iGEM 2019 — Team Bielefeld-CeBiTec

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# Team:Bielefeld-CeBiTec/Notebook

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Notebook

![](https://static.igem.org/mediawiki/2019/6/6b/T--Bielefeld-CeBiTec--header_laborbuch.jpg)

### 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 DH5ÃÂ± 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 ÃÂ¼l sterile H2O (final concentration: 20 ng/ÃÂµl); 10 min on 37ÃÂºC**
- 200 ÃÂµl competent E. coli DH5ÃÂ± (Ina, 6.5.19) + 1 ÃÂµl DNA (Cas13a) -> 30 min: ice
- 1 min 42ÃÂ°C
- 5 min ice
- + 500 ÃÂµl 37ÃÂ°C warm SOC-Medium
- 1 h 37ÃÂºC

### 2019-05-13 - 2019-05-19

### Transformation of E. coli DH5ÃÂ± with sfGFP

**Team:** CeDIS

**Investigators:** Isabel, Conze

**Superior experiment:** Labplasmid

**Procedure:**

- In the Parts Registry:
- 2-3 ng DNA
- +10 ÃÂµl sterile MQ --> Mix by pipetting
- Sit for 5 min
- DNA: sfGPF (BBa\_K1321337) (c=2 ng/10ÃÂµl --> 0.2 ng/ÃÂµl)
- 200 ÃÂµl competent E. coli DH5ÃÂ± (Ina, date: 6.5.19) + 1 ÃÂµl DNA (0.2 ng) -> 30 min: ice
- 1 min 42ÃÂ°C
- 5 min ice
- + 500 ÃÂµl 37ÃÂ°C warm SOC-Medium
- 1 h 37ÃÂºC

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

- Plate cell pellet on: LB-Cm
- Put plates at 37ÃÂ°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 ÃÂµl PCR-product + 4 ÃÂµl Purple Dye (NEB Biolabs) -> 10 ÃÂµl in gel
- 1 kb ladder (NEB) -> 5 ÃÂµl in gel
- Staining: 5 min in ethidium bromide
- Precultures for Plasmid-Prep & Sequencing:
- 10 ml LB + 10 ÃÂµl Amp -> LB-Amp
- 10 ml LB + 10 ÃÂµl Cm (34 mg ml-1) -> LB-Cm (cCm=34 ÃÂµg ml-1)
- 5 ml LB-Amp + E. coli DH5ÃÂ± (p2CT-His-MBP-Lsh-C13a-WT) Colony 2
- 5 ml LB-Amp + E. coli DH5ÃÂ± (p2CT-His-MBP-Lbu-C13a-WT) Colony 6
- 5 ml LB-Cm + E. coli DH5ÃÂ± (pSB1C3-His-SUMO-Lwa-C13a-WT) Colony 5
- 5 ml LB-Cm + E. coli DH5ÃÂ± (pSB1C3-sfGFP) Colony +

* At 37ÃÂ°C, rotating

### Overnight culture of BBa\_K2926048 and BBa\_J06504 in pTXB1

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Cloning mCherry expression-vectors (BBa\_K2926048 and BBa\_J06504 in pTXB1)

**Procedure:**

* 5 mL overnight cultures of BBa\_K2926048 (Cm) and BBa\_J06504 in pTXB1 (Amp)

### Glycerine culture and plasmid isolation of BBa\_K2926048 (Cm) and BBa\_J06504 in pTXB1

**Team:** Endocytosis

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Cloning mCherry expression-vectors (BBa\_K2926048 and BBa\_J06504 in pTXB1)

**Procedure:**

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

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

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Cloning mCherry\_p8 expression-vectors (BBa\_K2926052 and BBa\_K2926053)

**Procedure:**

* standard PCR-protocol (Q5 NEB)

+ primers:

- p8: 19 a-r (fwd) and 19 a-s (rev)
- Backbone BBa\_K2926048: 19 (fwd) and 19 a-j (rev)
- Backbone BBa\_J06504 in pTXB1: 19 a-e (fwd) and 19 a-j (rev)

+ standard PCR cleanup (Promega)

* analytic agarose gel (1 %, 80 V for backbones, 3 %, 80 V for p8-fragments)
* standard Gibson Assembly (1:3)
* standard transformation via heatshock (E. coli DH5 alpha)
* streaked out on agar plates (BBa\_K2926052: Amp; BBa\_K2926053: Cm)

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

**Team:** CeDIS and Endocytosis

**Investigators:** Isabel, Conze

**Superior experiment:** Cas13a (from Munich)

**Procedure:**

- Promega Wizard SV Gel and PCR Clean-Up System for:
- 50 ÃÂµl Lwa, Lbu, Lsh A

* c(Lwa) = 35,6 ng ÃÂµl-1
* c(Lbu) = 15 ng ÃÂµl-1
* c(Lsh) = 16,8 ng ÃÂµl-1

- 90 ÃÂµl Lwa, Lbu, Lsh B
- 110 ÃÂµl P8 Ptx (Johanna, 24.5.19.)
- 110 ÃÂµl P8 Ptx (Johanna, 24.5.19)

### 2019-05-27 - 2019-06-02

### Colony-PCR, overnight culture of positive colonies

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Cloning mCherry\_p8 expression-vectors (BBa\_K2926052 and BBa\_K2926053)

**Procedure:**

* standard PCR-protocol (Taq)

+ primers:

- BBa\_K2926052: 19 d-b (fwd) and 19 d-c (rev)
- BBa\_K2926053: 19 d-b (fwd) and 19 d-c (rev)

+ 5 mL overnight culture of BBa\_K2926052 in pTXB1 (Amp, clone 6) and BBa\_K2926053 in pSB1C3 (Cm, clone 6)

### Plasmid isolation, Sanger Sequencing

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Cloning mCherry\_p8 expression-vectors (BBa\_K2926052 and BBa\_K2926053)

**Procedure:**

* standard plasmid isolation (analytic jena)
* preparation for sanger sequencing

+ primers:

- BBa\_K2926052: 19 Seq1 and 19 Seq2
- BBa\_K2926053: 19 VF and VR

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

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Cloning Ligand\_mCherry\_p8 expression-vectors (BBa\_K2926054, BBa\_K2926055, BBa\_K2926057 and BBa\_K2926058)

**Procedure:**

* standard PCR protocol (Q5 NEB, BBa\_K2926055, BBa\_K2926058)

+ primers:

- BBa\_K2926055 in pTXB1: 19 c-o (fwd) and 19 c-p (rev)
- BBa\_K2926058 in pSB1C3: 19 c-n (fwd) and 19 c-p (rev)

+ Annealing of 19 c-j and 19 c-l and 19 c-j and 19 c-k to form a fragment of BBa\_K2926054 and BBa\_K2926055
+ equal amounts of 19 c-j and 19 c-l (for BBa\_K2926054) and 19 c-j and 19 c-k (for BBa\_K2926055)
+ heat to 98 Â°C
+ cool down to room temperature: 0.5 Â°C per second

* fill-in of 5' overhangs

+ standard Klenow-fragment protocol (Thermo scientific)

### PCR-Cleanup of gibson fragments

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Cloning Ligand\_mCherry\_p8 expression-vectors (BBa\_K2926054, BBa\_K2926055, BBa\_K2926057 and BBa\_K2926058)

**Procedure:**

* standard PCR-Cleanup (Promega) of fragments for BBa\_K2926055 in pTXB1 and BBa\_K2926058 in pSB1C3

### Transformation via heatshock

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** mCherry protein expression

**Procedure:**

* standard transformation protocol for BBa\_K2926052 and BBa\_K2926053 via heatshock in E. coli ER2566 (NEB)
* streaked out on agar plates (BBa\_K2926052: Amp; BBa\_K2926048: Cm)

### 2019-06-03 - 2019-06-09

### Gel-electrophoresis

**Team:** CeDIS

**Investigators:** Isabel, Conze

**Superior experiment:** Basic insert for Gibson in pSB1C3

**Procedure:**

- Gel-electrophoresis: restriction digest basic insert (Johanna)
- expected bands: 1528 bp & 1898 bp
- PCR:
- Template: Gibson-fragment from colony 8-basic insert
- Primer: pSB1C3\_BasicI\_f (19at, 58ÃÂ°C) & pSB1C3\_BasicI\_r (19av, 58ÃÂ°C)
- Fragment size: 3412 bp
- Polymerase: Q5 (NEB)
- Volume: 100 ÃÂµl
- DpnI-digest (over night)

### Generating Gibson fragments via PCR, template digestion (DpnI)

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Cloning Ligand\_mCherry\_p8 expression-vectors (BBa\_K2926054, BBa\_K2926055, BBa\_K2926057 and BBa\_K2926058)

**Procedure:**

* standard PCR protocol (Q5 NEB)

+ primers:

