Team:Bielefeld-CeBiTec/Notebook

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 DH5α 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 μl sterile H2O (final concentration: 20 ng/µl); 10 min on 37ºC
  • 200 µl competent E. coli DH5α (Ina, 6.5.19) + 1 µl DNA (Cas13a) -> 30 min: ice
  • 1 min 42°C
  • 5 min ice
  • + 500 µl 37°C warm SOC-Medium
  • 1 h 37ºC
  • 2019-05-13 - 2019-05-19

    Transformation of E. coli DH5α with sfGFP

    Team: CeDIS

    Investigators: Isabel, Conze

    Superior experiment: Labplasmid

    Procedure:

  • In the Parts Registry:
  • 2-3 ng DNA
  • +10 µl sterile MQ --> Mix by pipetting
  • Sit for 5 min
  • DNA: sfGPF (BBa_K1321337) (c=2 ng/10µl --> 0.2 ng/µl)
  • 200 µl competent E. coli DH5α (Ina, date: 6.5.19) + 1 µl DNA (0.2 ng) -> 30 min: ice
  • 1 min 42°C
  • 5 min ice
  • + 500 µl 37°C warm SOC-Medium
  • 1 h 37ºC
    • > Centrifuge (1 min, 9000 rpm = 7600 rcf);
  • Plate cell pellet on: LB-Cm
  • Put plates at 37°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 µl PCR-product + 4 µl Purple Dye (NEB Biolabs) -> 10 µl in gel
  • 1 kb ladder (NEB) -> 5 µl in gel
  • Staining: 5 min in ethidium bromide
  • Precultures for Plasmid-Prep & Sequencing:
  • 10 ml LB + 10 µl Amp -> LB-Amp
  • 10 ml LB + 10 µl Cm (34 mg ml-1) -> LB-Cm (cCm=34 µg ml-1)
  • 5 ml LB-Amp + E. coli DH5α (p2CT-His-MBP-Lsh-C13a-WT) Colony 2
  • 5 ml LB-Amp + E. coli DH5α (p2CT-His-MBP-Lbu-C13a-WT) Colony 6
  • 5 ml LB-Cm + E. coli DH5α (pSB1C3-His-SUMO-Lwa-C13a-WT) Colony 5
  • 5 ml LB-Cm + E. coli DH5α (pSB1C3-sfGFP) Colony +
    • At 37°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, Többer

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

    Procedure:

    • 350 µL Glycerine + 650 µL overnight culture
    • - 80 °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 µl Lwa, Lbu, Lsh A
    • c(Lwa) = 35,6 ng µl-1
    • c(Lbu) = 15 ng µl-1
    • c(Lsh) = 16,8 ng µl-1
  • 90 µl Lwa, Lbu, Lsh B
  • 110 µl P8 Ptx (Johanna, 24.5.19.)
  • 110 µl 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 °C
      • cool down to room temperature: 0.5 °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, 58°C) & pSB1C3_BasicI_r (19av, 58°C)
  • Fragment size: 3412 bp
  • Polymerase: Q5 (NEB)
  • Volume: 100 µl
  • 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 °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 DH5α (pSB1C3-Basic_Insert_Lwa)
  • Primer: VR (57°C) & Lwa_seq_5 (19ce, 59°C)
  • Fragment size: 2506 bp
  • Polymerase: GoTaq Hot Start (Promega)
  • Volume: 10 µl 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 °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 °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 °C
      • 17 °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 °C to 65 °C, 10 different temperatures
      • future annealing temperature: 64 °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 °C, 4500 rpm, 20 min)
    • cell mass:
      • BBa_J06504: 1.92 g
      • BBa_K2926048: 1.63 g
    • storage of the pellet at -80 °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 °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 °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 °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 °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 °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, 4°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 µg mCherry (131.33 µg/mL), 692.2 µg mCherryHis (76.9 µg/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 °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 °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 °C
      • 21 °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 °C, 4500 rpm, 20 min)
    • storage of the pellet at -80 °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 µL 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, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Primers:
      • f1 ori pSB1C3 GA f
      • f1 ori mC Prom GA r
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 5s
      • 59°C 20s
      • 72°C 15s
      • 72°C 2min
      • 4°C

    PCR mCherry V1

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Template: iGEM mCherry
    • Primers:
      • mC f1 ori RBS GA f
      • mC VIII GA r
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 5s
      • 59°C 20s
      • 72°C 15s
      • 72°C 2min
      • 4°C

    PCR mCherry V2

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Template: iGEM mCherry
    • Primers:
      • mC f1 ori RBS GA f
      • mC pSB1C3 GA r
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 5s
      • 59°C 20s
      • 72°C 15s
      • 72°C 2min
      • 4°C

