-lacZ, lane 16-25: pC5-lacZ

Restriction, Ligation and Transformation

GGBP + pC6, ADHII + pC6

15/09/10

Restriction, Ligation and Transformation

LacZ; PRBS-CsgA; T5-CsgA-ADHII; T5-CsgA-GFP-His; T5-CsgA-GGBP; T5-CsgA-SpyC

Colony PCR

Figure 28: GGBP + pC6 (lane 2-9) and ADHII + pC6 (lane 11-19)

15/09/11

Colony PCR

LacZ; PRBS-CsgA; T5-CsgA-ADHII; T5-CsgA-GFP-His; T5-CsgA-GGBP; T5-CsgA-SpyC

Miniprep

PRBS-CsgA

Overnightcultures

pC15, pC16, pC5, pC11, pC13
purification of SC-GFP-His (pC15) via Ni-column

Restriction, Ligation and Transformation

PRBS_csgA-Tag new c2,c3,c4,c14,c18

15/09/12

Colony PCR

pC20-pC23

Miniprep

pC15, pC16, pC5, pC11, pC13

Cryostock

pC 13-19

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