- backbone BBa\_K2926052 in pTXB1 for BBa\_K2926054: 19 c-h (fwd) and 19 c-m (rev)
- backbone BBa\_K2926052 in pTXB1 for BBa\_K2926055: 19 d-d (fwd) and 19 a-f (rev)
- backbone BBa\_K2926053 in pSB1C3 for BBa\_K2926057: 19 c-h (fwd) and 19 c-i (rev)
- backbone BBa\_K2926053 in pSB1C3 for BBa\_K2926058: 19 d-d (fwd) and 19 a-a (rev)

+ template digestion
+ standard DpnI digestion protocol (NEB)

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

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Cloning Ligand\_mCherry\_p8 expression-vectors (BBa\_K2926054, BBa\_K2926055, BBa\_K2926057 and BBa\_K2926058)

**Procedure:**

* DpnI inactivation (80 Â°C, 20 min)
* standard PCR-Cleanup of gibson fragments (Promega)
* standard gibson assembly protocol to generate BBa\_K2926054, BBa\_K2926055, BBa\_K2926057 and BBa\_K2926058
* standard transformation via heatshock

+ E. coli DH5 alpha
+ streaked out on agar plates (BBa\_K2926054 and BBa\_K2926057: Amp; BBa\_K2926055 and BBa\_K2926058: Cm)

### Colony-PCR: pSB1C3-Basic Insert-Cas13a

**Team:** CeDIS

**Investigators:** Isabel, Conze

**Superior experiment:** Construction: pSB1C3-Basic Insert

**Procedure:**

- Colony-PCR:
- Template: E. coli DH5ÃÂ± (pSB1C3-Basic\_Insert\_Lwa)
- Primer: VR (57ÃÂ°C) & Lwa\_seq\_5 (19ce, 59ÃÂ°C)
- Fragment size: 2506 bp
- Polymerase: GoTaq Hot Start (Promega)
- Volume: 10 ÃÂµl each

### Colony-PCR of BBa\_K2926055 and BBa\_K2926058

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Cloning Ligand\_mCherry\_p8 expression-vectors (BBa\_K2926054, BBa\_K2926055, BBa\_K2926057 and BBa\_K2926058)

**Procedure:**

* standard PCR-protocol (oneTaq, NEB)

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

### Pre-cultures

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** mCherry protein expression

**Procedure:**

* overnight cultures (25 mL LB, 37 Â°C) of BBa\_J06504 in pTXB1 and BBa\_K2926048 in pSB1C3 in E. coli ER2566

+ BBa\_K2926052 in pTXB1: Amp
+ BBa\_K2926053 in pSB1C3: Cm

### 2019-06-10 - 2019-06-16

### Expression cultures

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** mCherry protein expression

**Procedure:**

* standard expression protocol: expression culture (250 mL LB, 37 Â°C) of BBa\_J06504 in pTXB1 and BBa\_K2926048 in pSB1C3 in E. coli ER2566

+ BBa\_J06504 in pTXB1: Amp
+ BBa\_K2926048 in pSB1C3: Cm
+ start OD: 0.1
+ induction (0.4 mM IPTG) at OD 0.6-0.8
+ 3 min 37 Â°C
+ 17 Â°C over night

### Gibson Assembly, transformation via heatshock, colony PCR

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Cloning Ligand\_mCherry\_p8 expression-vectors (BBa\_K2926054, BBa\_K2926055, BBa\_K2926057 and BBa\_K2926058)

**Procedure:**

* standard Gibson Assembly protocol to generate BBa\_K2926054 and BBa\_K2926057 (1:3 backbone:insert)
* standard Transformation via heatshock (E. coli DH5 alpha)
* streaked out on agar plates

+ BBa\_K2926054 and BBa\_K2926055: Amp
+ BBa\_K2926057 and BBa\_K2926058: Cm

* colony PCR of BBa\_K2926058

+ standard colony PCR protocol (oneTaq, NEB)

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

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

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Cloning Ligand\_mCherry\_p8 expression-vectors (BBa\_K2926056 and BBa\_K2926067)

**Procedure:**

* standard PCR protocol (Q5, NEB)

+ primers:

- BBa\_K2926056: 19 c-w (rev) and 19 c-x (fwd)
- BBa\_K2926067: 19 c-r (fwd) and 19 c-s (rev)

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

### Overnight cultures of BBa\_K2926055 and BBa\_K2926058

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Cloning Ligand\_mCherry\_p8 expression-vectors (BBa\_K2926054, BBa\_K2926055, BBa\_K2926057 and BBa\_K2926058)

**Procedure:**

* 5 mL overnight culture of BBa\_K2926055 (Amp) and BBa\_K2926058 (Cm) in LB

### Cell harvest

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** mCherry protein expression

**Procedure:**

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

+ BBa\_J06504: 1.92 g
+ BBa\_K2926048: 1.63 g

* storage of the pellet at -80 Â°C

### Plasmid isolation of BBa\_K2926055 and BBa\_K2926058, colony PCR of BBa\_K2926054 and BBa\_K2926057

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Cloning Ligand\_mCherry\_p8 expression-vectors (BBa\_K2926054, BBa\_K2926055, BBa\_K2926057 and BBa\_K2926058)

**Procedure:**

* standard plasmid isolation of BBa\_K2926055 and BBa\_K2926058 (analytic jena)
* standard colony PCR protocol of BBa\_K2926054 and BBa\_K2926057 (oneTaq)

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

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

### First add-on PCR, analytic agarose gel

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Cloning Ligand\_mCherry\_p8 expression-vectors (BBa\_K2926056 and BBa\_K2926067)

**Procedure:**

* standard PCR protocol (Q5, NEB)

+ primers:

- BBa\_K2926056: 19 c-w (rev) and 19 c-x (fwd)
- BBa\_K2926067: 19 c-r (fwd) and 19 c-s (rev)

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

### 2019-06-17 - 2019-06-23

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

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Cloning Ligand\_mCherry\_p8 expression-vectors (BBa\_K2926056 and BBa\_K2926067)

**Procedure:**

* standard PCR protocol (Q5, NEB)

+ primers:

- BBa\_K2926056: 19 c-v (rev) and 19 c-t (fwd)
- BBa\_K2926067: 19 c-t (fwd) and 19 c-u (rev)

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

### overnight cultures of BBa\_K2926054 and BBa\_K2926057

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Cloning Ligand\_mCherry\_p8 expression-vectors (BBa\_K2926054, BBa\_K2926055, BBa\_K2926057 and BBa\_K2926058)

**Procedure:**

* overnight cultures of positive colonies

+ 5 mL overnight culture of BBa\_K2926054 (colony 6, Amp) and BBa\_K2926057 (colony 2, Cm) in LB

### Chemical transformation

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Expression of Ligand\_mCherry\_p8 (BBa\_K2926054, BBa\_K2926055, BBa\_K2926057 and BBa\_K2926058)

**Procedure:**

* standard heat shock transformation of BBa\_K2926054 and BBa\_K2926057 in E. coli ER2566
* streaked out on agar plates (BBa\_K2926054: Amp; BBa\_K2926057: Cm)

### Gradient PCR (second add-on PCR for BBa\_K2926056)

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Cloning Ligand\_mCherry\_p8 expression-vectors (BBa\_K2926056 and BBa\_K2926067)

**Procedure:**

* standard PCR protocol (Q5, NEB)

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

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

### Plasmid isolation of BBa\_K2926054 and BBa\_K2926058

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Cloning Ligand\_mCherry\_p8 expression-vectors (BBa\_K2926054 and BBa\_K2926058)

**Procedure:**

* standard plasmid isolation of BBa\_K2926054 and BBa\_K2926058 (analytic jena)

### Gradient PCR (second add-on PCR for BBa\_K2926067), first add-on PCR

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Cloning Ligand\_mCherry\_p8 expression-vectors (BBa\_K2926056 and BBa\_K2926067)

**Procedure:**

* standard PCR protocol (Q5, NEB)

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

* analytic agarose gel of PCR products (1 %, 1 kb ladder)
* standard PCR protocol (Q5, NEB) for first add-on PCR to generate BBa\_K2926056 and BBa\_K2926067

+ primers:

- BBa\_K2926056: 19 c-w (rev) and 19 c-x (fwd)
- BBa\_K2926067: 19 c-r (fwd) and 19 c-s (rev)

+ annealing temperature: 64 Â°C

### Analytical agarose gel of PCR products

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Cloning Ligand\_mCherry\_p8 expression-vectors (BBa\_K2926056 and BBa\_K2926067)

**Procedure:**

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

### 2019-06-24 - 2019-06-30

### Cell lysis, Protein purification

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** mCherry protein expression

**Procedure:**

* standard cell lysis (Ribolyzer)

+ resuspension of thawed cell pellet

- BBa\_J06504 in pTXB1: 35 mL BBa\_IMPACT Lysis Buffer
- BBa\_K2926048: 4 mL Macherey&Nagel LEW-Buffer
- addition of 1 mm Zirconia beads (Roth)
- Ribolyzer: 3x30 s; 8000 rpm

