Team:Bielefeld-CeBiTec/Notebook

2019-04-15 - 2019-04-21

- Plasmids pTXB1 and BBa_I746909 streaked out on agar plates

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Preparations to clone mCherry expression-vectors

Procedure:

pTXB1: Amp; pSB1C3: Cm

2019-04-22 - 2019-04-28

Chemical transformation

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Preparations to clone mCherry expression-vectors

Procedure:

standard chemical transformation of BBa_J06504 from the iGEM Plate (E. coli DH5 alpha)
streaked out on agar plate (Cm)

Overnight cultures to purify plasmid-DNA

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Preparations to clone mCherry expression-vectors

Procedure:

5 mL overnight cultures of pTXB1, BBa_J06504 and BBa_I746909

Plasmid isolation

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Preparations to clone mCherry expression-vectors

Procedure:

standard plasmid purification protocol (analytic jena) of pTXB1, BBa_J06504 and BBa_I746909

2019-04-29 - 2019-05-05

Generation of mCherry-fragments, gel extraction

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Cloning mCherry expression-vectors (BBa_K2926048 and BBa_J06504 in pTXB1)

Procedure:

standard PCR-protocol (Q5, NEB)

primers:

mCherry for pTXB1: 19 a-g (fwd) and 19 a-h (rev)
mCherry for pSB1C3: 19 a-c (fwd) and 19 a-d (rev)
pTXB1 for mCherry: 19 a-e (fwd) and 19 a-f (rev)
pSB1C3 for mCherryHis: 19 a-a (fwd) and 19 a-b (rev)

preparative agarose gel (1 %)

standard gel extraction (promega)

2019-05-06 - 2019-05-12

Generation of mCherry-fragments, gel extraction, Gibson-Assembly, chemical Transformation

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Cloning mCherry expression-vectors (BBa_K2926048 and BBa_J06504 in pTXB1)

Procedure:

standard Gibson Assembly
standard chemical transformation protocol (E. coli DH5 alpha)
streaked out on agar Plates (BBa_K2926048: Cm; BBa_J06504 in pTXB1: Amp)

Overnight cultures to amplify BBa_K2926048 and BBa_J06504 in pTXB1

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Cloning mCherry expression-vectors (BBa_K2926048 and BBa_J06504 in pTXB1)

Procedure:

5 mL overnight cultures of BBa_K2926048 (Cm) and BBa_J06504 in pTXB1 (Amp)

Plasmid isolation of BBa_K2926048 and BBa_J06504 in pTXB1, Sanger Sequencing

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Cloning mCherry expression-vectors (BBa_K2926048 and BBa_J06504 in pTXB1)

Procedure:

standard plasmid isolation (analytic jena Mini Prep)
preparation for sanger Sequencing

BBa_K2926048 (VF and VR)
BBa_J06504 in pTXB1 (Seq1 and Seq2)

Transformation of E. coli DH5ÃÂ± with Cas13a

Team: CeDIS

Investigators: Isabel, Conze

Superior experiment: Cas13a from Munich

Procedure:

Cas13a (from Munich, sent: 200 ng):

Lw Cas13a
Lsh Cas13a
Lbu Cas13a

Dissolved in 10 ÃÂ¼l sterile H2O (final concentration: 20 ng/ÃÂµl); 10 min on 37ÃÂºC

200 ÃÂµl competent E. coli DH5ÃÂ± (Ina, 6.5.19) + 1 ÃÂµl DNA (Cas13a) -> 30 min: ice

1 min 42ÃÂ°C

5 min ice

+ 500 ÃÂµl 37ÃÂ°C warm SOC-Medium

1 h 37ÃÂºC

2019-05-13 - 2019-05-19

Transformation of E. coli DH5ÃÂ± with sfGFP

Team: CeDIS

Investigators: Isabel, Conze

Superior experiment: Labplasmid

Procedure:

In the Parts Registry:

2-3 ng DNA

+10 ÃÂµl sterile MQ --> Mix by pipetting

Sit for 5 min

DNA: sfGPF (BBa_K1321337) (c=2 ng/10ÃÂµl --> 0.2 ng/ÃÂµl)

200 ÃÂµl competent E. coli DH5ÃÂ± (Ina, date: 6.5.19) + 1 ÃÂµl DNA (0.2 ng) -> 30 min: ice

1 min 42ÃÂ°C

5 min ice

+ 500 ÃÂµl 37ÃÂ°C warm SOC-Medium

1 h 37ÃÂºC

> Centrifuge (1 min, 9000 rpm = 7600 rcf);

Plate cell pellet on: LB-Cm

Put plates at 37ÃÂ°C (3 PM)

2019-05-20 - 2019-05-26

Preparation: Sequencing

Team: CeDIS

Investigators: Isabel, Conze

Superior experiment: Cas13a (from Munich) and Labplasmid

Procedure:

Cas13a-Lbu: Colony-PCR 17.05.2019, Ina

Cas13a-Lwa: Colony-PCR 17.05.2019, Ina

Cas13a-Lsh: Colony-PCR 17.05.2019, Ina

sfGFP: Colony-PCR 20.05.2019, Ina

20 ÃÂµl PCR-product + 4 ÃÂµl Purple Dye (NEB Biolabs) -> 10 ÃÂµl in gel

1 kb ladder (NEB) -> 5 ÃÂµl in gel

Staining: 5 min in ethidium bromide

Precultures for Plasmid-Prep & Sequencing:

10 ml LB + 10 ÃÂµl Amp -> LB-Amp

10 ml LB + 10 ÃÂµl Cm (34 mg ml-1) -> LB-Cm (cCm=34 ÃÂµg ml-1)

5 ml LB-Amp + E. coli DH5ÃÂ± (p2CT-His-MBP-Lsh-C13a-WT) Colony 2

5 ml LB-Amp + E. coli DH5ÃÂ± (p2CT-His-MBP-Lbu-C13a-WT) Colony 6

5 ml LB-Cm + E. coli DH5ÃÂ± (pSB1C3-His-SUMO-Lwa-C13a-WT) Colony 5

5 ml LB-Cm + E. coli DH5ÃÂ± (pSB1C3-sfGFP) Colony +

At 37ÃÂ°C, rotating

Overnight culture of BBa_K2926048 and BBa_J06504 in pTXB1

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Cloning mCherry expression-vectors (BBa_K2926048 and BBa_J06504 in pTXB1)

Procedure:

5 mL overnight cultures of BBa_K2926048 (Cm) and BBa_J06504 in pTXB1 (Amp)

Glycerine culture and plasmid isolation of BBa_K2926048 (Cm) and BBa_J06504 in pTXB1

Team: Endocytosis

Investigators: Astrid, TÃ¶bber

Superior experiment: Cloning mCherry expression-vectors (BBa_K2926048 and BBa_J06504 in pTXB1)

Procedure:

350 ÂµL Glycerine + 650 ÂµL overnight culture
- 80 Â°C
standard plasmid isolation (analytic jena)

generating gibson fragments via PCR, PCR-cleanup, analytic agarose gel, Gibson Assembly, Transformation via heatshock

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Cloning mCherry_p8 expression-vectors (BBa_K2926052 and BBa_K2926053)

Procedure:

standard PCR-protocol (Q5 NEB)

primers:

p8: 19 a-r (fwd) and 19 a-s (rev)
Backbone BBa_K2926048: 19 (fwd) and 19 a-j (rev)
Backbone BBa_J06504 in pTXB1: 19 a-e (fwd) and 19 a-j (rev)

standard PCR cleanup (Promega)

analytic agarose gel (1 %, 80 V for backbones, 3 %, 80 V for p8-fragments)
standard Gibson Assembly (1:3)
standard transformation via heatshock (E. coli DH5 alpha)
streaked out on agar plates (BBa_K2926052: Amp; BBa_K2926053: Cm)

PCR-clean up: Cas13a and P8 Ptx (Johanna, 24.5.19)

Team: CeDIS and Endocytosis

Investigators: Isabel, Conze

Superior experiment: Cas13a (from Munich)

Procedure:

Promega Wizard SV Gel and PCR Clean-Up System for:

50 ÃÂµl Lwa, Lbu, Lsh A

c(Lwa) = 35,6 ng ÃÂµl-1
c(Lbu) = 15 ng ÃÂµl-1
c(Lsh) = 16,8 ng ÃÂµl-1

90 ÃÂµl Lwa, Lbu, Lsh B

110 ÃÂµl P8 Ptx (Johanna, 24.5.19.)

110 ÃÂµl P8 Ptx (Johanna, 24.5.19)

2019-05-27 - 2019-06-02

Colony-PCR, overnight culture of positive colonies

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Cloning mCherry_p8 expression-vectors (BBa_K2926052 and BBa_K2926053)

Procedure:

standard PCR-protocol (Taq)

primers:

BBa_K2926052: 19 d-b (fwd) and 19 d-c (rev)
BBa_K2926053: 19 d-b (fwd) and 19 d-c (rev)

5 mL overnight culture of BBa_K2926052 in pTXB1 (Amp, clone 6) and BBa_K2926053 in pSB1C3 (Cm, clone 6)

Plasmid isolation, Sanger Sequencing

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Cloning mCherry_p8 expression-vectors (BBa_K2926052 and BBa_K2926053)

Procedure:

standard plasmid isolation (analytic jena)
preparation for sanger sequencing

primers:

BBa_K2926052: 19 Seq1 and 19 Seq2
BBa_K2926053: 19 VF and VR

Generating Gibson fragments via PCR and primer-annealing, fill-in of 5' overhangs

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Cloning Ligand_mCherry_p8 expression-vectors (BBa_K2926054, BBa_K2926055, BBa_K2926057 and BBa_K2926058)

Procedure:

standard PCR protocol (Q5 NEB, BBa_K2926055, BBa_K2926058)

primers:

BBa_K2926055 in pTXB1: 19 c-o (fwd) and 19 c-p (rev)
BBa_K2926058 in pSB1C3: 19 c-n (fwd) and 19 c-p (rev)

Annealing of 19 c-j and 19 c-l and 19 c-j and 19 c-k to form a fragment of BBa_K2926054 and BBa_K2926055
equal amounts of 19 c-j and 19 c-l (for BBa_K2926054) and 19 c-j and 19 c-k (for BBa_K2926055)
heat to 98 Â°C
cool down to room temperature: 0.5 Â°C per second

fill-in of 5' overhangs

standard Klenow-fragment protocol (Thermo scientific)

PCR-Cleanup of gibson fragments

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Cloning Ligand_mCherry_p8 expression-vectors (BBa_K2926054, BBa_K2926055, BBa_K2926057 and BBa_K2926058)

Procedure:

standard PCR-Cleanup (Promega) of fragments for BBa_K2926055 in pTXB1 and BBa_K2926058 in pSB1C3

Transformation via heatshock

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: mCherry protein expression

Procedure:

standard transformation protocol for BBa_K2926052 and BBa_K2926053 via heatshock in E. coli ER2566 (NEB)
streaked out on agar plates (BBa_K2926052: Amp; BBa_K2926048: Cm)

2019-06-03 - 2019-06-09

Gel-electrophoresis

Team: CeDIS

Investigators: Isabel, Conze

Superior experiment: Basic insert for Gibson in pSB1C3

Procedure:

Gel-electrophoresis: restriction digest basic insert (Johanna)

expected bands: 1528 bp & 1898 bp

PCR:

Template: Gibson-fragment from colony 8-basic insert

Primer: pSB1C3_BasicI_f (19at, 58ÃÂ°C) & pSB1C3_BasicI_r (19av, 58ÃÂ°C)

Fragment size: 3412 bp

Polymerase: Q5 (NEB)

Volume: 100 ÃÂµl

DpnI-digest (over night)

Generating Gibson fragments via PCR, template digestion (DpnI)

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Cloning Ligand_mCherry_p8 expression-vectors (BBa_K2926054, BBa_K2926055, BBa_K2926057 and BBa_K2926058)

Procedure:

standard PCR protocol (Q5 NEB)

primers:

backbone BBa_K2926052 in pTXB1 for BBa_K2926054: 19 c-h (fwd) and 19 c-m (rev)
backbone BBa_K2926052 in pTXB1 for BBa_K2926055: 19 d-d (fwd) and 19 a-f (rev)
backbone BBa_K2926053 in pSB1C3 for BBa_K2926057: 19 c-h (fwd) and 19 c-i (rev)
backbone BBa_K2926053 in pSB1C3 for BBa_K2926058: 19 d-d (fwd) and 19 a-a (rev)

template digestion
standard DpnI digestion protocol (NEB)