    PCR VIII

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Template: M13K07 NEB
    • Primers:
      • VIII mC GA f
      • VIII III GA r
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 5s
      • 60°C 10s
      • 72°C 10s
      • 72°C 2min
      • 4°C

    PCR III

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Template: M13K07 NEB
    • Primers:
      • III VIII GA f
      • III pSB1C3 GA r
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 5s
      • 59°C 15s
      • 72°C 15s
      • 72°C 2min
      • 4°C

    PCR pSB1C3

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Template: iGEM pSB1C3
    • Primers:
      • pSB3K3 split f
      • pSB3K3 split r
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 10s
      • 61°C 25s
      • 72°C 45s
      • 72°C 2min
      • 4°C

    Geleelectrophoresis all fragments

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    Gel Clean-Up all fragments

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    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, Többer

    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, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • DH5a

    Gibson Assembly Troygenic DNA V2

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Gibson Master Mix
    • pSB1C3
    • f1 ori
    • mCherry V2

    Transformation Troygenic DNA V2

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • DH5a

    Colony PCR Troygenic DNA V1

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Primers:
      • VF
      • VR
    • OneTaq Polymerase
      • 95°C 30s
      • 95°C 25s
      • 57°C 45s
      • 68°C 2:20min
      • 68°C 5min
      • 4°C

    Colony PCR Troygenic DNA V2

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Primers:
      • VF
      • VR
    • OneTaq Polymerase
      • 95°C 30s
      • 95°C 20s
      • 57°C 40s
      • 68°C 1:35min
      • 68°C 5min
      • 4°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 µL of each dilution (triplicates)
    • excitation: 570 nm, emission: 610 nm
    • gain: manual (100)

    PCR VIII

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Template: M13K07 NEB
    • Primers:
      • VIII mC GA f
      • VIII III GA r
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 5s
      • 60°C 10s
      • 72°C 10s
      • 72°C 2min
      • 4°C

    PCR III

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Template: M13K07 NEB
    • Primers:
      • III VIII GA f
      • III pSB1C3 GA r
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 5s
      • 59°C 15s
      • 72°C 15s
      • 72°C 2min
      • 4°C

    Geleelectrophoresis all fragments

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    Gel Clean-Up all fragments

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Monarch DNA Gel Extractions

    Gibson Assembly Troygenic DNA V1

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    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, Többer

    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 µL)
    • 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, Többer

    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, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • DH5a

    Gibson Assembly Troygenic DNA V2

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Gibson Master Mix
    • pSB1C3
    • f1 ori
    • mCherry V2

    Transformation Troygenic DNA V2

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • DH5a

    Overlap-PCR f1 ori - mCherry

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Templates: f1 ori, mCherry
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 10s
      • 70°C 20s
      • 72°C 35s
      • 72°C 2min
      • 4°C
    • Primers:
      • f1 ori pSB1C3 GA f
      • mC VIII GA r
      • 98°C 30s
      • 98°C 10s
      • 60°C 20s
      • 72°C 35s
      • 72°C 2min
      • 4°C

    Overlap-PCR VIII - III

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Templates: VIII, III
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 8s
      • 72°C 20s
      • 72°C 15s
      • 72°C 2min
      • 4°C
    • Primers:
      • VIII mC GA f
      • III pSB1C3 GA r
      • 98°C 30s
      • 98°C 8s
      • 59°C 20s
      • 72°C 15s
      • 72°C 2min
      • 4°C

    Geleelectrophoresis Overlap-PCRs

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    Gel Clean-Up Overlap-PCRs

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Monarch DNA Gel Extractions

    PCR III

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Template: M13K07 NEB
    • Primers:
      • III VIII GA f
      • III pSB1C3 GA r
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 5s
      • 59°C 15s
      • 72°C 15s
      • 72°C 2min
      • 4°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, Többer

    Superior experiment: Helperphage construction

    Procedure:

    • Template: iGEM pSB3K3
    • Primers:
      • pSB3K3 split f
      • pSB3K3 split r
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 10s
      • 61°C 25s
      • 72°C 1:10min
      • 72°C 2min
      • 4°C

    PCR Terminator V1

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Helperphage construction

    Procedure:

    • Template: M13K07 NEB
    • Primers:
      • M13 PCR f
      • M13 Term GA r
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 5s
      • 59°C 15s
      • 72°C 3s
      • 72°C 2min
      • 4°C

    PCR Terminator V2

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Helperphage construction

    Procedure:

    • Template: M13K07 NEB
    • Primers:
      • M13 PCR f
      • M13 Term GA r
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 5s
      • 59°C 15s
      • 72°C 3s
      • 72°C 2min
      • 4°C

    PCR f1 ori

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Primers:
      • f1 ori pSB1C3 GA f
      • f1 ori mC Prom GA r
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 5s
      • 59°C 20s
      • 72°C 15s
      • 72°C 2min
      • 4°C