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

* standard Ni-Ted purification protocol (Macherey&Nagel, BBa\_K2926048)

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

* standard IMPACT-purification (NEB)

### PCR cleanup, analytical agarose gel

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Cloning Ligand\_mCherry\_p8 expression-vectors (BBa\_K2926056 and BBa\_K2926067)

**Procedure:**

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

### IMPACT elution, bradford assay

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** mCherry protein expression

**Procedure:**

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

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

* standard bradford assay protocol (Roti-Nanoquant, Roth)

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

### Expression pre-cultures

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Expression of Ligand\_mCherry\_p8 (BBa\_K2926054 and BBa\_K2926055)

**Procedure:**

* overnight cultures of BBa\_K2926054 and BBa\_K2926055 in E. coli ER2566 (25 mL LB-Amp, 37 Â°C)

### Expression cultures

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Expression of Ligand\_mCherry\_p8 (BBa\_K2926054 and BBa\_K2926055)

**Procedure:**

* standard expression protocol: expression culture (250 mL LB-Amp, 37 Â°C) of BBa\_K2926054 and BBa\_K2926055 in pTXB1 in E. coli ER2566

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

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

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Cloning Ligand\_mCherry\_p8 expression-vectors (BBa\_K2926056 and BBa\_K2926067)

**Procedure:**

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

+ primers:

- BBa\_K2926056: 19 c-v (rev) and 19 c-t (fwd)
- BBa\_K2926067: 19 c-t (fwd) and 19 c-u (rev)

+ analytical agarose gel (1 %, 1 kb ladder)

* standard PCR cleanup (Promega)
* standard Gibson assembly (one fragment)
* standard transformation via heatshock (E. coli DH5 alpha)
* streaked out on agar plates (BBa\_K2926056: Amp, BBa\_K2926067: Cm)

### Cell harvest

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Expression of Ligand\_mCherry\_p8 (BBa\_K2926054 and BBa\_K2926055)

**Procedure:**

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

### SDS-PAGE of mCherry purification process

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** mCherry analysis

**Procedure:**

* standard SDS-PAGE protocol (12 %)

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

### Colony PCR, analytical agarose gel

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Cloning Ligand\_mCherry\_p8 expression-vectors (BBa\_K2926056 and BBa\_K2926067)

**Procedure:**

* standard PCR protocol (oneTaq) of BBa\_K2926067

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

* analytical agarose gel (1 %, 1 kb ladder)

### PCR f1 ori

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Primers:

+ f1 ori pSB1C3 GA f
+ f1 ori mC Prom GA r

* Q5 Polymerase

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

### PCR mCherry V1

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Template: iGEM mCherry
* Primers:

+ mC f1 ori RBS GA f
+ mC VIII GA r

* Q5 Polymerase

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

### PCR mCherry V2

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Template: iGEM mCherry
* Primers:

+ mC f1 ori RBS GA f
+ mC pSB1C3 GA r

* Q5 Polymerase

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

### PCR VIII

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Template: M13K07 NEB
* Primers:

+ VIII mC GA f
+ VIII III GA r

* Q5 Polymerase

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

### PCR III

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Template: M13K07 NEB
* Primers:

+ III VIII GA f
+ III pSB1C3 GA r

* Q5 Polymerase

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

### PCR pSB1C3

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Template: iGEM pSB1C3
* Primers:

+ pSB3K3 split f
+ pSB3K3 split r

* Q5 Polymerase

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

### Geleelectrophoresis all fragments

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

### Gel Clean-Up all fragments

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Monarch DNA Gel Extractions

### Overnight culture

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Cloning Ligand\_mCherry\_p8 expression-vectors (BBa\_K2926056 and BBa\_K2926067)

**Procedure:**

* overnight culture of one positive colony of BBa\_K2926067 (5 mL LB-Amp)

### 2019-07-01 - 2019-07-07

### Gibson Assembly Troygenic DNA V1

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Gibson Master Mix
* pSB1C3
* f1 ori
* mCherry V1
* VIII
* III

### Transformation Troygenic DNA V1

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* DH5a

### Gibson Assembly Troygenic DNA V2

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

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

### Transformation Troygenic DNA V2

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* DH5a

### Colony PCR Troygenic DNA V1

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Primers:

+ VF
+ VR

* OneTaq Polymerase

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

### Colony PCR Troygenic DNA V2

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Primers:

+ VF
+ VR

* OneTaq Polymerase

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

### Plasmid isolation

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Cloning Ligand\_mCherry\_p8 expression-vectors (BBa\_K2926056 and BBa\_K2926067)

**Procedure:**

* standard plasmid isolation protocol of BBa\_K2926067 (analytic jena)
* preparations for Sanger sequencing

+ primers: SEQ1 (fwd) and SEQ2 (rev)

### Establishing a fluorescence measurement method

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** mCherry characterization

**Procedure:**

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

### PCR VIII

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Template: M13K07 NEB
* Primers:

+ VIII mC GA f
+ VIII III GA r

* Q5 Polymerase

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

### PCR III

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Template: M13K07 NEB
* Primers:

+ III VIII GA f
+ III pSB1C3 GA r

* Q5 Polymerase

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

### Geleelectrophoresis all fragments

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

### Gel Clean-Up all fragments

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Monarch DNA Gel Extractions

### Gibson Assembly Troygenic DNA V1

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Gibson Master Mix
* pSB1C3
* f1 ori
* mCherry V1
* VIII
* III

### Transformation Troygenic DNA V1

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* DH5a

### Establishing a fluorescence measurement method

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** mCherry characterization

**Procedure:**

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

### Gibson Assembly Troygenic DNA V1

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Gibson Master Mix
* pSB1C3
* f1 ori
* mCherry V1
* VIII
* III

### Transformation Troygenic DNA V1

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* DH5a

### Gibson Assembly Troygenic DNA V2

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

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

### Transformation Troygenic DNA V2

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* DH5a

### Overlap-PCR f1 ori - mCherry

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Templates: f1 ori, mCherry
* Q5 Polymerase

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

* Primers:

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

### Overlap-PCR VIII - III

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Templates: VIII, III
* Q5 Polymerase

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

* Primers:

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

### Geleelectrophoresis Overlap-PCRs

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

### Gel Clean-Up Overlap-PCRs

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Monarch DNA Gel Extractions

### PCR III

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Template: M13K07 NEB
* Primers:

+ III VIII GA f
+ III pSB1C3 GA r

* Q5 Polymerase

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

### overnight cultures of BBa\_K2926058

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Cloning Ligand\_mCherry\_p8 expression-vectors (BBa\_K2926054, BBa\_K2926055, BBa\_K2926057 and BBa\_K2926058)

**Procedure:**

* overnight cultures of positive colonies

+ 5 mL overnight culture of BBa\_K2926058 (colony 2, 10 and 13, LB-Cm)

### PCR pSB3K3

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Helperphage construction

**Procedure:**

* Template: iGEM pSB3K3
* Primers:

+ pSB3K3 split f
+ pSB3K3 split r

* Q5 Polymerase

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

### PCR Terminator V1

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Helperphage construction

**Procedure:**

* Template: M13K07 NEB
* Primers:

+ M13 PCR f
+ M13 Term GA r

* Q5 Polymerase

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

### PCR Terminator V2

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Helperphage construction

**Procedure:**

* Template: M13K07 NEB
* Primers:

+ M13 PCR f
+ M13 Term GA r

* Q5 Polymerase

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

### PCR f1 ori

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Primers:

+ f1 ori pSB1C3 GA f
+ f1 ori mC Prom GA r

* Q5 Polymerase

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

### PCR mCherry

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Template: iGEM mCherry
* Primers:

+ mC f1 ori RBS GA f
+ mC VIII GA r

* Q5 Polymerase

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

### PCR f1 ori Overlap

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Primers:

+ f1 1C3 OP f
+ f1 mC Prom OP r

* Q5 Polymerase

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

### PCR mCherry Overlap

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Template: iGEM mCherry
* Primers:

+ mC ori OP f
+ mC VIII OP r

* Q5 Polymerase

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

### plasmid isolation of BBa\_K2926058

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Cloning Ligand\_mCherry\_p8 expression-vectors (BBa\_K2926054, BBa\_K2926055, BBa\_K2926057 and BBa\_K2926058)

**Procedure:**

* standard plasmid isolation protocol (analytic jena)

### Establishing a fluorescence measurement method, measurement of pH stability

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** mCherry characterization

**Procedure:**

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

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

### 2019-07-08 - 2019-07-14

### PCR II-VIII

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Helperphage construction

**Procedure:**

* Template: M13K07 NEB
* Primers:

+ M13 pSB3K3 f1
+ M13 Prom GA r

* Q5 Polymerase

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

### Overlap-PCR f1 ori - mCherry

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Templates: f1 ori Overlap, mCherry Overlap
* Q5 Polymerase