PCR-Cleanup of gibson fragments, Gibson Assembly, transformation via heatshock

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Cloning Ligand_mCherry_p8 expression-vectors (BBa_K2926054, BBa_K2926055, BBa_K2926057 and BBa_K2926058)

Procedure:

DpnI inactivation (80 Â°C, 20 min)
standard PCR-Cleanup of gibson fragments (Promega)
standard gibson assembly protocol to generate BBa_K2926054, BBa_K2926055, BBa_K2926057 and BBa_K2926058
standard transformation via heatshock

E. coli DH5 alpha
streaked out on agar plates (BBa_K2926054 and BBa_K2926057: Amp; BBa_K2926055 and BBa_K2926058: Cm)

Colony-PCR: pSB1C3-Basic Insert-Cas13a

Team: CeDIS

Investigators: Isabel, Conze

Superior experiment: Construction: pSB1C3-Basic Insert

Procedure:

Colony-PCR:

Template: E. coli DH5ÃÂ± (pSB1C3-Basic_Insert_Lwa)

Primer: VR (57ÃÂ°C) & Lwa_seq_5 (19ce, 59ÃÂ°C)

Fragment size: 2506 bp

Polymerase: GoTaq Hot Start (Promega)

Volume: 10 ÃÂµl each

Colony-PCR of BBa_K2926055 and BBa_K2926058

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Cloning Ligand_mCherry_p8 expression-vectors (BBa_K2926054, BBa_K2926055, BBa_K2926057 and BBa_K2926058)

Procedure:

standard PCR-protocol (oneTaq, NEB)

primers: 19 c-q (fwd) and 19 a-j (rev)

Pre-cultures

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: mCherry protein expression

Procedure:

overnight cultures (25 mL LB, 37 Â°C) of BBa_J06504 in pTXB1 and BBa_K2926048 in pSB1C3 in E. coli ER2566

BBa_K2926052 in pTXB1: Amp
BBa_K2926053 in pSB1C3: Cm

2019-06-10 - 2019-06-16

Expression cultures

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: mCherry protein expression

Procedure:

standard expression protocol: expression culture (250 mL LB, 37 Â°C) of BBa_J06504 in pTXB1 and BBa_K2926048 in pSB1C3 in E. coli ER2566

BBa_J06504 in pTXB1: Amp
BBa_K2926048 in pSB1C3: Cm
start OD: 0.1
induction (0.4 mM IPTG) at OD 0.6-0.8
3 min 37 Â°C
17 Â°C over night

Gibson Assembly, transformation via heatshock, colony PCR

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Cloning Ligand_mCherry_p8 expression-vectors (BBa_K2926054, BBa_K2926055, BBa_K2926057 and BBa_K2926058)

Procedure:

standard Gibson Assembly protocol to generate BBa_K2926054 and BBa_K2926057 (1:3 backbone:insert)
standard Transformation via heatshock (E. coli DH5 alpha)
streaked out on agar plates

BBa_K2926054 and BBa_K2926055: Amp
BBa_K2926057 and BBa_K2926058: Cm

colony PCR of BBa_K2926058

standard colony PCR protocol (oneTaq, NEB)

primers: 19 c-q (fwd) and 19 a-j (rev)

gradient PCR to determine the optimal annealing temperature for the first add-on PCR

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Cloning Ligand_mCherry_p8 expression-vectors (BBa_K2926056 and BBa_K2926067)

Procedure:

standard PCR protocol (Q5, NEB)

primers:

BBa_K2926056: 19 c-w (rev) and 19 c-x (fwd)
BBa_K2926067: 19 c-r (fwd) and 19 c-s (rev)

temperature gradient: 55 Â°C to 65 Â°C, 10 different temperatures
future annealing temperature: 64 Â°C

Overnight cultures of BBa_K2926055 and BBa_K2926058

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Cloning Ligand_mCherry_p8 expression-vectors (BBa_K2926054, BBa_K2926055, BBa_K2926057 and BBa_K2926058)

Procedure:

5 mL overnight culture of BBa_K2926055 (Amp) and BBa_K2926058 (Cm) in LB

Cell harvest

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: mCherry protein expression

Procedure:

cell harvest (4 Â°C, 4500 rpm, 20 min)
cell mass:

BBa_J06504: 1.92 g
BBa_K2926048: 1.63 g

storage of the pellet at -80 Â°C

Plasmid isolation of BBa_K2926055 and BBa_K2926058, colony PCR of BBa_K2926054 and BBa_K2926057

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Cloning Ligand_mCherry_p8 expression-vectors (BBa_K2926054, BBa_K2926055, BBa_K2926057 and BBa_K2926058)

Procedure:

standard plasmid isolation of BBa_K2926055 and BBa_K2926058 (analytic jena)
standard colony PCR protocol of BBa_K2926054 and BBa_K2926057 (oneTaq)

primers: 19 c-g (fwd) and 19 a-j (rev)

Analytic agarose gel (1 %, 1 kb ladder) of colony PCR products

First add-on PCR, analytic agarose gel

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Cloning Ligand_mCherry_p8 expression-vectors (BBa_K2926056 and BBa_K2926067)

Procedure:

standard PCR protocol (Q5, NEB)

primers:

BBa_K2926056: 19 c-w (rev) and 19 c-x (fwd)
BBa_K2926067: 19 c-r (fwd) and 19 c-s (rev)

analytic agarose gel of PCR products (1 %, 1 kb ladder)

2019-06-17 - 2019-06-23

PCR cleanup, second add-on PCR, analytic agarose gel

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Cloning Ligand_mCherry_p8 expression-vectors (BBa_K2926056 and BBa_K2926067)

Procedure:

standard PCR protocol (Q5, NEB)

primers:

BBa_K2926056: 19 c-v (rev) and 19 c-t (fwd)
BBa_K2926067: 19 c-t (fwd) and 19 c-u (rev)

analytic agarose gel of PCR products (1 %, 1 kb ladder)

overnight cultures of BBa_K2926054 and BBa_K2926057

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Cloning Ligand_mCherry_p8 expression-vectors (BBa_K2926054, BBa_K2926055, BBa_K2926057 and BBa_K2926058)

Procedure:

overnight cultures of positive colonies

5 mL overnight culture of BBa_K2926054 (colony 6, Amp) and BBa_K2926057 (colony 2, Cm) in LB

Chemical transformation

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Expression of Ligand_mCherry_p8 (BBa_K2926054, BBa_K2926055, BBa_K2926057 and BBa_K2926058)

Procedure:

standard heat shock transformation of BBa_K2926054 and BBa_K2926057 in E. coli ER2566
streaked out on agar plates (BBa_K2926054: Amp; BBa_K2926057: Cm)

Gradient PCR (second add-on PCR for BBa_K2926056)

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Cloning Ligand_mCherry_p8 expression-vectors (BBa_K2926056 and BBa_K2926067)

Procedure:

standard PCR protocol (Q5, NEB)

temperature gradient: 55-70 Â°C
primers: 19 c-v (rev) and 19 c-t (fwd)

analytic agarose gel of PCR products (1 %, 1 kb ladder)

Plasmid isolation of BBa_K2926054 and BBa_K2926058

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Cloning Ligand_mCherry_p8 expression-vectors (BBa_K2926054 and BBa_K2926058)

Procedure:

standard plasmid isolation of BBa_K2926054 and BBa_K2926058 (analytic jena)

Gradient PCR (second add-on PCR for BBa_K2926067), first add-on PCR

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Cloning Ligand_mCherry_p8 expression-vectors (BBa_K2926056 and BBa_K2926067)

Procedure:

standard PCR protocol (Q5, NEB)

temperature gradient: 55-70 Â°C
primers: 19 c-t (fwd) and 19 c-u (rev)

analytic agarose gel of PCR products (1 %, 1 kb ladder)
standard PCR protocol (Q5, NEB) for first add-on PCR to generate BBa_K2926056 and BBa_K2926067

primers:

BBa_K2926056: 19 c-w (rev) and 19 c-x (fwd)
BBa_K2926067: 19 c-r (fwd) and 19 c-s (rev)

annealing temperature: 64 Â°C

Analytical agarose gel of PCR products

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Cloning Ligand_mCherry_p8 expression-vectors (BBa_K2926056 and BBa_K2926067)

Procedure:

analytical agarose gel (1%, 1 kb ladder) of PCR fragments of first add-on PCR

2019-06-24 - 2019-06-30

Cell lysis, Protein purification

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: mCherry protein expression

Procedure:

standard cell lysis (Ribolyzer)

resuspension of thawed cell pellet

BBa_J06504 in pTXB1: 35 mL BBa_IMPACT Lysis Buffer
BBa_K2926048: 4 mL Macherey&Nagel LEW-Buffer
addition of 1 mm Zirconia beads (Roth)
Ribolyzer: 3x30 s; 8000 rpm

centrifugation of cell lysate: 1 h, 4500 rpm, 4 Â°C

standard Ni-Ted purification protocol (Macherey&Nagel, BBa_K2926048)

elution in 4.5 mL buffer, addition of 4.5 mL glycerine (86 %)
storage at -20 Â°C

standard IMPACT-purification (NEB)

PCR cleanup, analytical agarose gel

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Cloning Ligand_mCherry_p8 expression-vectors (BBa_K2926056 and BBa_K2926067)

Procedure:

standard PCR cleanup (Promega)
analytical agarose gel (1%, 1 kb ladder)

IMPACT elution, bradford assay

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: mCherry protein expression

Procedure:

elution of mCherry (standard protocol, 10 mL)
buffer change from elution buffer to column buffer (Zentricon, cut off 10 kDa, Merck)

centrifugation 3x, 15-60 min, 5000 rpm, 4Â°C
concentrated to 1.8 mL
addition of 1.8 mL glycerine (86 %)

standard bradford assay protocol (Roti-Nanoquant, Roth)

blank for protein: mCherry without bradford reagent
yield: 472.78 Âµg mCherry (131.33 Âµg/mL), 692.2 Âµg mCherryHis (76.9 Âµg/mL)

Expression pre-cultures

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Expression of Ligand_mCherry_p8 (BBa_K2926054 and BBa_K2926055)

Procedure:

overnight cultures of BBa_K2926054 and BBa_K2926055 in E. coli ER2566 (25 mL LB-Amp, 37 Â°C)

Expression cultures

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Expression of Ligand_mCherry_p8 (BBa_K2926054 and BBa_K2926055)

Procedure:

standard expression protocol: expression culture (250 mL LB-Amp, 37 Â°C) of BBa_K2926054 and BBa_K2926055 in pTXB1 in E. coli ER2566

start OD: 0.1
induction (0.4 mM IPTG) at OD 0.6-0.8
30 min 37 Â°C
21 Â°C over night

second add-on PCR, analytical agarose gel,PCR cleanup, Gibson assembly, transformation via heatshock

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Cloning Ligand_mCherry_p8 expression-vectors (BBa_K2926056 and BBa_K2926067)

Procedure:

standard PCR protocol (Q5, NEB) for second Add-on PCR

primers:

BBa_K2926056: 19 c-v (rev) and 19 c-t (fwd)
BBa_K2926067: 19 c-t (fwd) and 19 c-u (rev)

analytical agarose gel (1 %, 1 kb ladder)

standard PCR cleanup (Promega)
standard Gibson assembly (one fragment)
standard transformation via heatshock (E. coli DH5 alpha)
streaked out on agar plates (BBa_K2926056: Amp, BBa_K2926067: Cm)

Cell harvest

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Expression of Ligand_mCherry_p8 (BBa_K2926054 and BBa_K2926055)

Procedure:

cell harvest (4 Â°C, 4500 rpm, 20 min)
storage of the pellet at -80 Â°C

SDS-PAGE of mCherry purification process

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: mCherry analysis

Procedure:

standard SDS-PAGE protocol (12 %)

analysis of 10 ÂµL lysate, flow through, wash and purified protein of mCherry and mCherryHis
ladder: triple color protein standard (Serva)