    PCR mCherry

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Template: iGEM mCherry
    • Primers:
      • mC f1 ori RBS GA f
      • mC VIII GA r
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 5s
      • 59°C 20s
      • 72°C 15s
      • 72°C 2min
      • 4°C

    PCR f1 ori Overlap

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Primers:
      • f1 1C3 OP f
      • f1 mC Prom OP r
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 5s
      • 59°C 20s
      • 72°C 15s
      • 72°C 2min
      • 4°C

    PCR mCherry Overlap

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Template: iGEM mCherry
    • Primers:
      • mC ori OP f
      • mC VIII OP r
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 5s
      • 59°C 20s
      • 72°C 15s
      • 72°C 2min
      • 4°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, Többer

    Superior experiment: Helperphage construction

    Procedure:

    • Template: M13K07 NEB
    • Primers:
      • M13 pSB3K3 f1
      • M13 Prom GA r
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 8s
      • 56°C 25s
      • 72°C 45s
      • 72°C 2min
      • 4°C

    Overlap-PCR f1 ori - mCherry

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Templates: f1 ori Overlap, mCherry Overlap
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 10s
      • 70°C 20s
      • 72°C 35s
      • 72°C 2min
      • 4°C
    • Primers:
      • f1 1C3 OP f
      • mC VIII OP r
      • 98°C 30s
      • 98°C 10s
      • 60°C 20s
      • 72°C 35s
      • 72°C 2min
      • 4°C

    Geleelectrophoresis all fragments

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    PCR III-IV

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Helperphage construction

    Procedure:

    • Template: M13K07 NEB
    • Primers:
      • III VIII GA f
      • M13 pSB3K3 r2
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 10s
      • 57°C 25s
      • 72°C 1:15min
      • 72°C 2min
      • 4°C

    Geleelectrophoresis Overlap f1-mCherry, III-IV

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    Gel Clean-Up all fragments

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Monarch DNA Gel Extractions

    Gibson Assembly Helperphage V1

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Helperphage construction

    Procedure:

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

    Transformation Helperphage V1

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Helperphage construction

    Procedure:

    • DH5a

    Gibson Assembly Helperphage V2

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Helperphage construction

    Procedure:

    • Gibson Master Mix
    • pSB3K3
    • II-VIII
    • Terminator V2

    Transformation Helperphage V2

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Helperphage construction

    Procedure:

    • DH5a

    Gibson Assembly Troygenic DNA Overlap

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Gibson Master Mix
    • pSB1C3
    • f1 ori-mCherry
    • VIII-III

    Transformation Troygenic DNA Overlap

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    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 °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 °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 °C
      • RT over night

    Overlap-PCR f1 ori - mCherry

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Templates: f1 ori Overlap, mCherry Overlap
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 8s
      • 70°C 20s
      • 72°C 30s
      • 72°C 2min
      • 4°C
    • Primers:
      • f1 ori pSB1C3 GA f
      • mC VIII GA r
      • 98°C 30s
      • 98°C 8s
      • 60°C 20s
      • 72°C 30s
      • 72°C 2min
      • 4°C

    Overlap-PCR VIII - III

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Templates: VIII, III
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 8s
      • 70°C 20s
      • 72°C 30s
      • 72°C 2min
      • 4°C
    • Primers:
      • VIII mC GA f
      • III pSB1C3 GA r
      • 98°C 30s
      • 98°C 8s
      • 59°C 20s
      • 72°C 30s
      • 72°C 2min
      • 4°C

    PCR VIII Overlap

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Template: M13K07 NEB
    • Primers:
      • VIII mC OP f
      • VIII III OP r
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 5s
      • 59°C 15s
      • 72°C 10s
      • 72°C 2min
      • 4°C

    PCR III Overlap

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Template: M13K07 NEB
    • Primers:
      • III VIII OP f
      • III 1C3 OP r
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 5s
      • 59°C 15s
      • 72°C 10s
      • 72°C 2min
      • 4°C

    PCR pSB3K3

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Helperphage construction

    Procedure:

    • Template: iGEM pSB3K3
    • Primers:
      • pSB3K3 split f
      • pSB3K3 split r
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 10s
      • 52°C 25s
      • 72°C 1:15min
      • 72°C 2min
      • 4°C

    PCR II-VIII

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Helperphage construction

    Procedure:

    • Template: M13K07 NEB
    • Primers:
      • M13 pSB3K3 f1
      • M13 Prom GA r
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 10s
      • 56°C 25s
      • 72°C 45s
      • 72°C 2min
      • 4°C

    PCR III-IV

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Helperphage construction

    Procedure:

    • Template: M13K07 NEB
    • Primers:
      • III VIII GA f
      • M13 pSB3K3 r2
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 10s
      • 52°C 25s
      • 72°C 1:15min
      • 72°C 2min
      • 4°C

    Overlap-PCR f1 ori - mCherry

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Templates: f1 ori Overlap, mCherry Overlap
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 10s
      • 70°C 20s
      • 72°C 35s
      • 72°C 2min
      • 4°C
    • Primers:
      • f1 ori pSB1C3 OP f
      • mC VIII OP r
      • 98°C 30s
      • 98°C 10s
      • 60°C 20s
      • 72°C 35s
      • 72°C 2min
      • 4°C

    Overlap-PCR VIII - III

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Templates: VIII Overlap, III Overlap
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 8s
      • 72°C 20s
      • 72°C 15s
      • 72°C 2min
      • 4°C
    • Primers:
      • VIII mC OP f
      • III 1C3 OP r
      • 98°C 30s
      • 98°C 8s
      • 59°C 20s
      • 72°C 15s
      • 72°C 2min
      • 4°C

    Cell harvest

    Team: Endocytosis

    Investigators: Johanna, Opgenoorth

    Superior experiment: mCherry protein expression

    Procedure:

    • cell harvest (4 °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 °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 °C) of BBa_K2926067

    Geleelectrophoresis all fragments

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    Transformation pSB1C3 from iGEM plate

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    Transformation pSB3K3 from iGEM plate

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Helperphage construction

    Procedure:

    Gradient-PCR f1 ori

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Primers:
      • f1 ori pSB1C3 GA f
      • f1 ori mC Prom GA r
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 5s
      • 50-60°C 15s
      • 72°C 20s
      • 72°C 2min
      • 4°C

    PCR f1 ori Overlap

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Primers:
      • f1 1C3 OP f
      • f1 mC Prom OP r
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 5s
      • 59°C 15s
      • 72°C 20s
      • 72°C 2min
      • 4°C

    PCR mCherry

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Template: iGEM mCherry
    • Primers:
      • mC f1 ori RBS GA f
      • mC VIII GA r
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 5s
      • 59°C 15s
      • 72°C 20s
      • 72°C 2min
      • 4°C

    Gradient-PCR mCherry Overlap

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Template: iGEM mCherry
    • Primers:
      • mC ori OP f
      • mC VIII OP r
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 5s
      • 50-60°C 15s
      • 72°C 20s
      • 72°C 2min
      • 4°C

    PCR III

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Template: M13K07 NEB
    • Primers:
      • III VIII GA f
      • III pSB1C3 GA r
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 5s
      • 59°C 15s
      • 72°C 20s
      • 72°C 2min
      • 4°C

    PCR III Overlap

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Template: M13K07 NEB
    • Primers:
      • III VIII OP f
      • III 1C3 OP r
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 5s
      • 59°C 15s
      • 72°C 20s
      • 72°C 2min
      • 4°C

    Gradient-PCR pSB1C3

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Template: iGEM pSB1C3
    • Primers:
      • pSB3K3 split f
      • pSB3K3 split r
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 10s
      • 50-60°C 25s
      • 72°C 45s
      • 72°C 2min
      • 4°C

    Gradient-PCR II-VIII

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Helperphage construction

    Procedure:

    • Template: M13K07 NEB
    • Primers:
      • M13 pSB3K3 f1
      • M13 Prom GA r
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 10s
      • 50-60°C 25s
      • 72°C 45s
      • 72°C 2min
      • 4°C

    PCR VIII

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Template: M13K07 NEB
    • Primers:
      • VIII mC GA f
      • VIII III GA r
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 5s
      • 60°C 10s
      • 72°C 10s
      • 72°C 2min
      • 4°C

    Gradient-PCR VIII Overlap

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Template: M13K07 NEB
    • Primers:
      • VIII mC OP f
      • VIII III OP r
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 5s
      • 59-70°C 10s
      • 72°C 10s
      • 72°C 2min
      • 4°C

    PCR Terminator

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Helperphage construction

    Procedure:

    • Template: M13K07 NEB
    • Primers:
      • M13 PCR f
      • M13 Term GA r
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 5s
      • 59°C 10s
      • 72°C 10s
      • 72°C 2min
      • 4°C

    Gradient-PCR pSB3K3

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Helperphage construction

    Procedure:

    • Template: iGEM pSB3K3
    • Primers:
      • pSB3K3 split f
      • pSB3K3 split r
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 10s
      • 59°C 30s
      • 72°C 1:10min
      • 72°C 2min
      • 4°C

    Gradient-PCR III-IV

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Helperphage construction

    Procedure:

    • Template: M13K07 NEB
    • Primers:
      • III VIII GA f
      • M13 pSB3K3 r2
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 10s
      • 52°C 30s
      • 72°C 1:15min
      • 72°C 2min
      • 4°C

    Gradient-Overlap-PCR f1 ori - mCherry

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Templates: f1 ori, mCherry
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 8s
      • 50°C 20s
      • 72°C 35s
      • 72°C 2min
      • 4°C
    • Primers:
      • f1 ori pSB1C3 GA f
      • mC VIII GA r
      • 98°C 30s
      • 98°C 8s
      • 50-70°C 20s
      • 72°C 35s
      • 72°C 2min
      • 4°C

    Gradient-Overlap-PCR VIII - III

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Templates: VIII, III
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 5s
      • 50°C 15s
      • 72°C 15s
      • 72°C 2min
      • 4°C
    • Primers:
      • VIII mC GA f
      • III pSB1C3 GA r
      • 98°C 30s
      • 98°C 5s
      • 59-70°C 15s
      • 72°C 15s
      • 72°C 2min
      • 4°C

    Geleelectrophoresis all fragments

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    PCR Clean-Up all fragments

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Promega Wizard SV Gel and PCR Clean-Up System

    digest dnpI all fragments

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    - standard expression protocol: expression culture (250 mL LB, 37 °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 °C
      • 17 °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 °C over night)

    Gel Clean-Up all fragments

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Monarch DNA Gel Extractions

    PCR Clean-Up all fragments

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    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 °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 °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, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Primers:
      • f1 ori pSB1C3 GA f
      • f1 ori mC Prom GA r
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 5s
      • 51°C 15s
      • 72°C 20s
      • 72°C 2min
      • 4°C

    PCR mCherry

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Template: iGEM mCherry
    • Primers:
      • mC f1 ori RBS GA f
      • mC VIII GA r
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 5s
      • 59°C 15s
      • 72°C 20s
      • 72°C 2min
      • 4°C

    PCR mCherry Overlap

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Template: iGEM mCherry
    • Primers:
      • mC ori OP f
      • mC VIII OP r
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 5s
      • 55°C 15s
      • 72°C 20s
      • 72°C 2min
      • 4°C

    PCR III Overlap

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Template: M13K07 NEB
    • Primers:
      • III VIII OP f
      • III 1C3 OP r
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 5s
      • 59°C 15s
      • 72°C 20s
      • 72°C 2min
      • 4°C

    PCR pSB1C3

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Template: iGEM pSB1C3
    • Primers:
      • pSB3K3 split f
      • pSB3K3 split r
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 10s
      • 53°C 25s
      • 72°C 45s
      • 72°C 2min
      • 4°C

    PCR II-VIII

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Helperphage construction

    Procedure:

    • Template: M13K07 NEB
    • Primers:
      • M13 pSB3K3 f1
      • M13 Prom GA r
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 10s
      • 58°C 25s
      • 72°C 45s
      • 72°C 2min
      • 4°C

    PCR VIII Overlap

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Template: M13K07 NEB
    • Primers:
      • VIII mC OP f
      • VIII III OP r
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 5s
      • 66°C 10s
      • 72°C 10s
      • 72°C 2min
      • 4°C

    PCR pSB3K3

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Helperphage construction

    Procedure:

    • Template: iGEM pSB3K3
    • Primers:
      • pSB3K3 split f
      • pSB3K3 split r
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 10s
      • 59°C 30s
      • 70°C 1:10min
      • 72°C 2min
      • 4°C

    PCR III-IV

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Helperphage construction

    Procedure:

    • Template: M13K07 NEB
    • Primers:
      • III VIII GA f
      • M13 pSB3K3 r2
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 10s
      • 52°C 30s
      • 72°C 1:15min
      • 72°C 2min
      • 4°C

    Geleelectrophoresis all fragments

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    Gel Clean-Up all fragments

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Monarch DNA Gel Extractions

    PCR Clean-Up all fragments

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Promega Wizard SV Gel and PCR Clean-Up System

    Gibson Assembly Helperphage

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Helperphage construction

    Procedure:

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

    Transformation Helperphage

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Helperphage construction

    Procedure:

    • DH5a

    Gibson Assembly Troygenic DNA

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Gibson Master Mix
    • pSB1C3
    • f1 ori
    • mCherry
    • VIII
    • III

    Transformation Troygenic DNA

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    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, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Template: iGEM mCherry
    • Primers:
      • mC f1 ori RBS GA f
      • mC VIII GA r
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 5s
      • 59°C 15s
      • 72°C 20s
      • 72°C 2min
      • 4°C

    PCR mCherry Overlap

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Template: iGEM mCherry
    • Primers:
      • mC ori OP f
      • mC VIII OP r
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 5s
      • 55°C 15s
      • 72°C 20s
      • 72°C 2min
      • 4°C