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

* Primers:

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

### Geleelectrophoresis all fragments

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

### PCR III-IV

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Helperphage construction

**Procedure:**

* Template: M13K07 NEB
* Primers:

+ III VIII GA f
+ M13 pSB3K3 r2

* Q5 Polymerase

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

### Geleelectrophoresis Overlap f1-mCherry, III-IV

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

### Gel Clean-Up all fragments

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Monarch DNA Gel Extractions

### Gibson Assembly Helperphage V1

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Helperphage construction

**Procedure:**

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

### Transformation Helperphage V1

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Helperphage construction

**Procedure:**

* DH5a

### Gibson Assembly Helperphage V2

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Helperphage construction

**Procedure:**

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

### Transformation Helperphage V2

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Helperphage construction

**Procedure:**

* DH5a

### Gibson Assembly Troygenic DNA Overlap

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

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

### Transformation Troygenic DNA Overlap

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* DH5a

### Pre-cultures

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** mCherry protein expression

**Procedure:**

* overnight cultures (25 mL LB, 37 Â°C) of BBa\_J06504 in pTXB1 and BBa\_K2926048 in pSB1C3 in E. coli ER2566

+ BBa\_K2926052 in pTXB1: Amp
+ BBa\_K2926053 in pSB1C3: Cm

### Expression cultures

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** mCherry protein expression

**Procedure:**

* standard expression protocol: expression culture (250 mL LB, 37 Â°C) of BBa\_J06504 in pTXB1 and BBa\_K2926048 in pSB1C3 in E. coli ER2566

+ BBa\_J06504 in pTXB1: Amp
+ BBa\_K2926048 in pSB1C3: Cm
+ start OD: 0.1
+ induction (0.4 mM IPTG) at OD 0.6-0.8
+ 30 min 37 Â°C
+ RT over night

### Overlap-PCR f1 ori - mCherry

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Templates: f1 ori Overlap, mCherry Overlap
* Q5 Polymerase

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

* Primers:

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

### Overlap-PCR VIII - III

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Templates: VIII, III
* Q5 Polymerase

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

* Primers:

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

### PCR VIII Overlap

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Template: M13K07 NEB
* Primers:

+ VIII mC OP f
+ VIII III OP r

* Q5 Polymerase

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

### PCR III Overlap

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Template: M13K07 NEB
* Primers:

+ III VIII OP f
+ III 1C3 OP r

* Q5 Polymerase

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

### PCR pSB3K3

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Helperphage construction

**Procedure:**

* Template: iGEM pSB3K3
* Primers:

+ pSB3K3 split f
+ pSB3K3 split r

* Q5 Polymerase

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

### PCR II-VIII

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Helperphage construction

**Procedure:**

* Template: M13K07 NEB
* Primers:

+ M13 pSB3K3 f1
+ M13 Prom GA r

* Q5 Polymerase

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

### PCR III-IV

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Helperphage construction

**Procedure:**

* Template: M13K07 NEB
* Primers:

+ III VIII GA f
+ M13 pSB3K3 r2

* Q5 Polymerase

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

### Overlap-PCR f1 ori - mCherry

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Templates: f1 ori Overlap, mCherry Overlap
* Q5 Polymerase

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

* Primers:

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

### Overlap-PCR VIII - III

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Templates: VIII Overlap, III Overlap
* Q5 Polymerase

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

* Primers:

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

### Cell harvest

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** mCherry protein expression

**Procedure:**

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

+ mCherry: 2.45 g
+ mCherryHis: 1.39 g

### Cell lysis, new pre culture

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** mCherry and ligand\_mCherry\_p8 protein purification

**Procedure:**

* standard cell lysis (Ribolyzer)

+ lysis not successful

* new overnight pre cultures of BBa\_J06504, BBa\_K2926048, BBa\_K2926055 and BBa\_K2926054 in E. coli ER2566

+ 25 mL LB-Amp, 37 Â°C

### overnight culture of BBa\_K2926067

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Cloning Ligand\_mCherry\_p8 expression-vectors (BBa\_K2926056 and BBa\_K2926067)

**Procedure:**

* 5 mL overnight culture (LB-Cm, 37 Â°C) of BBa\_K2926067

### Geleelectrophoresis all fragments

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

### Transformation pSB1C3 from iGEM plate

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

### Transformation pSB3K3 from iGEM plate

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Helperphage construction

**Procedure:**

### Gradient-PCR f1 ori

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Primers:

+ f1 ori pSB1C3 GA f
+ f1 ori mC Prom GA r

* Q5 Polymerase

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

### PCR f1 ori Overlap

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Primers:

+ f1 1C3 OP f
+ f1 mC Prom OP r

* Q5 Polymerase

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

### PCR mCherry

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Template: iGEM mCherry
* Primers:

+ mC f1 ori RBS GA f
+ mC VIII GA r

* Q5 Polymerase

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

### Gradient-PCR mCherry Overlap

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Template: iGEM mCherry
* Primers:

+ mC ori OP f
+ mC VIII OP r

* Q5 Polymerase

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

### PCR III

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Template: M13K07 NEB
* Primers:

+ III VIII GA f
+ III pSB1C3 GA r

* Q5 Polymerase

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

### PCR III Overlap

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Template: M13K07 NEB
* Primers:

+ III VIII OP f
+ III 1C3 OP r

* Q5 Polymerase

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

### Gradient-PCR pSB1C3

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Template: iGEM pSB1C3
* Primers:

+ pSB3K3 split f
+ pSB3K3 split r

* Q5 Polymerase

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

### Gradient-PCR II-VIII

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Helperphage construction

**Procedure:**

* Template: M13K07 NEB
* Primers:

+ M13 pSB3K3 f1
+ M13 Prom GA r

* Q5 Polymerase

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

### PCR VIII

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Template: M13K07 NEB
* Primers:

+ VIII mC GA f
+ VIII III GA r

* Q5 Polymerase

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

### Gradient-PCR VIII Overlap

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Template: M13K07 NEB
* Primers:

+ VIII mC OP f
+ VIII III OP r

* Q5 Polymerase

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

### PCR Terminator

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Helperphage construction

**Procedure:**

* Template: M13K07 NEB
* Primers:

+ M13 PCR f
+ M13 Term GA r

* Q5 Polymerase

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

### Gradient-PCR pSB3K3

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Helperphage construction

**Procedure:**

* Template: iGEM pSB3K3
* Primers:

+ pSB3K3 split f
+ pSB3K3 split r

* Q5 Polymerase

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

### Gradient-PCR III-IV

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Helperphage construction

**Procedure:**

* Template: M13K07 NEB
* Primers:

+ III VIII GA f
+ M13 pSB3K3 r2

* Q5 Polymerase

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

### Gradient-Overlap-PCR f1 ori - mCherry

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Templates: f1 ori, mCherry
* Q5 Polymerase

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

* Primers:

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

### Gradient-Overlap-PCR VIII - III

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Templates: VIII, III
* Q5 Polymerase

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

* Primers:

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

### Geleelectrophoresis all fragments

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

### PCR Clean-Up all fragments

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Promega Wizard SV Gel and PCR Clean-Up System

### digest dnpI all fragments

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

### - standard expression protocol: expression culture (250 mL LB, 37 Â°C) of BBa\_J06504, BBa\_K2926055 and BBa\_K2926054 in pTXB1, BBa\_K2926048 in pSB1C3 in E. coli ER2566

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** mCherry and ligand\_mCherry\_p8 protein expression

**Procedure:**

* pTXB1: Amp

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

### plasmid isolation, PCR to generate gibson fragments, DpnI digestion

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Cloning Ligand\_mCherry\_p8 expression-vectors (BBa\_K2926056 and BBa\_K2926067)

**Procedure:**

* standard plasmid isolation protocol of BBa\_K2926067 (Zymo research)
* standard PCR protocol (Q5, NEB) to generate gibson fragment for BBa\_K2926056

+ template: BBa\_K2926067
+ primers: 19 i-g (fwd) and 19 i-h (rev)

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

### Gel Clean-Up all fragments

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Monarch DNA Gel Extractions

### PCR Clean-Up all fragments

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Promega Wizard SV Gel and PCR Clean-Up System

### Cell harvest

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** mCherry and ligand\_mCherry\_p8 protein expression

**Procedure:**

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

+ mCherry: 1.84 g
+ mCherryHis: 1.86 g
+ Flo\_mCherry\_p8: 1.79 g
+ Mat\_mCherry\_p8: 2.4 g

### Gel cleanup, Gibson assembly, Transformation via heatshock

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Cloning Ligand\_mCherry\_p8 expression-vectors (BBa\_K2926056 and BBa\_K2926067)

**Procedure:**

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

### Establishing a fluorescence standard for red fluorescing proteins

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** mCherry characterization - Texas Red

**Procedure:**

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

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

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

### 2019-07-15 - 2019-07-21

### PCR f1 ori

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Primers:

+ f1 ori pSB1C3 GA f
+ f1 ori mC Prom GA r

* Q5 Polymerase

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

### PCR mCherry

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Template: iGEM mCherry
* Primers:

+ mC f1 ori RBS GA f
+ mC VIII GA r

* Q5 Polymerase

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

### PCR mCherry Overlap

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Template: iGEM mCherry
* Primers:

+ mC ori OP f
+ mC VIII OP r

* Q5 Polymerase

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

### PCR III Overlap

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Template: M13K07 NEB
* Primers:

+ III VIII OP f
+ III 1C3 OP r

* Q5 Polymerase

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

### PCR pSB1C3

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Template: iGEM pSB1C3
* Primers:

+ pSB3K3 split f
+ pSB3K3 split r

* Q5 Polymerase

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

### PCR II-VIII

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Helperphage construction

**Procedure:**

* Template: M13K07 NEB
* Primers:

+ M13 pSB3K3 f1
+ M13 Prom GA r

* Q5 Polymerase

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

### PCR VIII Overlap

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Template: M13K07 NEB
* Primers:

+ VIII mC OP f
+ VIII III OP r

* Q5 Polymerase

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

### PCR pSB3K3

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Helperphage construction

**Procedure:**

* Template: iGEM pSB3K3
* Primers:

+ pSB3K3 split f
+ pSB3K3 split r

* Q5 Polymerase

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

### PCR III-IV

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Helperphage construction

**Procedure:**

* Template: M13K07 NEB
* Primers:

+ III VIII GA f
+ M13 pSB3K3 r2

* Q5 Polymerase

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

### Geleelectrophoresis all fragments

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

### Gel Clean-Up all fragments

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Monarch DNA Gel Extractions

### PCR Clean-Up all fragments

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Promega Wizard SV Gel and PCR Clean-Up System

### Gibson Assembly Helperphage

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Helperphage construction

**Procedure:**

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

### Transformation Helperphage

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Helperphage construction

**Procedure:**

* DH5a

### Gibson Assembly Troygenic DNA

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

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

### Transformation Troygenic DNA

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* DH5a

### Cell lysis, IMPACT purification

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** mCherry and ligand\_mCherry\_p8 protein purification (BBa\_J06504, BBa\_K2926055 and BBa\_K2926054 in pTXB1, BBa\_K2926048 in pSB1C3)

**Procedure:**

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

+ protein loss due to a buffer mistake

### PCR mCherry

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Template: iGEM mCherry
* Primers:

+ mC f1 ori RBS GA f
+ mC VIII GA r

* Q5 Polymerase

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

### PCR mCherry Overlap

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Template: iGEM mCherry
* Primers:

+ mC ori OP f
+ mC VIII OP r

* Q5 Polymerase

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

### Geleelectrophoresis mCherry

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

### Colony-PCR pSB1C3

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* GoTaq G2 DNA Polymerase

### Colony-PCR pSB3K3

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Helperphage construction

**Procedure:**

* GoTaq G2 DNA Polymerase

### Colony-PCR Helperphage V1

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Helperphage construction

**Procedure:**

* GoTaq G2 DNA Polymerase

### Colony-PCR Helperphage V2

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Helperphage construction

**Procedure:**

* GoTaq G2 DNA Polymerase

### Gradient-Overlap-PCR f1 ori - mCherry

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Templates: f1 ori, mCherry
* Q5 Polymerase

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

* Primers:

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

### Gradient-Overlap-PCR VIII - III

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Templates: VIII, III
* Q5 Polymerase

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

* Primers:

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

### Geleelectrophoresis all fragments

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Helperphage construction

**Procedure:**

### PCR Clean-Up mCherry

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Promega Wizard SV Gel and PCR Clean-Up System

### Gel Clean-Up mCherry

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Promega Wizard SV Gel and PCR Clean-Up System

### colony PCR, overnight cultures

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Cloning Ligand\_mCherry\_p8 expression-vectors (BBa\_K2926056 and BBa\_K2926067)

**Procedure:**

* standard colony PCR protocol (oneTaq, NEB)

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

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

### Elution, washing and concentrating

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** mCherry and ligand\_mCherry\_p8 protein purification (BBa\_J06504, BBa\_K2926055 and BBa\_K2926054 in pTXB1)

**Procedure:**

* elution of BBa\_J06504, BBa\_K2926055 and BBa\_K2926054 in 10 mL IMPACT elution buffer
* proteins washed with 3x10 mL PBS (Zentricon Merck, cut off 10 kDa)
* concentrated to around 1 mL
* addition of glycerine (86 %, 1:1)
* storage at -20 Â°C

### Gibson Assembly Troygenic DNA

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

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

### Transformation Troygenic DNA

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* DH5a

### Overlap-PCR f1 ori - mCherry

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Templates: f1 ori, mCherry
* Q5 Polymerase

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

* Primers:

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

### Overlap-PCR VIII - III

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Templates: VIII, III
* Q5 Polymerase

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

* Primers:

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

### Overlap-PCR f1 ori - mCherry

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Templates: f1 ori Overlap, mCherry Overlap
* Q5 Polymerase

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

* Primers:

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

### Overlap-PCR VIII - III

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Templates: VIII Overlap, III Overlap
* Q5 Polymerase

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

* Primers:

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

### Colony-PCR pSB1C3

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* GoTaq G2 DNA Polymerase

### plasmid isolation

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Cloning Ligand\_mCherry\_p8 expression-vectors (BBa\_K2926056 and BBa\_K2926067)

**Procedure:**

* standard plasmid isolation (Zymo research)
* prepared for Sanger sequencing

+ primers: SEQ1 (fwd) and SEQ2 (rev)

### Bradford assay, SDS-PAGE

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** mCherry and ligand\_mCherry\_p8 protein purification (BBa\_J06504, BBa\_K2926055 and BBa\_K2926054 in pTXB1)

**Procedure:**

* standard bradford assay protocol (Roth, Roti nanoquant)

+ mCherry: 53.6 ng/ÂµL; 125.85 Âµg
+ Flo\_mCherry\_p8: 2.86 ng/ÂµL; 7.74 Âµg
+ Mat\_mCherry\_p8: 2.45 ng/ÂµL; 6.53 Âµg

* standard SDS-PAGE protocol (12 %)

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

### TECAN-reader measurement

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** mCherry and ligand\_mCherry\_p8 protein characterization (BBa\_J06504, BBa\_K2926055 and BBa\_K2926054 in pTXB1)

**Procedure:**

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

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

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

### Dilution series mCherry

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Primers:

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

### Dilution series II-VIII

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Helperphage construction

**Procedure:**

* Primers:

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

### Dilution series pSB1C3

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Primers:

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

### Dilution series pSB3K3

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Helperphage construction

**Procedure:**

* Primers:

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

### Transformation pSB3K3 from iGEM plate

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Helperphage construction

**Procedure:**

* 10Âµl Kanamycin on plate

### Inoculation pSB1C3

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

### PCR Clean-Up all fragments

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Promega Wizard SV Gel and PCR Clean-Up System

### Gel Clean-Up all fragments

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Promega Wizard SV Gel and PCR Clean-Up System

### Inoculation pSB3K3

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Helperphage construction

**Procedure:**

### Geleelectrophoresis all fragments

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

### PCR Clean-Up pSB3K3

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Helperphage construction

**Procedure:**

* Promega Wizard SV Gel and PCR Clean-Up System

### Gel Clean-Up pSB3K3

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Promega Wizard SV Gel and PCR Clean-Up System

### Plasmid Miniprep pSB1C3

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* ZymoPURE Plasmid Miniprep

### PCR mCherry

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Template: iGEM mCherry
* Primers:

+ mC f1 ori RBS GA f
+ mC VIII GA r

* Q5 Polymerase

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

### PCR mCherry Overlap

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Template: iGEM mCherry
* Primers:

+ mC ori OP f
+ mC VIII OP r

* Q5 Polymerase

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

### PCR Clean-Up mCherry

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Helperphage construction

**Procedure:**

* Promega Wizard SV Gel and PCR Clean-Up System

### PCR pSB1C3

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

* Primers:

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

### Geleelectrophoresis mCherry

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

### Gel Clean-Up mCherry

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Helperphage construction

**Procedure:**

* Promega Wizard SV Gel and PCR Clean-Up System

### Pre-cultures

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** mCherry and ligand\_mCherry\_p8 protein expression

**Procedure:**

* overnight cultures (25 mL LB, 37 Â°C) of BBa\_J06504, BBa\_K2926055 and BBa\_K2926054 in pTXB1, BBa\_K2926048 in E. coli ER2566

+ pTXB1: Amp
+ pSB1C3: Cm

### Geleelectrophoresis all fragments

**Team:** Troygenic Assembly

**Investigators:** Astrid, TÃ¶bber

**Superior experiment:** Troygenic DNA construction

**Procedure:**

### protein expression

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** mCherry and ligand\_mCherry\_p8 protein expression