Colony PCR, analytical agarose gel

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Cloning Ligand_mCherry_p8 expression-vectors (BBa_K2926056 and BBa_K2926067)

Procedure:

standard PCR protocol (oneTaq) of BBa_K2926067

primers: 19 a-j (rev) and 19 c-y (fwd)

analytical agarose gel (1 %, 1 kb ladder)

PCR f1 ori

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Primers:

f1 ori pSB1C3 GA f
f1 ori mC Prom GA r

Q5 Polymerase

98Â°C 30s
98Â°C 5s
59Â°C 20s
72Â°C 15s
72Â°C 2min
4Â°C

PCR mCherry V1

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Template: iGEM mCherry
Primers:

mC f1 ori RBS GA f
mC VIII GA r

Q5 Polymerase

98Â°C 30s
98Â°C 5s
59Â°C 20s
72Â°C 15s
72Â°C 2min
4Â°C

PCR mCherry V2

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Template: iGEM mCherry
Primers:

mC f1 ori RBS GA f
mC pSB1C3 GA r

Q5 Polymerase

98Â°C 30s
98Â°C 5s
59Â°C 20s
72Â°C 15s
72Â°C 2min
4Â°C

PCR VIII

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Template: M13K07 NEB
Primers:

VIII mC GA f
VIII III GA r

Q5 Polymerase

98Â°C 30s
98Â°C 5s
60Â°C 10s
72Â°C 10s
72Â°C 2min
4Â°C

PCR III

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Template: M13K07 NEB
Primers:

III VIII GA f
III pSB1C3 GA r

Q5 Polymerase

98Â°C 30s
98Â°C 5s
59Â°C 15s
72Â°C 15s
72Â°C 2min
4Â°C

PCR pSB1C3

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Template: iGEM pSB1C3
Primers:

pSB3K3 split f
pSB3K3 split r

Q5 Polymerase

98Â°C 30s
98Â°C 10s
61Â°C 25s
72Â°C 45s
72Â°C 2min
4Â°C

Geleelectrophoresis all fragments

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Gel Clean-Up all fragments

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Monarch DNA Gel Extractions

Overnight culture

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Cloning Ligand_mCherry_p8 expression-vectors (BBa_K2926056 and BBa_K2926067)

Procedure:

overnight culture of one positive colony of BBa_K2926067 (5 mL LB-Amp)

2019-07-01 - 2019-07-07

Gibson Assembly Troygenic DNA V1

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Gibson Master Mix
pSB1C3
f1 ori
mCherry V1
VIII
III

Transformation Troygenic DNA V1

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

DH5a

Gibson Assembly Troygenic DNA V2

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Gibson Master Mix
pSB1C3
f1 ori
mCherry V2

Transformation Troygenic DNA V2

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

DH5a

Colony PCR Troygenic DNA V1

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Primers:

VF
VR

OneTaq Polymerase

95Â°C 30s
95Â°C 25s
57Â°C 45s
68Â°C 2:20min
68Â°C 5min
4Â°C

Colony PCR Troygenic DNA V2

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Primers:

VF
VR

OneTaq Polymerase

95Â°C 30s
95Â°C 20s
57Â°C 40s
68Â°C 1:35min
68Â°C 5min
4Â°C

Plasmid isolation

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Cloning Ligand_mCherry_p8 expression-vectors (BBa_K2926056 and BBa_K2926067)

Procedure:

standard plasmid isolation protocol of BBa_K2926067 (analytic jena)
preparations for Sanger sequencing

primers: SEQ1 (fwd) and SEQ2 (rev)

Establishing a fluorescence measurement method

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: mCherry characterization

Procedure:

preparation of a dilution series of mCherry and mCherryHis: 1:1, 1:10, 1:50, 1:100, 1:1000
preparation of a dilution series of IMPACT-buffer with 86 % glycerine (1:1) and His Elution Buffer with 86 % glycerine (1:1): 1:1, 1:10, 1:50, 1:100, 1:1000
measurement (TECAN-reader) of 100 ÂµL of each dilution (triplicates)
excitation: 570 nm, emission: 610 nm
gain: manual (100)

PCR VIII

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Template: M13K07 NEB
Primers:

VIII mC GA f
VIII III GA r

Q5 Polymerase

98Â°C 30s
98Â°C 5s
60Â°C 10s
72Â°C 10s
72Â°C 2min
4Â°C

PCR III

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Template: M13K07 NEB
Primers:

III VIII GA f
III pSB1C3 GA r

Q5 Polymerase

98Â°C 30s
98Â°C 5s
59Â°C 15s
72Â°C 15s
72Â°C 2min
4Â°C

Geleelectrophoresis all fragments

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Gel Clean-Up all fragments

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Monarch DNA Gel Extractions

Gibson Assembly Troygenic DNA V1

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Gibson Master Mix
pSB1C3
f1 ori
mCherry V1
VIII
III

Transformation Troygenic DNA V1

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

DH5a

Establishing a fluorescence measurement method

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: mCherry characterization

Procedure:

comparison of the fluorescence of mCherry and mCherryHis (200 ng protein in 100 ÂµL)
measurement (TECAN-reader), excitation: 570 nm, emission: 610 nm
measurement of fluorescence and absorption spectra of both proteins
gain: manual (100)

Gibson Assembly Troygenic DNA V1

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Gibson Master Mix
pSB1C3
f1 ori
mCherry V1
VIII
III

Transformation Troygenic DNA V1

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

DH5a

Gibson Assembly Troygenic DNA V2

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Gibson Master Mix
pSB1C3
f1 ori
mCherry V2

Transformation Troygenic DNA V2

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

DH5a

Overlap-PCR f1 ori - mCherry

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Templates: f1 ori, mCherry
Q5 Polymerase

98Â°C 30s
98Â°C 10s
70Â°C 20s
72Â°C 35s
72Â°C 2min
4Â°C

Primers:

f1 ori pSB1C3 GA f
mC VIII GA r
98Â°C 30s
98Â°C 10s
60Â°C 20s
72Â°C 35s
72Â°C 2min
4Â°C

Overlap-PCR VIII - III

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Templates: VIII, III
Q5 Polymerase

98Â°C 30s
98Â°C 8s
72Â°C 20s
72Â°C 15s
72Â°C 2min
4Â°C

Primers:

VIII mC GA f
III pSB1C3 GA r
98Â°C 30s
98Â°C 8s
59Â°C 20s
72Â°C 15s
72Â°C 2min
4Â°C

Geleelectrophoresis Overlap-PCRs

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Gel Clean-Up Overlap-PCRs

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Monarch DNA Gel Extractions

PCR III

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Template: M13K07 NEB
Primers:

III VIII GA f
III pSB1C3 GA r

Q5 Polymerase

98Â°C 30s
98Â°C 5s
59Â°C 15s
72Â°C 15s
72Â°C 2min
4Â°C

overnight cultures of BBa_K2926058

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Cloning Ligand_mCherry_p8 expression-vectors (BBa_K2926054, BBa_K2926055, BBa_K2926057 and BBa_K2926058)

Procedure:

overnight cultures of positive colonies

5 mL overnight culture of BBa_K2926058 (colony 2, 10 and 13, LB-Cm)

PCR pSB3K3

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Helperphage construction

Procedure:

Template: iGEM pSB3K3
Primers:

pSB3K3 split f
pSB3K3 split r

Q5 Polymerase

98Â°C 30s
98Â°C 10s
61Â°C 25s
72Â°C 1:10min
72Â°C 2min
4Â°C

PCR Terminator V1

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Helperphage construction

Procedure:

Template: M13K07 NEB
Primers:

M13 PCR f
M13 Term GA r

Q5 Polymerase

98Â°C 30s
98Â°C 5s
59Â°C 15s
72Â°C 3s
72Â°C 2min
4Â°C

PCR Terminator V2

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Helperphage construction

Procedure:

Template: M13K07 NEB
Primers:

M13 PCR f
M13 Term GA r

Q5 Polymerase

98Â°C 30s
98Â°C 5s
59Â°C 15s
72Â°C 3s
72Â°C 2min
4Â°C

PCR f1 ori

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Primers:

f1 ori pSB1C3 GA f
f1 ori mC Prom GA r

Q5 Polymerase

98Â°C 30s
98Â°C 5s
59Â°C 20s
72Â°C 15s
72Â°C 2min
4Â°C

PCR mCherry

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Template: iGEM mCherry
Primers:

mC f1 ori RBS GA f
mC VIII GA r

Q5 Polymerase

98Â°C 30s
98Â°C 5s
59Â°C 20s
72Â°C 15s
72Â°C 2min
4Â°C

PCR f1 ori Overlap

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Primers:

f1 1C3 OP f
f1 mC Prom OP r

Q5 Polymerase

98Â°C 30s
98Â°C 5s
59Â°C 20s
72Â°C 15s
72Â°C 2min
4Â°C

PCR mCherry Overlap

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Template: iGEM mCherry
Primers:

mC ori OP f
mC VIII OP r

Q5 Polymerase

98Â°C 30s
98Â°C 5s
59Â°C 20s
72Â°C 15s
72Â°C 2min
4Â°C

plasmid isolation of BBa_K2926058

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Cloning Ligand_mCherry_p8 expression-vectors (BBa_K2926054, BBa_K2926055, BBa_K2926057 and BBa_K2926058)

Procedure:

standard plasmid isolation protocol (analytic jena)

Establishing a fluorescence measurement method, measurement of pH stability

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: mCherry characterization

Procedure:

measurement of the fluorescence intensity of mCherry and mCherryHis-dilution series (200 ng, 150 ng, 100 ng, 50 ng, 20 ng, 10 ng)
measurement (TECAN-reader), excitation: 570 nm, emission: 610 nm, gain: manual (100)
measurement of fluorescence and absorption spectra of both proteins
measurement of fluorescence intensity, fluorescence spectrum (600 nm to 620 nm) and absorbance spectrum (300 nm to 850 nm) (200 ng mCherry and mCherryHis) incubated for 15 minutes in different buffers

PBS (pH 1.2, 2.2, 2.9, 3.9, 5.3, 6.1, 6.9)
TBS (pH 8.1, 8.9, 10.3, 11.2, 11.9, 12.7)

2019-07-08 - 2019-07-14

PCR II-VIII

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Helperphage construction

Procedure:

Template: M13K07 NEB
Primers:

M13 pSB3K3 f1
M13 Prom GA r

Q5 Polymerase

98Â°C 30s
98Â°C 8s
56Â°C 25s
72Â°C 45s
72Â°C 2min
4Â°C

Overlap-PCR f1 ori - mCherry

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Templates: f1 ori Overlap, mCherry Overlap
Q5 Polymerase

98Â°C 30s
98Â°C 10s
70Â°C 20s
72Â°C 35s
72Â°C 2min
4Â°C

Primers:

f1 1C3 OP f
mC VIII OP r
98Â°C 30s
98Â°C 10s
60Â°C 20s
72Â°C 35s
72Â°C 2min
4Â°C

Geleelectrophoresis all fragments

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

PCR III-IV

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Helperphage construction

Procedure:

Template: M13K07 NEB
Primers:

III VIII GA f
M13 pSB3K3 r2

Q5 Polymerase

98Â°C 30s
98Â°C 10s
57Â°C 25s
72Â°C 1:15min
72Â°C 2min
4Â°C

Geleelectrophoresis Overlap f1-mCherry, III-IV

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Gel Clean-Up all fragments

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Monarch DNA Gel Extractions

Gibson Assembly Helperphage V1

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Helperphage construction

Procedure:

Gibson Master Mix
pSB3K3
II-VIII
Terminator V1
III-IV

Transformation Helperphage V1

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Helperphage construction

Procedure:

DH5a

Gibson Assembly Helperphage V2

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Helperphage construction

Procedure:

Gibson Master Mix
pSB3K3
II-VIII
Terminator V2

Transformation Helperphage V2

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Helperphage construction

Procedure:

DH5a

Gibson Assembly Troygenic DNA Overlap

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Gibson Master Mix
pSB1C3
f1 ori-mCherry
VIII-III

Transformation Troygenic DNA Overlap

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

DH5a

Pre-cultures

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: mCherry protein expression

Procedure:

overnight cultures (25 mL LB, 37 Â°C) of BBa_J06504 in pTXB1 and BBa_K2926048 in pSB1C3 in E. coli ER2566