    Geleelectrophoresis mCherry

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    Colony-PCR pSB1C3

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • GoTaq G2 DNA Polymerase

    Colony-PCR pSB3K3

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Helperphage construction

    Procedure:

    • GoTaq G2 DNA Polymerase

    Colony-PCR Helperphage V1

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Helperphage construction

    Procedure:

    • GoTaq G2 DNA Polymerase

    Colony-PCR Helperphage V2

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Helperphage construction

    Procedure:

    • GoTaq G2 DNA Polymerase

    Gradient-Overlap-PCR f1 ori - mCherry

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Templates: f1 ori, mCherry
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 8s
      • 50°C 20s
      • 72°C 35s
      • 72°C 2min
      • 4°C
    • Primers:
      • f1 ori pSB1C3 GA f
      • mC VIII GA r
      • 98°C 30s
      • 98°C 8s
      • 58-70°C 20s
      • 72°C 35s
      • 72°C 2min
      • 4°C

    Gradient-Overlap-PCR VIII - III

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Templates: VIII, III
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 5s
      • 50°C 15s
      • 72°C 15s
      • 72°C 2min
      • 4°C
    • Primers:
      • VIII mC GA f
      • III pSB1C3 GA r
      • 98°C 30s
      • 98°C 5s
      • 50-70°C 15s
      • 72°C 15s
      • 72°C 2min
      • 4°C

    Geleelectrophoresis all fragments

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Helperphage construction

    Procedure:

    PCR Clean-Up mCherry

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Promega Wizard SV Gel and PCR Clean-Up System

    Gel Clean-Up mCherry

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    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 °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 °C

    Gibson Assembly Troygenic DNA

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Gibson Master Mix
    • pSB1C3
    • f1 ori
    • mCherry
    • VIII
    • III

    Transformation Troygenic DNA

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • DH5a

    Overlap-PCR f1 ori - mCherry

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Templates: f1 ori, mCherry
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 8s
      • 50°C 20s
      • 72°C 35s
      • 72°C 2min
      • 4°C
    • Primers:
      • f1 ori pSB1C3 GA f
      • mC VIII GA r
      • 98°C 30s
      • 98°C 8s
      • 61°C 20s
      • 72°C 35s
      • 72°C 2min
      • 4°C

    Overlap-PCR VIII - III

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Templates: VIII, III
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 5s
      • 50°C 15s
      • 72°C 15s
      • 72°C 2min
      • 4°C
    • Primers:
      • VIII mC GA f
      • III pSB1C3 GA r
      • 98°C 30s
      • 98°C 5s
      • 53°C 15s
      • 72°C 15s
      • 72°C 2min
      • 4°C

    Overlap-PCR f1 ori - mCherry

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Templates: f1 ori Overlap, mCherry Overlap
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 10s
      • 70°C 20s
      • 72°C 35s
      • 72°C 2min
      • 4°C
    • Primers:
      • f1 ori pSB1C3 OP f
      • mC VIII OP r
      • 98°C 30s
      • 98°C 10s
      • 61°C 20s
      • 72°C 35s
      • 72°C 2min
      • 4°C

    Overlap-PCR VIII - III

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Templates: VIII Overlap, III Overlap
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 8s
      • 72°C 20s
      • 72°C 15s
      • 72°C 2min
      • 4°C
    • Primers:
      • VIII mC OP f
      • III 1C3 OP r
      • 98°C 30s
      • 98°C 8s
      • 53°C 20s
      • 72°C 15s
      • 72°C 2min
      • 4°C

    Colony-PCR pSB1C3

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    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/µL; 125.85 µg
      • Flo_mCherry_p8: 2.86 ng/µL; 7.74 µg
      • Mat_mCherry_p8: 2.45 ng/µL; 6.53 µg
    • standard SDS-PAGE protocol (12 %)
      • analysis of 10 µL 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 µM)
    • measurement of fluorescence intensity of mCherry dilution series and fusion proteins (200 µL)
      • 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, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

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

    Dilution series II-VIII

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Helperphage construction

    Procedure:

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

    Dilution series pSB1C3

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

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

    Dilution series pSB3K3

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Helperphage construction

    Procedure:

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

    Transformation pSB3K3 from iGEM plate

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Helperphage construction

    Procedure:

    • 10µl Kanamycin on plate

    Inoculation pSB1C3

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    PCR Clean-Up all fragments

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    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, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Promega Wizard SV Gel and PCR Clean-Up System

    Inoculation pSB3K3

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Helperphage construction

    Procedure:

    Geleelectrophoresis all fragments

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    PCR Clean-Up pSB3K3

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Helperphage construction

    Procedure:

    • Promega Wizard SV Gel and PCR Clean-Up System

    Gel Clean-Up pSB3K3

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Promega Wizard SV Gel and PCR Clean-Up System

    Plasmid Miniprep pSB1C3

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • ZymoPURE Plasmid Miniprep

    PCR mCherry

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Template: iGEM mCherry
    • Primers:
      • mC f1 ori RBS GA f
      • mC VIII GA r
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 5s
      • 55°C 20s
      • 72°C 20s
      • 72°C 2min
      • 4°C

    PCR mCherry Overlap

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    • Template: iGEM mCherry
    • Primers:
      • mC ori OP f
      • mC VIII OP r
    • Q5 Polymerase
      • 98°C 30s
      • 98°C 5s
      • 55°C 20s
      • 72°C 20s
      • 72°C 2min
      • 4°C

    PCR Clean-Up mCherry

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Helperphage construction

    Procedure:

    • Promega Wizard SV Gel and PCR Clean-Up System

    PCR pSB1C3

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

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

    Geleelectrophoresis mCherry

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    Superior experiment: Troygenic DNA construction

    Procedure:

    Gel Clean-Up mCherry

    Team: Troygenic Assembly

    Investigators: Astrid, Többer

    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 °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, Többer

    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 °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 °C
      • 17 °C over night

    Cell harvest

    Team: Endocytosis

    Investigators: Johanna, Opgenoorth

    Superior experiment: mCherry and ligand_mCherry_p8 protein expression

    Procedure:

    • cell harvest (4 °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 °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 °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/µL; 1.43 mg
      • mCherryHis: 14.15 ng/µL; 45.28 µg
      • Flo_mCherry_p8: 6.68 ng/µL; 8 µg
      • Mat_mCherry_p8: 10 ng/µL; 13.6 µg
    • standard SDS-PAGE protocol (12 %)
      • analysis of 10 µL 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 °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 °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 °C
      • 17 °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 µM, 1 µM, 0.5 µM, 0.25 µM, 0.1 µM
    • protein dilution series
      • 0.5 µM, 0.25 µM, 0.1 µM, 0.05 µM, 0.025 µM, 0.01 µM
    • 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 °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 °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 µL 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 µM, 1 µM, 0.5 µM, 0.25 µM, 0.1 µM
    • protein dilution series
      • 0.5 µM, 0.25 µM, 0.1 µM, 0.05 µM, 0.025 µM, 0.01 µM
    • 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 °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 °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 °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 °C
      • 17 °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 °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 °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 °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/µL; 503 µg
      • Mat_mCherry: 445.71 ng/µL; 356.57 µg
      • Opy_mCherry: 196.95 ng/µL; 236.38 µg
    • standard SDS-PAGE protocol (12 %)
      • analysis of 10 µL 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 µM, 1 µM, 0.5 µM, 0.25 µM, 0.1 µM
    • protein dilution series
      • 0.5 µM, 0.25 µM, 0.1 µM, 0.05 µM, 0.025 µM, 0.01 µM
    • measurement of fluorescence intensity of dilution series (quadruplicates)
      • TECAN reader, gain: calculated from well with 2.5 µM, 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 µM 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 µM Texas Red, excitation: 570 nm, emission: 610 nm
    • pH stability assay
      • 0.2 µM 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 µM 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 °C)

    Pre culture

    Team: Endocytosis

    Investigators: Johanna, Opgenoorth

    Superior experiment: Endocytosis assay S. cerevisiae

    Procedure:

    • overnight culture of S. cerevisiae (10 mL YPD, 30 °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 °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 µM)
    • negative control: every fusion protein in minimal media
    • incubation (30 °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 µM 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 °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 µL YPD
    • addition of fusion protein (0.2 µM)
    • incubation (15 min, 30 °C, 450 rpm, dark)
    • centrifugation (1 min, 12 000 rpm, RT)
    • resuspended in 10 µL minimal media
    • fluorescence microscopy of 5 µL (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 °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 µM)
    • negative control: every fusion protein in minimal media
    • incubation (30 °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 µM 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 °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 µL SD media
      • addition of fusion protein (1 µM)
      • incubation (15 min, 30 °C, 450 rpm, dark)
      • centrifugation (1 min, 12 000 rpm, RT)
      • washing step (20 µL PBS, centrifugation 1 min, 12 000 rpm, RT)
      • resuspended in 10 µL minimal media
      • fluorescence microscopy of 5 µL (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 °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 °C, 450 rpm, dark)
      • centrifugation (1 min, 12 000 rpm, RT)
      • washing step (20 µL spheroblast buffer, centrifugation 1 min, 12 000 rpm, RT)
      • resuspended in 10 µL spheroblast buffer
      • fluorescence microscopy of 5 µL (LSM 700 (Zeiss), magnification: 100x, Texas Red [?(ex)=555 nm, ?(Em)=570 nm to 800 nm])
    • trying out higher concentrated proteins
      • resuspension in 60 µL YPD
      • addition of fusion protein (5 µM)
      • incubation (15 min, 30 °C, 450 rpm, dark)
      • centrifugation (1 min, 12 000 rpm, RT)
      • washing step (20 µL PBS, centrifugation 1 min, 12 000 rpm, RT)
      • resuspended in 10 µL minimal media
      • fluorescence microscopy of 5 µL (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 µM and 2 µM, Mat_mCherry and Opy_mCherry, 0 min, 15 min, 30 min, 45 min)
    • centrifugation (1 min, 12 000 rpm, RT)
    • washing step (500 µL PBS, 1 min, 12 000 rpm, RT)
    • resuspension (500 µL 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 µM)
    • negative control: every fusion protein in minimal media
    • incubation (30 °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 µM 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 °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 °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 °C
      • 17 °C over night