**Procedure:**

* standard expression protocol: expression culture (250 mL LB, 37 Â°C) of BBa\_J06504, BBa\_K2926055 and BBa\_K2926054 in pTXB1, BBa\_K2926048 in pSB1C3 in E. coli ER2566

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

### Cell harvest

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** mCherry and ligand\_mCherry\_p8 protein expression

**Procedure:**

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

+ mCherry: 2.13 g
+ mCherryHis: 1.92 g
+ Flo\_mCherry\_p8: 2.09 g
+ Mat\_mCherry\_p8: 2.11 g

### 2019-07-22 - 2019-07-28

### Cell lysis, protein purification

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** mCherry and ligand\_mCherry\_p8 protein purification (BBa\_J06504, BBa\_K2926055 and BBa\_K2926054 in pTXB1, BBa\_K2926048 in pSB1C3)

**Procedure:**

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

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

### Elution, washing and concentrating

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** mCherry and ligand\_mCherry\_p8 protein purification (BBa\_J06504, BBa\_K2926055, BBa\_K2926054, BBa\_J06504)

**Procedure:**

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

### Bradford assay, SDS-PAGE

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** mCherry and ligand\_mCherry\_p8 protein purification (BBa\_J06504, BBa\_K2926055, BBa\_K2926054, BBa\_J06504)

**Procedure:**

* standard bradford assay protocol (Roth, Roti nanoquant)

+ mCherry: 916.99 ng/ÂµL; 1.43 mg
+ mCherryHis: 14.15 ng/ÂµL; 45.28 Âµg
+ Flo\_mCherry\_p8: 6.68 ng/ÂµL; 8 Âµg
+ Mat\_mCherry\_p8: 10 ng/ÂµL; 13.6 Âµg

* standard SDS-PAGE protocol (12 %)

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

### transformation via heatshock

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Cloning Ligand\_mCherry\_p8 expression-vectors (BBa\_K2926056 and BBa\_K2926067)

**Procedure:**

* transformation of BBa\_K2926056 via heatshock in E. coli ER2566
* streaked out on agar plates (Amp)

### Pre-cultures

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Ligand\_mCherry\_p8 protein expression

**Procedure:**

* overnight cultures (25 mL LB-Amp, 37 Â°C) of BBa\_K2926056 in pTXB1 in E. coli ER2566

### protein expression

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Ligand\_mCherry\_p8 protein expression

**Procedure:**

* standard expression protocol: expression culture (250 mL LB-Amp, 37 Â°C) of BBa\_K2926056 in pTXB1 in E. coli ER2566

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

### TECAN-reader measurement

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** mCherry, mCherryHis and ligand\_mCherry\_p8 protein characterization

**Procedure:**

* Texas Red standard

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

* protein dilution series

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

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

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

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

### Cell harvest

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** ligand\_mCherry\_p8 protein expression (BBa\_K2926056)

**Procedure:**

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

### 2019-07-29 - 2019-08-04

### cell lysis, IMPACT purification

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** ligand\_mCherry\_p8 protein purification

**Procedure:**

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

### Elution, washing and concentrating

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** ligand\_mCherry\_p8 protein purification

**Procedure:**

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

### Bradford assay

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** ligand\_mCherry\_p8 protein purification

**Procedure:**

* standard bradford assay protocol (Roth, Roti nanoquant)

+ Opy\_mCherry\_p8

### SDS-PAGE

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** ligand\_mCherry\_p8 protein purification

**Procedure:**

* standard SDS-PAGE protocol (12 %)

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

### TECAN-reader measurement

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** mCherry and ligand\_mCherry\_p8 protein characterization

**Procedure:**

* Texas Red standard

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

* protein dilution series

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

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

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

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

### PCR, PCR cleanup

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Cloning ligand\_mCherry fusion proteins

**Procedure:**

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

+ BBa\_K292605:

- template: BBa\_J06504 in pTXB1
- primers: 19 a-f (rev) and 19 d-d (fwd)

+ BBa\_K2926054:

- template: BBa\_J06504 in pTXB1
- primers: 19 c-h (fwd) and 19 c-m (rev)

+ BBa\_K2926051

- template: BBa\_K2926067 in pSB1C3
- primers: 19 a-h (rev) and 19 i-g (fwd)

+ standard PCR cleanup protocol (Macherey&Nagel)

### 2019-08-05 - 2019-08-11

### Analytical agarose gel, Gibson assembly, transformation via heatshock

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Cloning ligand\_mCherry fusion proteins

**Procedure:**

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

+ BBa\_K292605: PCR fragment and gene synthesis
+ BBa\_K2926054: PCR fragment and primer dimer
+ BBa\_K2926051: PCR fragment and pTXB1 linerarized

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

### Colony PCR, overnight culture

**Team:** Endocytosis

**Investigators:** Alexander, Schulze

**Superior experiment:** Cloning ligand\_mCherry fusion proteins

**Procedure:**

* standard colony PCR protocol (GoTaq, Promega)

+ primers: 19 e-j (rev) and 19 c-q (fwd, BBa\_K292605), 19 c-g (fwd, BBa\_K2926054) or 19 c-y (fwd, BBa\_K2926051)

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

+ BBa\_K292605: colony 15
+ BBa\_K2926054: colony 16
+ BBa\_K2926051: colony 9

### plasmid isolation, preparations for Sanger sequencing

**Team:** Endocytosis

**Investigators:** Alexander, Schulze

**Superior experiment:** Cloning ligand\_mCherry fusion proteins

**Procedure:**

* standard plasmid preparation protocol (Qiagen)
* preparations for Sanger sequencing

+ primers: SEQ1 (fwd) and SEQ2 (rev)

### 2019-08-12 - 2019-08-18

### transformation via heatshock, overnight cultures

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** ligand\_mCherry fusion protein expression

**Procedure:**

* standard transformation of BBa\_K2926050, BBa\_K2926049 and BBa\_K2926051 in pTXB1 via heatshock in E. coli ER2566
* streaked out on agar plates (Amp)
* overnight cultures (25 mL LB-Amp, 37 Â°C) of BBa\_K2926050, BBa\_K2926049 and BBa\_K2926051 in pTXB1 in E. coli ER2566

### protein expression

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** ligand\_mCherry fusion protein expression

**Procedure:**

* standard expression protocol: expression culture (250 mL LB-Amp, 37 Â°C) of BBa\_K2926050, BBa\_K2926049 and BBa\_K2926051 in pTXB1 in E. coli ER2566

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

### cell harvest, cell lysis, IMPACT purification

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** ligand\_mCherry fusion protein expression and purification

**Procedure:**

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

+ BBa\_K2926050: 2.17 g
+ BBa\_K2926049: 1.46 g
+ BBa\_K2926051: 1.3 g

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

### cell harvest, cell lysis, IMPACT purification

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** ligand\_mCherry fusion protein expression

**Procedure:**

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

+ BBa\_K2926068: 1.83 g

### Elution, washing and concentrating

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** ligand\_mCherry fusion protein purification

**Procedure:**

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

### Bradford assay, SDS-PAGE

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** ligand\_mCherry fusion protein purification

**Procedure:**

* standard bradford assay protocol (Roth, Roti nanoquant)

+ Flo\_mCherry: 470.3 ng/ÂµL; 503 Âµg
+ Mat\_mCherry: 445.71 ng/ÂµL; 356.57 Âµg
+ Opy\_mCherry: 196.95 ng/ÂµL; 236.38 Âµg

* standard SDS-PAGE protocol (12 %)

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

### TECAN-reader measurement

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** ligand\_mCherry fusion protein characterization

**Procedure:**

* Texas Red standard

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

* protein dilution series

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

* measurement of fluorescence intensity of dilution series (quadruplicates)

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

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

### TECAN-reader measurement

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** mCherry protein characterization

**Procedure:**

* light stability assay

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

* pH stability assay

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

### 2019-08-19 - 2019-08-25

### Pre culture

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Endocytosis assay S. cerevisiae

**Procedure:**

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

### Pre culture

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Endocytosis assay S. cerevisiae

**Procedure:**

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

### S. cerevisiae in minimal media

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Endocytosis assay S. cerevisiae

**Procedure:**

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

### Fluorescence measurement of supernatant

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Endocytosis assay S. cerevisiae

**Procedure:**

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

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

### 2019-08-26 - 2019-09-01

### Pre culture

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Fluorescence microscopy S. cerevisiae

**Procedure:**

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

### Fluorescence microscopy

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Fluorescence microscopy S. cerevisiae

**Procedure:**

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

### Pre culture

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Endocytosis assay S. cerevisiae

**Procedure:**

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

### Main culture, fluorescence measurement of supernatant

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Endocytosis assay S. cerevisiae

**Procedure:**

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

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

### 2019-09-02 - 2019-09-08

### Pre culture

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Fluorescence microscopy S. cerevisiae