BBa_K2926052 in pTXB1: Amp
BBa_K2926053 in pSB1C3: Cm

Expression cultures

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: mCherry protein expression

Procedure:

standard expression protocol: expression culture (250 mL LB, 37 Â°C) of BBa_J06504 in pTXB1 and BBa_K2926048 in pSB1C3 in E. coli ER2566

BBa_J06504 in pTXB1: Amp
BBa_K2926048 in pSB1C3: Cm
start OD: 0.1
induction (0.4 mM IPTG) at OD 0.6-0.8
30 min 37 Â°C
RT over night

Overlap-PCR f1 ori - mCherry

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Templates: f1 ori Overlap, mCherry Overlap
Q5 Polymerase

98Â°C 30s
98Â°C 8s
70Â°C 20s
72Â°C 30s
72Â°C 2min
4Â°C

Primers:

f1 ori pSB1C3 GA f
mC VIII GA r
98Â°C 30s
98Â°C 8s
60Â°C 20s
72Â°C 30s
72Â°C 2min
4Â°C

Overlap-PCR VIII - III

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Templates: VIII, III
Q5 Polymerase

98Â°C 30s
98Â°C 8s
70Â°C 20s
72Â°C 30s
72Â°C 2min
4Â°C

Primers:

VIII mC GA f
III pSB1C3 GA r
98Â°C 30s
98Â°C 8s
59Â°C 20s
72Â°C 30s
72Â°C 2min
4Â°C

PCR VIII Overlap

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Template: M13K07 NEB
Primers:

VIII mC OP f
VIII III OP r

Q5 Polymerase

98Â°C 30s
98Â°C 5s
59Â°C 15s
72Â°C 10s
72Â°C 2min
4Â°C

PCR III Overlap

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Template: M13K07 NEB
Primers:

III VIII OP f
III 1C3 OP r

Q5 Polymerase

98Â°C 30s
98Â°C 5s
59Â°C 15s
72Â°C 10s
72Â°C 2min
4Â°C

PCR pSB3K3

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Helperphage construction

Procedure:

Template: iGEM pSB3K3
Primers:

pSB3K3 split f
pSB3K3 split r

Q5 Polymerase

98Â°C 30s
98Â°C 10s
52Â°C 25s
72Â°C 1:15min
72Â°C 2min
4Â°C

PCR II-VIII

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Helperphage construction

Procedure:

Template: M13K07 NEB
Primers:

M13 pSB3K3 f1
M13 Prom GA r

Q5 Polymerase

98Â°C 30s
98Â°C 10s
56Â°C 25s
72Â°C 45s
72Â°C 2min
4Â°C

PCR III-IV

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Helperphage construction

Procedure:

Template: M13K07 NEB
Primers:

III VIII GA f
M13 pSB3K3 r2

Q5 Polymerase

98Â°C 30s
98Â°C 10s
52Â°C 25s
72Â°C 1:15min
72Â°C 2min
4Â°C

Overlap-PCR f1 ori - mCherry

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Templates: f1 ori Overlap, mCherry Overlap
Q5 Polymerase

98Â°C 30s
98Â°C 10s
70Â°C 20s
72Â°C 35s
72Â°C 2min
4Â°C

Primers:

f1 ori pSB1C3 OP f
mC VIII OP r
98Â°C 30s
98Â°C 10s
60Â°C 20s
72Â°C 35s
72Â°C 2min
4Â°C

Overlap-PCR VIII - III

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Templates: VIII Overlap, III Overlap
Q5 Polymerase

98Â°C 30s
98Â°C 8s
72Â°C 20s
72Â°C 15s
72Â°C 2min
4Â°C

Primers:

VIII mC OP f
III 1C3 OP r
98Â°C 30s
98Â°C 8s
59Â°C 20s
72Â°C 15s
72Â°C 2min
4Â°C

Cell harvest

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: mCherry protein expression

Procedure:

cell harvest (4 Â°C, 4500 rpm, 20 min)
cell mass:

mCherry: 2.45 g
mCherryHis: 1.39 g

Cell lysis, new pre culture

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: mCherry and ligand_mCherry_p8 protein purification

Procedure:

standard cell lysis (Ribolyzer)

lysis not successful

new overnight pre cultures of BBa_J06504, BBa_K2926048, BBa_K2926055 and BBa_K2926054 in E. coli ER2566

25 mL LB-Amp, 37 Â°C

overnight culture of BBa_K2926067

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Cloning Ligand_mCherry_p8 expression-vectors (BBa_K2926056 and BBa_K2926067)

Procedure:

5 mL overnight culture (LB-Cm, 37 Â°C) of BBa_K2926067

Geleelectrophoresis all fragments

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Transformation pSB1C3 from iGEM plate

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Transformation pSB3K3 from iGEM plate

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Helperphage construction

Procedure:

Gradient-PCR f1 ori

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Primers:

f1 ori pSB1C3 GA f
f1 ori mC Prom GA r

Q5 Polymerase

98Â°C 30s
98Â°C 5s
50-60Â°C 15s
72Â°C 20s
72Â°C 2min
4Â°C

PCR f1 ori Overlap

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Primers:

f1 1C3 OP f
f1 mC Prom OP r

Q5 Polymerase

98Â°C 30s
98Â°C 5s
59Â°C 15s
72Â°C 20s
72Â°C 2min
4Â°C

PCR mCherry

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Template: iGEM mCherry
Primers:

mC f1 ori RBS GA f
mC VIII GA r

Q5 Polymerase

98Â°C 30s
98Â°C 5s
59Â°C 15s
72Â°C 20s
72Â°C 2min
4Â°C

Gradient-PCR mCherry Overlap

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Template: iGEM mCherry
Primers:

mC ori OP f
mC VIII OP r

Q5 Polymerase

98Â°C 30s
98Â°C 5s
50-60Â°C 15s
72Â°C 20s
72Â°C 2min
4Â°C

PCR III

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Template: M13K07 NEB
Primers:

III VIII GA f
III pSB1C3 GA r

Q5 Polymerase

98Â°C 30s
98Â°C 5s
59Â°C 15s
72Â°C 20s
72Â°C 2min
4Â°C

PCR III Overlap

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Template: M13K07 NEB
Primers:

III VIII OP f
III 1C3 OP r

Q5 Polymerase

98Â°C 30s
98Â°C 5s
59Â°C 15s
72Â°C 20s
72Â°C 2min
4Â°C

Gradient-PCR pSB1C3

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Template: iGEM pSB1C3
Primers:

pSB3K3 split f
pSB3K3 split r

Q5 Polymerase

98Â°C 30s
98Â°C 10s
50-60Â°C 25s
72Â°C 45s
72Â°C 2min
4Â°C

Gradient-PCR II-VIII

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Helperphage construction

Procedure:

Template: M13K07 NEB
Primers:

M13 pSB3K3 f1
M13 Prom GA r

Q5 Polymerase

98Â°C 30s
98Â°C 10s
50-60Â°C 25s
72Â°C 45s
72Â°C 2min
4Â°C

PCR VIII

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Template: M13K07 NEB
Primers:

VIII mC GA f
VIII III GA r

Q5 Polymerase

98Â°C 30s
98Â°C 5s
60Â°C 10s
72Â°C 10s
72Â°C 2min
4Â°C

Gradient-PCR VIII Overlap

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Template: M13K07 NEB
Primers:

VIII mC OP f
VIII III OP r

Q5 Polymerase

98Â°C 30s
98Â°C 5s
59-70Â°C 10s
72Â°C 10s
72Â°C 2min
4Â°C

PCR Terminator

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Helperphage construction

Procedure:

Template: M13K07 NEB
Primers:

M13 PCR f
M13 Term GA r

Q5 Polymerase

98Â°C 30s
98Â°C 5s
59Â°C 10s
72Â°C 10s
72Â°C 2min
4Â°C

Gradient-PCR pSB3K3

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Helperphage construction

Procedure:

Template: iGEM pSB3K3
Primers:

pSB3K3 split f
pSB3K3 split r

Q5 Polymerase

98Â°C 30s
98Â°C 10s
59Â°C 30s
72Â°C 1:10min
72Â°C 2min
4Â°C

Gradient-PCR III-IV

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Helperphage construction

Procedure:

Template: M13K07 NEB
Primers:

III VIII GA f
M13 pSB3K3 r2

Q5 Polymerase

98Â°C 30s
98Â°C 10s
52Â°C 30s
72Â°C 1:15min
72Â°C 2min
4Â°C

Gradient-Overlap-PCR f1 ori - mCherry

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Templates: f1 ori, mCherry
Q5 Polymerase

98Â°C 30s
98Â°C 8s
50Â°C 20s
72Â°C 35s
72Â°C 2min
4Â°C

Primers:

f1 ori pSB1C3 GA f
mC VIII GA r
98Â°C 30s
98Â°C 8s
50-70Â°C 20s
72Â°C 35s
72Â°C 2min
4Â°C

Gradient-Overlap-PCR VIII - III

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Templates: VIII, III
Q5 Polymerase

98Â°C 30s
98Â°C 5s
50Â°C 15s
72Â°C 15s
72Â°C 2min
4Â°C

Primers:

VIII mC GA f
III pSB1C3 GA r
98Â°C 30s
98Â°C 5s
59-70Â°C 15s
72Â°C 15s
72Â°C 2min
4Â°C

Geleelectrophoresis all fragments

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

PCR Clean-Up all fragments

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Promega Wizard SV Gel and PCR Clean-Up System

digest dnpI all fragments

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

- standard expression protocol: expression culture (250 mL LB, 37 Â°C) of BBa_J06504, BBa_K2926055 and BBa_K2926054 in pTXB1, BBa_K2926048 in pSB1C3 in E. coli ER2566

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: mCherry and ligand_mCherry_p8 protein expression

Procedure:

pTXB1: Amp

pSB1C3: Cm
start OD: 0.1
induction (0.4 mM IPTG) at OD 0.6-0.8
30 min 37 Â°C
17 Â°C over night

plasmid isolation, PCR to generate gibson fragments, DpnI digestion

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Cloning Ligand_mCherry_p8 expression-vectors (BBa_K2926056 and BBa_K2926067)

Procedure:

standard plasmid isolation protocol of BBa_K2926067 (Zymo research)
standard PCR protocol (Q5, NEB) to generate gibson fragment for BBa_K2926056

template: BBa_K2926067
primers: 19 i-g (fwd) and 19 i-h (rev)

DpnI digestion of PCR product (NEB, 37 Â°C over night)

Gel Clean-Up all fragments

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Monarch DNA Gel Extractions

PCR Clean-Up all fragments

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Promega Wizard SV Gel and PCR Clean-Up System

Cell harvest

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: mCherry and ligand_mCherry_p8 protein expression

Procedure:

cell harvest (4 Â°C, 4500 rpm, 20 min)
cell mass:

mCherry: 1.84 g
mCherryHis: 1.86 g
Flo_mCherry_p8: 1.79 g
Mat_mCherry_p8: 2.4 g

Gel cleanup, Gibson assembly, Transformation via heatshock

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Cloning Ligand_mCherry_p8 expression-vectors (BBa_K2926056 and BBa_K2926067)

Procedure:

DpnI inactication (20 min 80 Â°C)
analytical agarose gel (1 %, 1 kb ladder)
preparative agarose gel (1 %, 1 kb ladder)
standard gel cleanup protocol (Promega)
standard gibson assembly of PCR fragment and pTXB1 fragment generated in May
standard transformation via heatshock in E. coli DH5 alpha
streaked out on agar plates (Amp)

Establishing a fluorescence standard for red fluorescing proteins

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: mCherry characterization - Texas Red

Procedure:

measurement: fluorescence intensity of Fluorescein and Texas Red (standard iGEM protocol for plate reader calibration)

TECAN-reader, gain: calculated from well with highest fluorescence dye concentration
Fluorescein: excitation: 494 nm, emission: 525 nm
Texas red: excitation: 570 nm, emission: 610 nm

measurement: absorption spectrum (350 nm to 800 nm) and emission spectrum (510 nm to 850 nm Fluorescein, 600 nm to 850 nm Texas Red) of both dyes