    - overnight culture (25 mL LB-Amp, 37 °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 °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 °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 °C
      • 17 °C over night

    - overnight culture (25 mL LB-Amp, 37 °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 °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 °C
      • 17 °C over night

    cell harvest

    Team: Endocytosis

    Investigators: Johanna, Opgenoorth

    Superior experiment: mCherry and ligand_mCherry fusion protein expression

    Procedure:

    • cell harvest (4 °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 °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 °C
    • standard bradford assay protocol (Roth, Roti nanoquant)
      • Flo_mCherry: 256 ng/µL; 409 µg
      • Mat_mCherry: 1.96 µg/µL; 2.352 mg
      • Opy_mCherry: 1.24 µg/µL; 1.488 mg
      • mCherry: 2.42 µg/µL; 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 °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 µM)
    • negative control: every fusion protein in minimal media
    • incubation (30 °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 µM 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 °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 µM)
    • negative control: every fusion protein in minimal media
    • incubation (30 °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 µM 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 °C)

    Pre culture

    Team: Endocytosis

    Investigators: Johanna, Opgenoorth

    Superior experiment: Fluorescence microscopy S. cerevisiae

    Procedure:

    • overnight culture of S. cerevisiae (10 mL YPD, 30 °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 µL SD media
      • addition of fusion protein (1 µM)
      • incubation (30 min, 30 °C, 450 rpm, dark)
      • centrifugation (1 min, 12 000 rpm, RT)
      • washing step (50 µL PBS, centrifugation 1 min, 12 000 rpm, RT)
      • resuspended in 10 µL minimal media
      • fluorescence microscopy of 5 µL (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 °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 °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 °C
      • 17 °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 °C
    • standard bradford assay protocol (Roth, Roti nanoquant)
      • Pro_mCherry: 70.7 ng/µL; 67.9 µg

    SDS-PAGE

    Team: Endocytosis

    Investigators: Johanna, Opgenoorth

    Superior experiment: ligand_mCherry fusion protein purification

    Procedure:

    • standard SDS-PAGE protocol (12 %)
      • analysis of 10 µL 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 µM, 1 µM, 0.5 µM, 0.25 µM, 0.1 µM
    • protein dilution series
      • 0.5 µM, 0.25 µM, 0.1 µM, 0.05 µM, 0.025 µM, 0.01 µM
    • measurement of fluorescence intensity of dilution series (quadruplicates)
      • TECAN reader, gain: calculated from well with 2.5 µM 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 °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 °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 °C
      • 17 °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 °C, 4500 rpm, 20 min)
    • cell mass:
      • BBa_K2926068: 1.57 g
    • storage at -80 °C

    Pre culture

    Team: Endocytosis

    Investigators: Johanna, Opgenoorth

    Superior experiment: Endocytosis assay A. niger

    Procedure:

    • 100 µL 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 µL A. niger culture in minimal media incubated with 0.5 µM Pro_mCherry or mCherry (30 °C, 450 rpm, dark)
    • negative control: 800 µL minimal media with 0.5 µM Pro_mCherry or mCherry
    • measurement of fluorescence intensity in the supernatant after 0 min and 60 min
      • centrifugation of 400 µL mL culture (1 min, 12 000 rpm)
      • measurement of fluorescence in supernatant (TECAN reader, gain: calculated from well with 2.5 µM 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 µM Mat_mCherry (30 °C, 450 rpm, dark)
    • negative control: 1 mL minimal media with 0.5 µM Mat_mCherry
    • measurement of fluorescence intensity in the supernatant after 0 min and 60 min
      • centrifugation of 400 µL mL culture (1 min, 12 000 rpm)
      • measurement of fluorescence in supernatant (TECAN reader, gain: calculated from well with 2.5 µM Teas Red, excitation: 570 nm, emission: 610 nm)