**Procedure:**

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

### Fluorescence microscopy

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Fluorescence microscopy S. cerevisiae

**Procedure:**

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

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

* to potentially enhance uptake: digestion of cell wall

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

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

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

* trying out higher concentrated proteins

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

### Main culture, fluorescence measurement of pellet

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Endocytosis assay S. cerevisiae

**Procedure:**

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

### Main culture, fluorescence measurement of supernatant

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Endocytosis assay S. cerevisiae

**Procedure:**

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

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

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

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Cloning ligand\_mCherry proteins for Aspergillus niger

**Procedure:**

* standard PCR protocol (Q5, NEB)

+ primers: 19 p-m (fwd) and 19 p-n (rev)
+ template: BBa\_J06504 in pTXB1

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

### overnight cultures

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** mCherry and ligand\_mCherry fusion protein expression

**Procedure:**

* overnight cultures (25 mL LB-Amp, 37 Â°C) of BBa\_K2969051 and BBa\_J06504 in pTXB1 in E. coli ER2566

### protein expression

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** mCherry and ligand\_mCherry fusion protein expression

**Procedure:**

* standard expression protocol: expression culture (250 mL LB-Amp, 37 Â°C) of BBa\_K2969051 and BBa\_J06504 in pTXB1 in E. coli ER2566

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

### - overnight culture (25 mL LB-Amp, 37 Â°C) of BBa\_K2969050 in pTXB1 in E. coli ER2566

**Team:** Endocytosis

**Investigators:** overnight, cultures

**Superior experiment:** ligand\_mCherry fusion protein purification

**Procedure:**

### 2019-09-09 - 2019-09-15

### cell harvest

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** mCherry and ligand\_mCherry fusion protein expression

**Procedure:**

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

+ BBa\_K2926051: 1.71 g
+ BBa\_J06504: 1.61 g

### protein expression

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** mCherry and ligand\_mCherry fusion protein expression

**Procedure:**

* standard expression protocol: expression culture (250 mL LB-Amp, 37 Â°C) of BBa\_K2969050 in pTXB1 in E. coli ER2566

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

### - overnight culture (25 mL LB-Amp, 37 Â°C) of BBa\_K2969049 in pTXB1 in E. coli ER2566

**Team:** Endocytosis

**Investigators:** overnight, cultures

**Superior experiment:** ligand\_mCherry fusion protein purification

**Procedure:**

### cell lysis, IMPACT purification

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** mCherry and ligand\_mCherry fusion protein expression and purification

**Procedure:**

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

### protein expression

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** mCherry and ligand\_mCherry fusion protein expression

**Procedure:**

* standard expression protocol: expression culture (250 mL LB-Amp, 37 Â°C) of BBa\_K2969049 in pTXB1 in E. coli ER2566

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

### cell harvest

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** mCherry and ligand\_mCherry fusion protein expression

**Procedure:**

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

+ BBa\_K2926050: 1.66 g

### Elution, washing and concentrating

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** mCherry and ligand\_mCherry fusion protein purification

**Procedure:**

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

### cell lysis, IMPACT purification

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** ligand\_mCherry fusion protein purification

**Procedure:**

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

### Elution, washing and concentrating, Bradford assay

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** ligand\_mCherry fusion protein purification

**Procedure:**

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

+ Flo\_mCherry: 256 ng/ÂµL; 409 Âµg
+ Mat\_mCherry: 1.96 Âµg/ÂµL; 2.352 mg
+ Opy\_mCherry: 1.24 Âµg/ÂµL; 1.488 mg
+ mCherry: 2.42 Âµg/ÂµL; 3.315 mg

### Endocytosis assay S. cerevisiae

**Team:** Endocytosis

**Investigators:** Endocytosis

**Superior experiment:** >2019-08-29

**Procedure:**

- Johanna Opgenoorth
- Pre culture

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

### Main culture, fluorescence measurement of supernatant

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Endocytosis assay S. cerevisiae

**Procedure:**

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

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

### 2019-09-16 - 2019-09-22

### Pre culture

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Endocytosis assay S. cerevisiae

**Procedure:**

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

### Main culture, fluorescence measurement of supernatant

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Endocytosis assay S. cerevisiae

**Procedure:**

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

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

### colony PCR, analytical agarose gel, overnight culture

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Cloning ligand\_mCherry proteins for Aspergillus niger

**Procedure:**

* standard colony PCR protocol (GoTaq, Promega)

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

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

### Pre culture

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Fluorescence microscopy S. cerevisiae

**Procedure:**

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

### Plasmid isolation, transformation via heatshock

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Cloning ligand\_mCherry proteins for Aspergillus niger

**Procedure:**

* standard plasmid isolation (Qiagen)
* preparation for sanger sequencing

+ primers: SEQ1 (fwd) and SEQ2 (rev)

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

### - start of main culture (OD 0.2) in YPD

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Fluorescence microscopy S. cerevisiae

**Procedure:**

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

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

### 2019-09-23 - 2019-09-29

### overnight culture

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** ligand\_mCherry fusion protein expression

**Procedure:**

* overnight culture (25 mL LB-Amp, 37 Â°C) of BBa\_K2926068 in pTXB1 in E. coli ER2566

### protein expression

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** ligand\_mCherry fusion protein expression

**Procedure:**

* standard expression protocol: expression culture (250 mL LB-Amp, 37 Â°C) of BBa\_K2926068 in pTXB1 in E. coli ER2566

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

### 2019-09-30 - 2019-10-06

### cell lysis, protein purification

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** ligand\_mCherry fusion protein purification

**Procedure:**

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

### Elution, washing and concentrating

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** ligand\_mCherry fusion protein purification, Bradford assay

**Procedure:**

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

+ Pro\_mCherry: 70.7 ng/ÂµL; 67.9 Âµg

### SDS-PAGE

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** ligand\_mCherry fusion protein purification

**Procedure:**

* standard SDS-PAGE protocol (12 %)

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

### TECAN-reader measurement

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** ligand\_mCherry fusion protein characterization

**Procedure:**

* Texas Red standard

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

* protein dilution series

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

* measurement of fluorescence intensity of dilution series (quadruplicates)

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

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

### overnight culture

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** ligand\_mCherry fusion protein expression

**Procedure:**

* overnight culture (25 mL LB-Amp, 37 Â°C) of BBa\_K2926068 in pTXB1 in E. coli ER2566

### protein expression

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** ligand\_mCherry fusion protein expression

**Procedure:**

* standard expression protocol: expression culture (250 mL LB-Amp, 37 Â°C) of BBa\_K2926068 in pTXB1 in E. coli ER2566

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

### cell harvest, cell lysis, IMPACT purification

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** ligand\_mCherry fusion protein expression

**Procedure:**

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

+ BBa\_K2926068: 1.57 g

* storage at -80 Â°C

### Pre culture

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Endocytosis assay A. niger

**Procedure:**

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

### preparations for MALDI-ToF

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Protein characterization

**Procedure:**

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

### 2019-10-07 - 2019-10-13

### preparations for MALDI-ToF

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Protein characterization

**Procedure:**

* washing and tryptic digestion of SDS-PAGE protein bonds

### MALDI-Tof

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Protein characterization

**Procedure:**

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

### Endocytosis assay A. niger

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Endocytosis assay A. niger

**Procedure:**

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

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

### Endocytosis assay A. niger

**Team:** Endocytosis

**Investigators:** Johanna, Opgenoorth

**Superior experiment:** Endocytosis assay A. niger

**Procedure:**

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

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


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# Team:Bielefeld-CeBiTec/Experiments

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  + [Bielefeld](https://2019.igem.org/Team%3ABielefeld-CeBiTec/Bielefeld)
* [Partner](https://2019.igem.org/Team%3ABielefeld-CeBiTec/Partner)
* [Judging Form](https://igem.org/2019_Judging_Form?team=Bielefeld-CeBiTec)

Progress Indicator Animation

Protocols

![](https://static.igem.org/mediawiki/2019/f/fc/T--Bielefeld-CeBiTec--header_pipetten.jpg)

# Protocols

This page
contains a list of various methods applied during our experimental time in the
laboratory. A list of enzymes, polymerases, and other kinds of enzymes is also provided below.