2019-07-15 - 2019-07-21

PCR f1 ori

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Primers:

f1 ori pSB1C3 GA f
f1 ori mC Prom GA r

Q5 Polymerase

98Â°C 30s
98Â°C 5s
51Â°C 15s
72Â°C 20s
72Â°C 2min
4Â°C

PCR mCherry

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Template: iGEM mCherry
Primers:

mC f1 ori RBS GA f
mC VIII GA r

Q5 Polymerase

98Â°C 30s
98Â°C 5s
59Â°C 15s
72Â°C 20s
72Â°C 2min
4Â°C

PCR mCherry Overlap

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Template: iGEM mCherry
Primers:

mC ori OP f
mC VIII OP r

Q5 Polymerase

98Â°C 30s
98Â°C 5s
55Â°C 15s
72Â°C 20s
72Â°C 2min
4Â°C

PCR III Overlap

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Template: M13K07 NEB
Primers:

III VIII OP f
III 1C3 OP r

Q5 Polymerase

98Â°C 30s
98Â°C 5s
59Â°C 15s
72Â°C 20s
72Â°C 2min
4Â°C

PCR pSB1C3

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Template: iGEM pSB1C3
Primers:

pSB3K3 split f
pSB3K3 split r

Q5 Polymerase

98Â°C 30s
98Â°C 10s
53Â°C 25s
72Â°C 45s
72Â°C 2min
4Â°C

PCR II-VIII

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Helperphage construction

Procedure:

Template: M13K07 NEB
Primers:

M13 pSB3K3 f1
M13 Prom GA r

Q5 Polymerase

98Â°C 30s
98Â°C 10s
58Â°C 25s
72Â°C 45s
72Â°C 2min
4Â°C

PCR VIII Overlap

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Template: M13K07 NEB
Primers:

VIII mC OP f
VIII III OP r

Q5 Polymerase

98Â°C 30s
98Â°C 5s
66Â°C 10s
72Â°C 10s
72Â°C 2min
4Â°C

PCR pSB3K3

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Helperphage construction

Procedure:

Template: iGEM pSB3K3
Primers:

pSB3K3 split f
pSB3K3 split r

Q5 Polymerase

98Â°C 30s
98Â°C 10s
59Â°C 30s
70Â°C 1:10min
72Â°C 2min
4Â°C

PCR III-IV

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Helperphage construction

Procedure:

Template: M13K07 NEB
Primers:

III VIII GA f
M13 pSB3K3 r2

Q5 Polymerase

98Â°C 30s
98Â°C 10s
52Â°C 30s
72Â°C 1:15min
72Â°C 2min
4Â°C

Geleelectrophoresis all fragments

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Gel Clean-Up all fragments

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Monarch DNA Gel Extractions

PCR Clean-Up all fragments

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Promega Wizard SV Gel and PCR Clean-Up System

Gibson Assembly Helperphage

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Helperphage construction

Procedure:

Gibson Master Mix
pSB3K3
II-VIII
Terminator
III-IV

Transformation Helperphage

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Helperphage construction

Procedure:

DH5a

Gibson Assembly Troygenic DNA

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Gibson Master Mix
pSB1C3
f1 ori
mCherry
VIII
III

Transformation Troygenic DNA

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

DH5a

Cell lysis, IMPACT purification

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: mCherry and ligand_mCherry_p8 protein purification (BBa_J06504, BBa_K2926055 and BBa_K2926054 in pTXB1, BBa_K2926048 in pSB1C3)

Procedure:

standard cell lysis (Ribolyzer)
standard purification protocol (IMPACT, NEB)
standard purification protocol (Ni-TED, Macherey&Nagel)

protein loss due to a buffer mistake

PCR mCherry

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Template: iGEM mCherry
Primers:

mC f1 ori RBS GA f
mC VIII GA r

Q5 Polymerase

98Â°C 30s
98Â°C 5s
59Â°C 15s
72Â°C 20s
72Â°C 2min
4Â°C

PCR mCherry Overlap

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Template: iGEM mCherry
Primers:

mC ori OP f
mC VIII OP r

Q5 Polymerase

98Â°C 30s
98Â°C 5s
55Â°C 15s
72Â°C 20s
72Â°C 2min
4Â°C

Geleelectrophoresis mCherry

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Colony-PCR pSB1C3

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

GoTaq G2 DNA Polymerase

Colony-PCR pSB3K3

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Helperphage construction

Procedure:

GoTaq G2 DNA Polymerase

Colony-PCR Helperphage V1

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Helperphage construction

Procedure:

GoTaq G2 DNA Polymerase

Colony-PCR Helperphage V2

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Helperphage construction

Procedure:

GoTaq G2 DNA Polymerase

Gradient-Overlap-PCR f1 ori - mCherry

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Templates: f1 ori, mCherry
Q5 Polymerase

98Â°C 30s
98Â°C 8s
50Â°C 20s
72Â°C 35s
72Â°C 2min
4Â°C

Primers:

f1 ori pSB1C3 GA f
mC VIII GA r
98Â°C 30s
98Â°C 8s
58-70Â°C 20s
72Â°C 35s
72Â°C 2min
4Â°C

Gradient-Overlap-PCR VIII - III

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Templates: VIII, III
Q5 Polymerase

98Â°C 30s
98Â°C 5s
50Â°C 15s
72Â°C 15s
72Â°C 2min
4Â°C

Primers:

VIII mC GA f
III pSB1C3 GA r
98Â°C 30s
98Â°C 5s
50-70Â°C 15s
72Â°C 15s
72Â°C 2min
4Â°C

Geleelectrophoresis all fragments

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Helperphage construction

Procedure:

PCR Clean-Up mCherry

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Promega Wizard SV Gel and PCR Clean-Up System

Gel Clean-Up mCherry

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Promega Wizard SV Gel and PCR Clean-Up System

colony PCR, overnight cultures

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Cloning Ligand_mCherry_p8 expression-vectors (BBa_K2926056 and BBa_K2926067)

Procedure:

standard colony PCR protocol (oneTaq, NEB)

primers: 10 a-j (rev) and 19 c-y (fwd)

analytical agarose gel (1 %)
overnight cultures of positive colonies (5 mL LB-Amp, 37 Â°C over night)

Elution, washing and concentrating

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: mCherry and ligand_mCherry_p8 protein purification (BBa_J06504, BBa_K2926055 and BBa_K2926054 in pTXB1)

Procedure:

elution of BBa_J06504, BBa_K2926055 and BBa_K2926054 in 10 mL IMPACT elution buffer
proteins washed with 3x10 mL PBS (Zentricon Merck, cut off 10 kDa)
concentrated to around 1 mL
addition of glycerine (86 %, 1:1)
storage at -20 Â°C

Gibson Assembly Troygenic DNA

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Gibson Master Mix
pSB1C3
f1 ori
mCherry
VIII
III

Transformation Troygenic DNA

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

DH5a

Overlap-PCR f1 ori - mCherry

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Templates: f1 ori, mCherry
Q5 Polymerase

98Â°C 30s
98Â°C 8s
50Â°C 20s
72Â°C 35s
72Â°C 2min
4Â°C

Primers:

f1 ori pSB1C3 GA f
mC VIII GA r
98Â°C 30s
98Â°C 8s
61Â°C 20s
72Â°C 35s
72Â°C 2min
4Â°C

Overlap-PCR VIII - III

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Templates: VIII, III
Q5 Polymerase

98Â°C 30s
98Â°C 5s
50Â°C 15s
72Â°C 15s
72Â°C 2min
4Â°C

Primers:

VIII mC GA f
III pSB1C3 GA r
98Â°C 30s
98Â°C 5s
53Â°C 15s
72Â°C 15s
72Â°C 2min
4Â°C

Overlap-PCR f1 ori - mCherry

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Templates: f1 ori Overlap, mCherry Overlap
Q5 Polymerase

98Â°C 30s
98Â°C 10s
70Â°C 20s
72Â°C 35s
72Â°C 2min
4Â°C

Primers:

f1 ori pSB1C3 OP f
mC VIII OP r
98Â°C 30s
98Â°C 10s
61Â°C 20s
72Â°C 35s
72Â°C 2min
4Â°C

Overlap-PCR VIII - III

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Templates: VIII Overlap, III Overlap
Q5 Polymerase

98Â°C 30s
98Â°C 8s
72Â°C 20s
72Â°C 15s
72Â°C 2min
4Â°C

Primers:

VIII mC OP f
III 1C3 OP r
98Â°C 30s
98Â°C 8s
53Â°C 20s
72Â°C 15s
72Â°C 2min
4Â°C

Colony-PCR pSB1C3

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

GoTaq G2 DNA Polymerase

plasmid isolation

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Cloning Ligand_mCherry_p8 expression-vectors (BBa_K2926056 and BBa_K2926067)

Procedure:

standard plasmid isolation (Zymo research)
prepared for Sanger sequencing

primers: SEQ1 (fwd) and SEQ2 (rev)

Bradford assay, SDS-PAGE

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: mCherry and ligand_mCherry_p8 protein purification (BBa_J06504, BBa_K2926055 and BBa_K2926054 in pTXB1)

Procedure:

standard bradford assay protocol (Roth, Roti nanoquant)

mCherry: 53.6 ng/ÂµL; 125.85 Âµg
Flo_mCherry_p8: 2.86 ng/ÂµL; 7.74 Âµg
Mat_mCherry_p8: 2.45 ng/ÂµL; 6.53 Âµg

standard SDS-PAGE protocol (12 %)

analysis of 10 ÂµL lysate, flow through, wash and purified protein
ladder: triple color protein standard (Serva)

TECAN-reader measurement

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: mCherry and ligand_mCherry_p8 protein characterization (BBa_J06504, BBa_K2926055 and BBa_K2926054 in pTXB1)

Procedure:

dilution series of mCherry and mCherryHis (diluted 1:1 down from 1 ÂµM)
measurement of fluorescence intensity of mCherry dilution series and fusion proteins (200 ÂµL)

TECAN reader, gain: calculated from well with highest concentration, excitation: 570 nm, emission: 610 nm

measurement of emission spectrum (600 nm to 850 nm) and absorption spectrum (300 nm to 850 nm) of all four proteins

Dilution series mCherry

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Primers:

mC f1ori RBS GA f
mC VIII GA r
98Â°C 30s
98Â°C 5s
55Â°C 20s
72Â°C 20s
72Â°C 2min
4Â°C

Dilution series II-VIII

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Helperphage construction

Procedure:

Primers:

M13 pSB3K3 f1
M13 Prom GA r
98Â°C 30s
98Â°C 10s
59Â°C 30s
72Â°C 1min
72Â°C 2min
4Â°C

Dilution series pSB1C3

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Primers:

pSB3K3 split f
pSB3K3 split r
98Â°C 30s
98Â°C 10s
53Â°C 25s
72Â°C 45s
72Â°C 2min
4Â°C

Dilution series pSB3K3

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Helperphage construction

Procedure:

Primers:

pSB3K3 split f
pSB3K3 split r
98Â°C 30s
98Â°C 10s
61Â°C 25s
72Â°C 1:10min
72Â°C 2min
4Â°C

Transformation pSB3K3 from iGEM plate

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Helperphage construction

Procedure:

10Âµl Kanamycin on plate

Inoculation pSB1C3

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

PCR Clean-Up all fragments

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Promega Wizard SV Gel and PCR Clean-Up System

Gel Clean-Up all fragments

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Promega Wizard SV Gel and PCR Clean-Up System

Inoculation pSB3K3

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Helperphage construction

Procedure:

Geleelectrophoresis all fragments

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

PCR Clean-Up pSB3K3

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Helperphage construction

Procedure:

Promega Wizard SV Gel and PCR Clean-Up System

Gel Clean-Up pSB3K3

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Promega Wizard SV Gel and PCR Clean-Up System

Plasmid Miniprep pSB1C3

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

ZymoPURE Plasmid Miniprep

PCR mCherry

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Template: iGEM mCherry
Primers:

mC f1 ori RBS GA f
mC VIII GA r

Q5 Polymerase

98Â°C 30s
98Â°C 5s
55Â°C 20s
72Â°C 20s
72Â°C 2min
4Â°C

PCR mCherry Overlap

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Template: iGEM mCherry
Primers:

mC ori OP f
mC VIII OP r

Q5 Polymerase

98Â°C 30s
98Â°C 5s
55Â°C 20s
72Â°C 20s
72Â°C 2min
4Â°C

PCR Clean-Up mCherry

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Helperphage construction

Procedure:

Promega Wizard SV Gel and PCR Clean-Up System

PCR pSB1C3

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Primers:

pSB3K3 split f
pSB3K3 split r
98Â°C 30s
98Â°C 10s
53Â°C 25s
72Â°C 45s
72Â°C 2min
4Â°C

Geleelectrophoresis mCherry

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

Gel Clean-Up mCherry

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Helperphage construction

Procedure:

Promega Wizard SV Gel and PCR Clean-Up System

Pre-cultures

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: mCherry and ligand_mCherry_p8 protein expression

Procedure:

overnight cultures (25 mL LB, 37 Â°C) of BBa_J06504, BBa_K2926055 and BBa_K2926054 in pTXB1, BBa_K2926048 in E. coli ER2566

pTXB1: Amp
pSB1C3: Cm

Geleelectrophoresis all fragments

Team: Troygenic Assembly

Investigators: Astrid, TÃ¶bber

Superior experiment: Troygenic DNA construction

Procedure:

protein expression

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: mCherry and ligand_mCherry_p8 protein expression

Procedure:

standard expression protocol: expression culture (250 mL LB, 37 Â°C) of BBa_J06504, BBa_K2926055 and BBa_K2926054 in pTXB1, BBa_K2926048 in pSB1C3 in E. coli ER2566

pTXB1: Amp
pSB1C3: Cm
start OD: 0.1
induction (0.4 mM IPTG) at OD 0.6-0.8
30 min 37 Â°C
17 Â°C over night

Cell harvest

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: mCherry and ligand_mCherry_p8 protein expression

Procedure:

cell harvest (4 Â°C, 4500 rpm, 20 min)
cell mass:

mCherry: 2.13 g
mCherryHis: 1.92 g
Flo_mCherry_p8: 2.09 g
Mat_mCherry_p8: 2.11 g

2019-07-22 - 2019-07-28

Cell lysis, protein purification

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: mCherry and ligand_mCherry_p8 protein purification (BBa_J06504, BBa_K2926055 and BBa_K2926054 in pTXB1, BBa_K2926048 in pSB1C3)

Procedure:

standard cell lysis (Ribolyzer, addition of sand instead of metal beads)
standard purification protocol (IMPACT, NEB)
standard purification protocol (Ni-TED, Macherey&Nagel)

washed 3 times with PBS, concentrated (Zentricon Merck, cut off 10 kDa) and mixed 1:1 with glycerine (86%)
stored at -20 Â°C

Elution, washing and concentrating

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: mCherry and ligand_mCherry_p8 protein purification (BBa_J06504, BBa_K2926055, BBa_K2926054, BBa_J06504)

Procedure:

elution in 10 mL IMPACT elution buffer
proteins washed with 3x10 mL PBS (Zentricon Merck, cut off 10 kDa)
concentrated to around 1 mL
addition of glycerine (86 %, 1:1)
storage at -20 Â°C

Bradford assay, SDS-PAGE

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: mCherry and ligand_mCherry_p8 protein purification (BBa_J06504, BBa_K2926055, BBa_K2926054, BBa_J06504)

Procedure:

standard bradford assay protocol (Roth, Roti nanoquant)

mCherry: 916.99 ng/ÂµL; 1.43 mg
mCherryHis: 14.15 ng/ÂµL; 45.28 Âµg
Flo_mCherry_p8: 6.68 ng/ÂµL; 8 Âµg
Mat_mCherry_p8: 10 ng/ÂµL; 13.6 Âµg

standard SDS-PAGE protocol (12 %)

analysis of 10 ÂµL lysate, flow through, wash and purified protein
ladder: triple color protein standard (Serva)

transformation via heatshock

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Cloning Ligand_mCherry_p8 expression-vectors (BBa_K2926056 and BBa_K2926067)

Procedure:

transformation of BBa_K2926056 via heatshock in E. coli ER2566
streaked out on agar plates (Amp)

Pre-cultures

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Ligand_mCherry_p8 protein expression

Procedure:

overnight cultures (25 mL LB-Amp, 37 Â°C) of BBa_K2926056 in pTXB1 in E. coli ER2566

protein expression

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Ligand_mCherry_p8 protein expression

Procedure:

standard expression protocol: expression culture (250 mL LB-Amp, 37 Â°C) of BBa_K2926056 in pTXB1 in E. coli ER2566

start OD: 0.1
induction (0.4 mM IPTG) at OD 0.6-0.8
30 min 37 Â°C
17 Â°C over night

TECAN-reader measurement

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: mCherry, mCherryHis and ligand_mCherry_p8 protein characterization

Procedure:

Texas Red standard

2.5 ÂµM, 1 ÂµM, 0.5 ÂµM, 0.25 ÂµM, 0.1 ÂµM

protein dilution series

0.5 ÂµM, 0.25 ÂµM, 0.1 ÂµM, 0.05 ÂµM, 0.025 ÂµM, 0.01 ÂµM

measurement of fluorescence intensity of mCherry dilution series and fusion proteins (quadruplicates)

TECAN reader, gain: calculated from well with highest concentration, excitation: 570 nm, emission: 610 nm

measurement of emission spectrum (600 nm to 850 nm) and absorption spectrum (300 nm to 850 nm) of all four proteins

Cell harvest

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: ligand_mCherry_p8 protein expression (BBa_K2926056)

Procedure:

cell harvest (4 Â°C, 4500 rpm, 20 min)
cell mass: 1.18 g

2019-07-29 - 2019-08-04

cell lysis, IMPACT purification

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: ligand_mCherry_p8 protein purification

Procedure:

standard cell lysis (Ribolyzer, addition of sand instead of metal beads)
standard purification protocol (IMPACT, NEB)

Elution, washing and concentrating

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: ligand_mCherry_p8 protein purification

Procedure:

elution in 10 mL IMPACT elution buffer
proteins washed with 3x10 mL PBS (Zentricon Merck, cut off 10 kDa)
addition of glycerine (86 %, 1:1)
storage at -20 Â°C

Bradford assay

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: ligand_mCherry_p8 protein purification

Procedure:

standard bradford assay protocol (Roth, Roti nanoquant)

Opy_mCherry_p8

SDS-PAGE

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: ligand_mCherry_p8 protein purification

Procedure:

standard SDS-PAGE protocol (12 %)

analysis of 10 ÂµL lysate, flow through, wash and purified protein
ladder: triple color protein standard (Serva)

TECAN-reader measurement

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: mCherry and ligand_mCherry_p8 protein characterization

Procedure:

Texas Red standard

2.5 ÂµM, 1 ÂµM, 0.5 ÂµM, 0.25 ÂµM, 0.1 ÂµM

protein dilution series

0.5 ÂµM, 0.25 ÂµM, 0.1 ÂµM, 0.05 ÂµM, 0.025 ÂµM, 0.01 ÂµM

measurement of fluorescence intensity of mCherry dilution series and fusion proteins (quadruplicates)

TECAN reader, gain: calculated from well with highest concentration, excitation: 570 nm, emission: 610 nm

measurement of emission spectrum (600 nm to 850 nm) and absorption spectrum (300 nm to 850 nm) of all four proteins

PCR, PCR cleanup

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Cloning ligand_mCherry fusion proteins

Procedure:

standard PCR protocol (Q5, NEB) to generate gibson fragments

BBa_K292605:

template: BBa_J06504 in pTXB1
primers: 19 a-f (rev) and 19 d-d (fwd)

BBa_K2926054:

template: BBa_J06504 in pTXB1
primers: 19 c-h (fwd) and 19 c-m (rev)

BBa_K2926051

template: BBa_K2926067 in pSB1C3
primers: 19 a-h (rev) and 19 i-g (fwd)

standard PCR cleanup protocol (Macherey&Nagel)

2019-08-05 - 2019-08-11

Analytical agarose gel, Gibson assembly, transformation via heatshock

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Cloning ligand_mCherry fusion proteins

Procedure:

analytical agarose gel of PCR fragments (1 %, 1 kb ladder)
standard gibson assembly

BBa_K292605: PCR fragment and gene synthesis
BBa_K2926054: PCR fragment and primer dimer
BBa_K2926051: PCR fragment and pTXB1 linerarized

standars transformation via heatshock in E. coli DH5 alpha
streaked out on agar plates (Amp)

Colony PCR, overnight culture

Team: Endocytosis

Investigators: Alexander, Schulze

Superior experiment: Cloning ligand_mCherry fusion proteins

Procedure:

standard colony PCR protocol (GoTaq, Promega)

primers: 19 e-j (rev) and 19 c-q (fwd, BBa_K292605), 19 c-g (fwd, BBa_K2926054) or 19 c-y (fwd, BBa_K2926051)

overnight culture of positive colonies (5 mL LB-Amp, 37 Â°C)

BBa_K292605: colony 15
BBa_K2926054: colony 16
BBa_K2926051: colony 9

plasmid isolation, preparations for Sanger sequencing

Team: Endocytosis

Investigators: Alexander, Schulze

Superior experiment: Cloning ligand_mCherry fusion proteins

Procedure:

standard plasmid preparation protocol (Qiagen)
preparations for Sanger sequencing

primers: SEQ1 (fwd) and SEQ2 (rev)

2019-08-12 - 2019-08-18

transformation via heatshock, overnight cultures

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: ligand_mCherry fusion protein expression

Procedure:

standard transformation of BBa_K2926050, BBa_K2926049 and BBa_K2926051 in pTXB1 via heatshock in E. coli ER2566
streaked out on agar plates (Amp)
overnight cultures (25 mL LB-Amp, 37 Â°C) of BBa_K2926050, BBa_K2926049 and BBa_K2926051 in pTXB1 in E. coli ER2566

protein expression

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: ligand_mCherry fusion protein expression

Procedure:

standard expression protocol: expression culture (250 mL LB-Amp, 37 Â°C) of BBa_K2926050, BBa_K2926049 and BBa_K2926051 in pTXB1 in E. coli ER2566

start OD: 0.1
induction (0.4 mM IPTG) at OD 0.6-0.8
30 min 37 Â°C
17 Â°C over night

cell harvest, cell lysis, IMPACT purification

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: ligand_mCherry fusion protein expression and purification

Procedure:

cell harvest (4 Â°C, 4500 rpm, 20 min)
cell mass:

BBa_K2926050: 2.17 g
BBa_K2926049: 1.46 g
BBa_K2926051: 1.3 g

standard cell lysis (French Press, 2x, 16 000 psi, ca 1 mL/min)
standard purification protocol (IMPACT, NEB)

cell harvest, cell lysis, IMPACT purification

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: ligand_mCherry fusion protein expression

Procedure:

cell harvest (4 Â°C, 4500 rpm, 20 min)
cell mass:

BBa_K2926068: 1.83 g

Elution, washing and concentrating

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: ligand_mCherry fusion protein purification

Procedure:

elution in 10 mL IMPACT elution buffer
proteins washed with 3x10 mL PBS (Zentricon Merck, cut off 10 kDa)
addition of glycerine (86 %, 1:1)
storage at -20 Â°C

Bradford assay, SDS-PAGE

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: ligand_mCherry fusion protein purification

Procedure:

standard bradford assay protocol (Roth, Roti nanoquant)

Flo_mCherry: 470.3 ng/ÂµL; 503 Âµg
Mat_mCherry: 445.71 ng/ÂµL; 356.57 Âµg
Opy_mCherry: 196.95 ng/ÂµL; 236.38 Âµg

standard SDS-PAGE protocol (12 %)

analysis of 10 ÂµL lysate, flow through, wash and purified protein
ladder: triple color protein standard (Serva)

TECAN-reader measurement

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: ligand_mCherry fusion protein characterization

Procedure:

Texas Red standard

2.5 ÂµM, 1 ÂµM, 0.5 ÂµM, 0.25 ÂµM, 0.1 ÂµM

protein dilution series

0.5 ÂµM, 0.25 ÂµM, 0.1 ÂµM, 0.05 ÂµM, 0.025 ÂµM, 0.01 ÂµM

measurement of fluorescence intensity of dilution series (quadruplicates)

TECAN reader, gain: calculated from well with 2.5 ÂµM, excitation: 570 nm, emission: 610 nm

measurement of emission spectrum (600 nm to 850 nm) and absorption spectrum (300 nm to 850 nm) of all three proteins

TECAN-reader measurement

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: mCherry protein characterization

Procedure:

light stability assay

0.2 ÂµM mCherry
fluorescence intensity measured after 0 min, 15 min, 30 min, 1 h, 2 h, 3 h
TECAN reader, gain: calculated from well with 2.5 ÂµM Texas Red, excitation: 570 nm, emission: 610 nm

pH stability assay

0.2 ÂµM mCherry incubated for 5 min in buffers with different pH
fluorescence intensiy and emission maximum measured
TECAN reader, gain: calculated from well with 2.5 ÂµM Texas Red, excitation: 570 nm, emission: 610 nm

2019-08-19 - 2019-08-25

Pre culture

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Endocytosis assay S. cerevisiae

Procedure:

overnight culture of S. cerevisiae (10 mL YPD, 30 Â°C)

Pre culture

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Endocytosis assay S. cerevisiae

Procedure:

overnight culture of S. cerevisiae (10 mL YPD, 30 Â°C)

S. cerevisiae in minimal media

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Endocytosis assay S. cerevisiae

Procedure:

50 mL culture of S. cerevisiae in minimal media (OD 0.2, 30 Â°C)

Fluorescence measurement of supernatant

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Endocytosis assay S. cerevisiae

Procedure:

division of 50 mL culture to four 5 mL cultures
addition of 1x no fusion protein, 1x mCherry, 1x Opy_mCherry, 1x Mat_mCherry (0.2 ÂµM)
negative control: every fusion protein in minimal media
incubation (30 Â°C, 180 rpm, dark)
measurement of fluorescence every 15 minutes

centrifugation of 1 mL culture (1 min, 12 000 rpm)
measurement of fluorescence in supernatant (TECAN reader, gain: calculated from well with 2.5 ÂµM Teas Red, excitation: 570 nm, emission: 610 nm)

2019-08-26 - 2019-09-01

Pre culture

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Fluorescence microscopy S. cerevisiae

Procedure:

pre culture of S. cerevisiae (10 mL YPD, 30 Â°C, 180 rpm over night)

Fluorescence microscopy

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Fluorescence microscopy S. cerevisiae

Procedure:

start of main culture (OD 0.2) in minimal media and YPD
at OD around 0.5: harvest of 0.35 OD (1 min, 12 000 rpm, RT)
resuspension in 60 ÂµL YPD
addition of fusion protein (0.2 ÂµM)
incubation (15 min, 30 Â°C, 450 rpm, dark)
centrifugation (1 min, 12 000 rpm, RT)
resuspended in 10 ÂµL minimal media
fluorescence microscopy of 5 ÂµL (DM5000 B (Leica), magnification: 100x, Texas Red [?(ex)=555 nm, ?(Em)=570 nm to 800 nm])

Pre culture

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Endocytosis assay S. cerevisiae

Procedure:

overnight culture of S. cerevisiae (10 mL YPD, 30 Â°C, 180 rpm)

Main culture, fluorescence measurement of supernatant

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Endocytosis assay S. cerevisiae

Procedure:

main culture in minimal media (OD ca 0.2)
at OD 4.5: divided into 5x5 mL culture
addition of 1x no fusion protein, 1x mCherry, 1x Opy_mCherry, 1x Mat_mCherry, 1x Flo_mCherry (0.2 ÂµM)
negative control: every fusion protein in minimal media
incubation (30 Â°C, 180 rpm, dark)
measurement of fluorescence every 10 minutes

centrifugation of 1 mL culture (1 min, 12 000 rpm)
measurement of fluorescence in supernatant (TECAN reader, gain: calculated from well with 2.5 ÂµM Teas Red, excitation: 570 nm, emission: 610 nm)

2019-09-02 - 2019-09-08

Pre culture

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Fluorescence microscopy S. cerevisiae

Procedure:

overnight culture of S. cerevisiae (10 mL YPD, 30 Â°C, 180 rpm)

Fluorescence microscopy

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Fluorescence microscopy S. cerevisiae

Procedure:

start of main culture (OD 0.2) in YPD
at OD around 0.5: harvest of 0.35 OD (1 min, 12 000 rpm, RT)

resuspension in 60 ÂµL SD media
addition of fusion protein (1 ÂµM)
incubation (15 min, 30 Â°C, 450 rpm, dark)
centrifugation (1 min, 12 000 rpm, RT)
washing step (20 ÂµL PBS, centrifugation 1 min, 12 000 rpm, RT)
resuspended in 10 ÂµL minimal media
fluorescence microscopy of 5 ÂµL (LSM 700 (Zeiss), magnification: 100x, Texas Red [?(ex)=555 nm, ?(Em)=570 nm to 800 nm])

to potentially enhance uptake: digestion of cell wall

incubation of S. cerevisiae (0.35 OD) in speroblast buffer (15 min, 30 Â°C, 450 rpm, dark)

10 mL spheroblast buffer contain: 6 mL Sorbitol (2 M), 0.5 mL K3PO4 (pH 7.4, 1 M), 0.1 mL MgCl2 (0.1 M)

after 15 min: addition of fusion proteins (1 mM)
further incubation (15 min, 30 Â°C, 450 rpm, dark)
centrifugation (1 min, 12 000 rpm, RT)
washing step (20 ÂµL spheroblast buffer, centrifugation 1 min, 12 000 rpm, RT)
resuspended in 10 ÂµL spheroblast buffer
fluorescence microscopy of 5 ÂµL (LSM 700 (Zeiss), magnification: 100x, Texas Red [?(ex)=555 nm, ?(Em)=570 nm to 800 nm])

trying out higher concentrated proteins

resuspension in 60 ÂµL YPD
addition of fusion protein (5 ÂµM)
incubation (15 min, 30 Â°C, 450 rpm, dark)
centrifugation (1 min, 12 000 rpm, RT)
washing step (20 ÂµL PBS, centrifugation 1 min, 12 000 rpm, RT)
resuspended in 10 ÂµL minimal media
fluorescence microscopy of 5 ÂµL (LSM 700 (Zeiss), magnification: 100x, filters: Texas Red [?(ex)=555 nm, ?(Em)=570 nm to 800 nm], transmitted light

Main culture, fluorescence measurement of pellet

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Endocytosis assay S. cerevisiae

Procedure:

main culture of S. cerevisiae in YPD (OD 0.2)
at OD 0.7: incubation of 1 mL culture with fusion protein (0.2 ÂµM and 2 ÂµM, Mat_mCherry and Opy_mCherry, 0 min, 15 min, 30 min, 45 min)
centrifugation (1 min, 12 000 rpm, RT)
washing step (500 ÂµL PBS, 1 min, 12 000 rpm, RT)
resuspension (500 ÂµL PBS)
measurement of fluorescence intensity (TECAN reader, gain: manual (100), excitation: 570 nm, emission: 610 nm)

Main culture, fluorescence measurement of supernatant

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Endocytosis assay S. cerevisiae

Procedure:

main culture in minimal media (OD ca 0.2)
at OD 4.5: divided into 4x5 mL culture
addition of 1x no fusion protein, 1x mCherry, 1x Opy_mCherry, 1x Mat_mCherry, 1 ÂµM)
negative control: every fusion protein in minimal media
incubation (30 Â°C, 180 rpm, dark)
measurement of fluorescence every 15 minutes

centrifugation of 1 mL culture (1 min, 12 000 rpm)
measurement of fluorescence in supernatant (TECAN reader, gain: calculated from well with 2.5 ÂµM Teas Red, excitation: 570 nm, emission: 610 nm)

PCR to generate gibson fragments, PCR cleanup, Gibson assembly, transformation via heatshock

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Cloning ligand_mCherry proteins for Aspergillus niger

Procedure:

standard PCR protocol (Q5, NEB)

primers: 19 p-m (fwd) and 19 p-n (rev)
template: BBa_J06504 in pTXB1

standard PCR cleanup (Qiagen)
standard gibson assembly (one fragment)
transformation via heatshock in E. coli DH5 alpha
streaked out on agar plates (Amp)

overnight cultures

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: mCherry and ligand_mCherry fusion protein expression

Procedure:

overnight cultures (25 mL LB-Amp, 37 Â°C) of BBa_K2969051 and BBa_J06504 in pTXB1 in E. coli ER2566

protein expression

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: mCherry and ligand_mCherry fusion protein expression

Procedure:

standard expression protocol: expression culture (250 mL LB-Amp, 37 Â°C) of BBa_K2969051 and BBa_J06504 in pTXB1 in E. coli ER2566

start OD: 0.1
induction (0.4 mM IPTG) at OD 0.6-0.8
30 min 37 Â°C
17 Â°C over night

- overnight culture (25 mL LB-Amp, 37 Â°C) of BBa_K2969050 in pTXB1 in E. coli ER2566

Team: Endocytosis

Investigators: overnight, cultures

Superior experiment: ligand_mCherry fusion protein purification

Procedure:

2019-09-09 - 2019-09-15

cell harvest

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: mCherry and ligand_mCherry fusion protein expression

Procedure:

cell harvest (4 Â°C, 4500 rpm, 20 min)
cell mass:

BBa_K2926051: 1.71 g
BBa_J06504: 1.61 g

protein expression

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: mCherry and ligand_mCherry fusion protein expression

Procedure:

standard expression protocol: expression culture (250 mL LB-Amp, 37 Â°C) of BBa_K2969050 in pTXB1 in E. coli ER2566

start OD: 0.1
induction (0.4 mM IPTG) at OD 0.6-0.8
30 min 37 Â°C
17 Â°C over night

- overnight culture (25 mL LB-Amp, 37 Â°C) of BBa_K2969049 in pTXB1 in E. coli ER2566

Team: Endocytosis

Investigators: overnight, cultures

Superior experiment: ligand_mCherry fusion protein purification

Procedure:

cell lysis, IMPACT purification

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: mCherry and ligand_mCherry fusion protein expression and purification

Procedure:

standard cell lysis (French Press, 2x, 16 000 psi, ca 1 mL/min)
standard purification protocol (IMPACT, NEB)

protein expression

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: mCherry and ligand_mCherry fusion protein expression

Procedure:

standard expression protocol: expression culture (250 mL LB-Amp, 37 Â°C) of BBa_K2969049 in pTXB1 in E. coli ER2566

start OD: 0.1
induction (0.4 mM IPTG) at OD 0.6-0.8
30 min 37 Â°C
17 Â°C over night

cell harvest

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: mCherry and ligand_mCherry fusion protein expression

Procedure:

cell harvest (4 Â°C, 4500 rpm, 20 min)
cell mass:

BBa_K2926050: 1.66 g

Elution, washing and concentrating

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: mCherry and ligand_mCherry fusion protein purification

Procedure:

elution in 10 mL IMPACT elution buffer
proteins washed with 3x10 mL PBS (Zentricon Merck, cut off 10 kDa)
addition of glycerine (86 %, 1:1)
storage at -20 Â°C

cell lysis, IMPACT purification

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: ligand_mCherry fusion protein purification

Procedure:

standard cell lysis (French Press, 2x, 16 000 psi, ca 1 mL/min)
standard purification protocol (IMPACT, NEB)

Elution, washing and concentrating, Bradford assay

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: ligand_mCherry fusion protein purification

Procedure:

elution in 10 mL IMPACT elution buffer
proteins washed with 3x10 mL PBS (Zentricon Merck, cut off 10 kDa)
addition of glycerine (86 %, 1:1)
storage at -20 Â°C
standard bradford assay protocol (Roth, Roti nanoquant)

Flo_mCherry: 256 ng/ÂµL; 409 Âµg
Mat_mCherry: 1.96 Âµg/ÂµL; 2.352 mg
Opy_mCherry: 1.24 Âµg/ÂµL; 1.488 mg
mCherry: 2.42 Âµg/ÂµL; 3.315 mg

Endocytosis assay S. cerevisiae

Team: Endocytosis

Investigators: Endocytosis

Superior experiment: >2019-08-29

Procedure:

Johanna Opgenoorth

Pre culture

overnight culture of S. cerevisiae (10 mL YPD, 30 Â°C, 180 rpm)

Main culture, fluorescence measurement of supernatant

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Endocytosis assay S. cerevisiae

Procedure:

main culture in minimal media (OD ca 0.2)
at OD 4.5: divided into 5x5 mL culture
addition of 1x no fusion protein, 1x mCherry, 1x Opy_mCherry, 1x Mat_mCherry, 1x Flo_mCherry (1 ÂµM)
negative control: every fusion protein in minimal media
incubation (30 Â°C, 180 rpm, dark)
measurement of fluorescence every 15 minutes

centrifugation of 1 mL culture (1 min, 12 000 rpm)
measurement of fluorescence in supernatant (TECAN reader, gain: calculated from well with 2.5 ÂµM Teas Red, excitation: 570 nm, emission: 610 nm)

2019-09-16 - 2019-09-22

Pre culture

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Endocytosis assay S. cerevisiae

Procedure:

overnight culture of S. cerevisiae (10 mL YPD, 30 Â°C, 180 rpm)

Main culture, fluorescence measurement of supernatant

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Endocytosis assay S. cerevisiae

Procedure:

main culture in minimal media (OD ca 0.2)
at OD 4.5: divided into 5x5 mL culture
addition of 1x no fusion protein, 1x mCherry, 1x Opy_mCherry, 1x Mat_mCherry, 1x Flo_mCherry (1 ÂµM)
negative control: every fusion protein in minimal media
incubation (30 Â°C, 180 rpm, dark)
measurement of fluorescence every 15 minutes

centrifugation of 1 mL culture (1 min, 12 000 rpm)
measurement of fluorescence in supernatant (TECAN reader, gain: calculated from well with 2.5 ÂµM Teas Red, excitation: 570 nm, emission: 610 nm)

colony PCR, analytical agarose gel, overnight culture

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Cloning ligand_mCherry proteins for Aspergillus niger

Procedure:

standard colony PCR protocol (GoTaq, Promega)

primers: 19 p-t (fwd) and 19 a-j (rev)

analytical agarose gel (1 %, 1 kb ladder)
overnight culture of positive colony 6 (5 mL LB-Amp, 37 Â°C)

Pre culture

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Fluorescence microscopy S. cerevisiae

Procedure:

overnight culture of S. cerevisiae (10 mL YPD, 30 Â°C, 180 rpm)

Plasmid isolation, transformation via heatshock

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Cloning ligand_mCherry proteins for Aspergillus niger

Procedure:

standard plasmid isolation (Qiagen)
preparation for sanger sequencing

primers: SEQ1 (fwd) and SEQ2 (rev)

transformation in E. coli ER2566 via heat shock
streaked out on agar plates (Amp)

- start of main culture (OD 0.2) in YPD

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Fluorescence microscopy S. cerevisiae

Procedure:

at OD around 0.5: harvest of 0.35 OD (1 min, 12 000 rpm, RT)

resuspension in 60 ÂµL SD media
addition of fusion protein (1 ÂµM)
incubation (30 min, 30 Â°C, 450 rpm, dark)
centrifugation (1 min, 12 000 rpm, RT)
washing step (50 ÂµL PBS, centrifugation 1 min, 12 000 rpm, RT)
resuspended in 10 ÂµL minimal media
fluorescence microscopy of 5 ÂµL (LSM 700 (Zeiss), magnification: 100x, Texas Red [?(ex)=555 nm, ?(Em)=570 nm to 800 nm])

2019-09-23 - 2019-09-29

overnight culture

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: ligand_mCherry fusion protein expression

Procedure:

overnight culture (25 mL LB-Amp, 37 Â°C) of BBa_K2926068 in pTXB1 in E. coli ER2566

protein expression

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: ligand_mCherry fusion protein expression

Procedure:

standard expression protocol: expression culture (250 mL LB-Amp, 37 Â°C) of BBa_K2926068 in pTXB1 in E. coli ER2566

start OD: 0.1
induction (0.4 mM IPTG) at OD 0.6-0.8
30 min 37 Â°C
17 Â°C over night

2019-09-30 - 2019-10-06

cell lysis, protein purification

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: ligand_mCherry fusion protein purification

Procedure:

standard cell lysis (French Press, 2x, 16 000 psi, ca 1 mL/min)
standard purification protocol (IMPACT, NEB)

Elution, washing and concentrating

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: ligand_mCherry fusion protein purification, Bradford assay

Procedure:

elution in 10 mL IMPACT elution buffer
proteins washed with 3x10 mL PBS (Zentricon Merck, cut off 10 kDa)
addition of glycerine (86 %, 1:1)
storage at -20 Â°C
standard bradford assay protocol (Roth, Roti nanoquant)

Pro_mCherry: 70.7 ng/ÂµL; 67.9 Âµg

SDS-PAGE

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: ligand_mCherry fusion protein purification

Procedure:

standard SDS-PAGE protocol (12 %)

analysis of 10 ÂµL lysate, flow through, wash and purified protein
ladder: triple color protein standard (Serva)

TECAN-reader measurement

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: ligand_mCherry fusion protein characterization

Procedure:

Texas Red standard

2.5 ÂµM, 1 ÂµM, 0.5 ÂµM, 0.25 ÂµM, 0.1 ÂµM

protein dilution series

0.5 ÂµM, 0.25 ÂµM, 0.1 ÂµM, 0.05 ÂµM, 0.025 ÂµM, 0.01 ÂµM

measurement of fluorescence intensity of dilution series (quadruplicates)

TECAN reader, gain: calculated from well with 2.5 ÂµM Texas Red, excitation: 570 nm, emission: 610 nm

measurement of emission spectrum (600 nm to 850 nm) and absorption spectrum (300 nm to 850 nm)

overnight culture

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: ligand_mCherry fusion protein expression

Procedure:

overnight culture (25 mL LB-Amp, 37 Â°C) of BBa_K2926068 in pTXB1 in E. coli ER2566

protein expression

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: ligand_mCherry fusion protein expression

Procedure:

standard expression protocol: expression culture (250 mL LB-Amp, 37 Â°C) of BBa_K2926068 in pTXB1 in E. coli ER2566

start OD: 0.1
induction (0.4 mM IPTG) at OD 0.6-0.8
30 min 37 Â°C
17 Â°C over night

cell harvest, cell lysis, IMPACT purification

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: ligand_mCherry fusion protein expression

Procedure:

cell harvest (4 Â°C, 4500 rpm, 20 min)
cell mass:

BBa_K2926068: 1.57 g

storage at -80 Â°C

Pre culture

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Endocytosis assay A. niger

Procedure:

100 ÂµL of A. niger culture transferred to 10 mL minimal media

preparations for MALDI-ToF

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Protein characterization

Procedure:

isolation of desired protein bonds from SDS-PAGEs in pre-washed reaction tubes

2019-10-07 - 2019-10-13

preparations for MALDI-ToF

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Protein characterization

Procedure:

washing and tryptic digestion of SDS-PAGE protein bonds

MALDI-Tof

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Protein characterization

Procedure:

measurement of tryptic digested fusion proteins and mCherry in MALDI-ToF

Endocytosis assay A. niger

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Endocytosis assay A. niger

Procedure:

800 ÂµL A. niger culture in minimal media incubated with 0.5 ÂµM Pro_mCherry or mCherry (30 Â°C, 450 rpm, dark)
negative control: 800 ÂµL minimal media with 0.5 ÂµM Pro_mCherry or mCherry
measurement of fluorescence intensity in the supernatant after 0 min and 60 min

centrifugation of 400 ÂµL mL culture (1 min, 12 000 rpm)
measurement of fluorescence in supernatant (TECAN reader, gain: calculated from well with 2.5 ÂµM Teas Red, excitation: 570 nm, emission: 610 nm)

Endocytosis assay A. niger

Team: Endocytosis

Investigators: Johanna, Opgenoorth

Superior experiment: Endocytosis assay A. niger

Procedure:

1 mL A. niger culture in minimal media incubated with 0.5 ÂµM Mat_mCherry (30 Â°C, 450 rpm, dark)
negative control: 1 mL minimal media with 0.5 ÂµM Mat_mCherry
measurement of fluorescence intensity in the supernatant after 0 min and 60 min

centrifugation of 400 ÂµL mL culture (1 min, 12 000 rpm)
measurement of fluorescence in supernatant (TECAN reader, gain: calculated from well with 2.5 ÂµM Teas Red, excitation: 570 nm, emission: 610 nm)

Team:Bielefeld-CeBiTec/Notebook

2019-04-15 - 2019-04-21

- Plasmids pTXB1 and BBa_I746909 streaked out on agar plates

2019-04-22 - 2019-04-28

Chemical transformation

Overnight cultures to purify plasmid-DNA

Plasmid isolation

2019-04-29 - 2019-05-05

Generation of mCherry-fragments, gel extraction

2019-05-06 - 2019-05-12

Generation of mCherry-fragments, gel extraction, Gibson-Assembly, chemical Transformation

Overnight cultures to amplify BBa_K2926048 and BBa_J06504 in pTXB1

Plasmid isolation of BBa_K2926048 and BBa_J06504 in pTXB1, Sanger Sequencing

Transformation of E. coli DH5ÃÂ± with Cas13a

2019-05-13 - 2019-05-19

Transformation of E. coli DH5ÃÂ± with sfGFP

2019-05-20 - 2019-05-26

Preparation: Sequencing

Overnight culture of BBa_K2926048 and BBa_J06504 in pTXB1

Glycerine culture and plasmid isolation of BBa_K2926048 (Cm) and BBa_J06504 in pTXB1

generating gibson fragments via PCR, PCR-cleanup, analytic agarose gel, Gibson Assembly, Transformation via heatshock

PCR-clean up: Cas13a and P8 Ptx (Johanna, 24.5.19)

2019-05-27 - 2019-06-02

Colony-PCR, overnight culture of positive colonies

Plasmid isolation, Sanger Sequencing

Generating Gibson fragments via PCR and primer-annealing, fill-in of 5' overhangs

PCR-Cleanup of gibson fragments

Transformation via heatshock

2019-06-03 - 2019-06-09

Gel-electrophoresis

Generating Gibson fragments via PCR, template digestion (DpnI)

PCR-Cleanup of gibson fragments, Gibson Assembly, transformation via heatshock

Colony-PCR: pSB1C3-Basic Insert-Cas13a

Colony-PCR of BBa_K2926055 and BBa_K2926058

Pre-cultures

2019-06-10 - 2019-06-16

Expression cultures

Gibson Assembly, transformation via heatshock, colony PCR

gradient PCR to determine the optimal annealing temperature for the first add-on PCR

Overnight cultures of BBa_K2926055 and BBa_K2926058

Cell harvest

Plasmid isolation of BBa_K2926055 and BBa_K2926058, colony PCR of BBa_K2926054 and BBa_K2926057

First add-on PCR, analytic agarose gel

2019-06-17 - 2019-06-23

PCR cleanup, second add-on PCR, analytic agarose gel

overnight cultures of BBa_K2926054 and BBa_K2926057

Chemical transformation

Gradient PCR (second add-on PCR for BBa_K2926056)

Plasmid isolation of BBa_K2926054 and BBa_K2926058

Gradient PCR (second add-on PCR for BBa_K2926067), first add-on PCR

Analytical agarose gel of PCR products

2019-06-24 - 2019-06-30

Cell lysis, Protein purification

PCR cleanup, analytical agarose gel

IMPACT elution, bradford assay

Expression pre-cultures

Expression cultures

second add-on PCR, analytical agarose gel,PCR cleanup, Gibson assembly, transformation via heatshock

Cell harvest

SDS-PAGE of mCherry purification process

Colony PCR, analytical agarose gel

PCR f1 ori

PCR mCherry V1

PCR mCherry V2

PCR VIII

PCR III

PCR pSB1C3

Geleelectrophoresis all fragments

Gel Clean-Up all fragments

Overnight culture

2019-07-01 - 2019-07-07

Gibson Assembly Troygenic DNA V1

Transformation Troygenic DNA V1

Gibson Assembly Troygenic DNA V2

Transformation Troygenic DNA V2

Colony PCR Troygenic DNA V1

Colony PCR Troygenic DNA V2

Plasmid isolation

Establishing a fluorescence measurement method

PCR VIII

Transformation of E. coli DH5ÃÂ± with Cas13a

Transformation of E. coli DH5ÃÂ± with sfGFP