* [50x TAE](https://static.igem.org/mediawiki/2019/4/49/T--Bielefeld-CeBiTec--50xTAE_Protocol.pdf)
* [Bradford Assay with ROTI® Nanoquant](https://static.igem.org/mediawiki/2019/0/0e/T--Bielefeld-CeBiTec--BradfordAssay_Protocol.pdf)
* [Cas13a Activity Assay](https://static.igem.org/mediawiki/2019/b/b2/T--Bielefeld-CeBiTec--Cas13a_Assay_Protocol.pdf)
* [Cell Lysis French Press](https://static.igem.org/mediawiki/2019/2/2e/T--Bielefeld-CeBiTec--FrenchPress_Protocol.pdf)
* [Cell Lysis Ribolyzer](https://static.igem.org/mediawiki/2019/5/5b/T--Bielefeld-CeBiTec--Ribolyzer_Protocol.pdf)
* [Chemically Competent Cells](https://static.igem.org/mediawiki/2019/a/a0/T--Bielefeld-CeBiTec--ChemCompetentCells_Protocol.pdf)
* [CM Medium (Aspergillus)](https://static.igem.org/mediawiki/2019/1/13/T--Bielefeld-CeBiTec--CMMedium_Protocol.pdf)* [Colony PCR with GoTaq (Promega)](https://static.igem.org/mediawiki/2019/9/9b/T--Bielefeld-CeBiTec--GoTaq_ColPCR_Protocol.pdf)
  * [Colony PCR with LongAmp (NEB)](https://static.igem.org/mediawiki/2019/1/1c/T--Bielefeld-CeBiTec--LongAmp_ColPCR_Protocol.pdf)
  * [Colony PCR with Taq Polymerase](https://static.igem.org/mediawiki/2019/9/97/T--Bielefeld-CeBiTec--Taq_ColPCR_Protocol.pdf)
  * [ddPCR](https://static.igem.org/mediawiki/2019/3/3c/T--Bielefeld-CeBiTec--ddPCR_Protocol.pdf)
  * [Endocytosis Test](https://static.igem.org/mediawiki/2019/f/f9/T--Bielefeld-CeBiTec--EndocytosisTest_Protocol.pdf)
  * [Fluorescence Microscopy](https://static.igem.org/mediawiki/2019/4/40/T--Bielefeld-CeBiTec--FluorescenceMicroscope_Protocol.pdf)
  * [Fungicide Simulation](https://static.igem.org/mediawiki/2019/2/29/T--Bielefeld-CeBiTec--FungicideSimulation_Protocol.pdf)
  * [Gel and PCR Cleanup Kit (Promega)](https://static.igem.org/mediawiki/2019/4/42/T--Bielefeld-CeBiTec--Promega_PCRCleanup_Protocol.pdf)
  * [Gel and PCR Cleanup Kit (Qiagen)](https://static.igem.org/mediawiki/2019/e/ef/T--Bielefeld-CeBiTec--Qiagen_PCRCleanup_Protocol.pdf)
  * [Gibson Assembly](https://static.igem.org/mediawiki/2019/a/a6/T--Bielefeld-CeBiTec--GibsonAssembly_Protocol.pdf)
  * [Golden Gate Assembly](https://static.igem.org/mediawiki/2019/7/75/T--Bielefeld-CeBiTec--GoldenGate_Protocol.pdf)
  * [Heat Shock Transformation](https://static.igem.org/mediawiki/2019/b/b3/T--Bielefeld-CeBiTec--HeatShock_Transformation_Protocol.pdf)
  * [His-Tag Purification](https://static.igem.org/mediawiki/2019/9/90/T--Bielefeld-CeBiTec--HisTag_Purification_Protocol.pdf)
  * [IMPACT Protein Purification](https://static.igem.org/mediawiki/2019/8/8a/T--Bielefeld-CeBiTec--ImpactKit_Protocol.pdf)
  * [Lab Application Assay](https://static.igem.org/mediawiki/2019/5/5b/T--Bielefeld-CeBiTec--LabApplication_Assay_Protocol.pdf)

* [LB Medium](https://static.igem.org/mediawiki/2019/8/8f/T--Bielefeld-CeBiTec--LBMedium_Protocol.pdf)
* [Light Stability Assay](https://static.igem.org/mediawiki/2019/8/8a/T--Bielefeld-CeBiTec--LightStability_Assay_Protocol.pdf)
* [Macherey Nagel Gel and PCR Cleanup Kit](https://static.igem.org/mediawiki/2019/0/0f/T--Bielefeld-CeBiTec--MachereyNagel_PCRCleanup_Protocol.pdf)
* [MALDI-ToF](https://static.igem.org/mediawiki/2019/c/c1/T--Bielefeld-CeBiTec--MALDITOF_Protocol.pdf)
* [Merck Protein Ultrafiltration](https://static.igem.org/mediawiki/2019/5/54/T--Bielefeld-CeBiTec--Merck_Ultrafiltration_Protocol.pdf)
* [Nanopore Sequencing](https://static.igem.org/mediawiki/2019/9/9f/T--Bielefeld-CeBiTec--NanoporeSequencing_Protocol.pdf)
* [PCR with Q5 Polymerase (NEB)](https://static.igem.org/mediawiki/2019/b/bf/T--Bielefeld-CeBiTec--Q5PCR_Protocol.pdf)
* [PBS Buffer](https://static.igem.org/mediawiki/2019/0/07/T--Bielefeld-CeBiTec--PBSBuffer_Protocol.pdf)
* [pH Sensitivity Assay](https://static.igem.org/mediawiki/2019/2/27/T--Bielefeld-CeBiTec--pH_Sensitivity_Assay_Protocol.pdf)
* [Phage Plaque Assay](https://static.igem.org/mediawiki/2019/3/3e/T--Bielefeld-CeBiTec--PhagePlaqueAssay_Protocol.pdf)
* [Phage Purification](https://static.igem.org/mediawiki/2019/c/c5/T--Bielefeld-CeBiTec--Isolation_PhageDNA_Protocol.pdf)
* [Phage Purification: Protein](https://static.igem.org/mediawiki/2019/3/32/T--Bielefeld-CeBiTec--PhageProtein_Protocol.pdf)
* [Phage Purification: NEB Protocol](https://static.igem.org/mediawiki/2019/3/32/T--Bielefeld-CeBiTec--PhagePurification_NEB_Protocol.pdf)
* [Plasmid Purification Kit (Qiagen)](https://static.igem.org/mediawiki/2019/f/fd/T--Bielefeld-CeBiTec--Qiagen_PlasmidKit_Protocol.pdf)
* [Plasmid Purification Kit (Zymo)](https://static.igem.org/mediawiki/2019/6/6b/T--Bielefeld-CeBiTec--Zymo_PlasmidKit_Protocol.pdf)
* [Restriction Ligation Cloning](https://static.igem.org/mediawiki/2019/a/a6/T--Bielefeld-CeBiTec--RestrictionLigation_Cloning_P)
* [SD Medium](https://static.igem.org/mediawiki/2019/8/8a/T--Bielefeld-CeBiTec--SDMedium_Protocol.pdf)
* [SDS PAGE](https://static.igem.org/mediawiki/2019/c/c4/T--Bielefeld-CeBiTec--SDSPAGE_Protocol.pdf)
* [SOC Medium](https://static.igem.org/mediawiki/2019/d/dd/T--Bielefeld-CeBiTec--SOCMedium_Protocol.pdf)
* [Yeast Transformation](https://static.igem.org/mediawiki/2019/d/d5/T--Bielefeld-CeBiTec--YeastCells_Transformation_Protocol.pdf)
* [YT Medium](https://static.igem.org/mediawiki/2019/7/7f/T--Bielefeld-CeBiTec--YTMedium_Protocol.pdf)
* [YPD Medium](https://static.igem.org/mediawiki/2019/9/94/T--Bielefeld-CeBiTec--YPDMedium_Protocol.pdf)

# **Enzymes**

## **Restriction Enzymes**

| **Enzyme** | **Manufacturer** |
| --- | --- |
| [BamHI (High Fidelity)](https://international.neb.com/products/r3136-bamhi-hf) | NEB |
| [BbsI](https://international.neb.com/products/r0539-bbsi) | NEB |
| [BbsI (High Fidelity)](https://international.neb.com/products/r3539-bbsi-hf#Product%20Information) | NEB |
| [DpnI](https://international.neb.com/products/r0176-dpni#Product%20Information) | NEB |
| [EcoRI (High Fidelity)](https://international.neb.com/products/r3101-ecori-hf#Product%20Information) | NEB |
| [MfeI (High Fidelity)](https://international.neb.com/products/r3589-mfei-hf#Product%20Information) | NEB |
| [MunI (FastDigest)](https://www.thermofisher.com/order/catalog/product/FD0753) | ThermoFischer |
| [NcoI (High Fidelity)](https://international.neb.com/products/r3193-ncoi-hf#Product%20Information) | NEB |
| [NheI (High Fidelity)](https://international.neb.com/products/r3131-nhei-hf#Product%20Information) | NEB |
| [NotI (High Fidelity)](http://www.neb.com/products/r3189-noti-hf#Product%20Information) | NEB |

## **Polymerase**

| **Polymerase** | **Manufacturer** |
| --- | --- |
| [GoTaq DNA Polymerase](https://www.promega.de/products/pcr/taq-polymerase/gotaq-dna-polymerase/) | Promega |
| [LongAmp Taq DNA Polymerase](https://international.neb.com/products/m0323-longamp-taq-dna-polymerase#Product%20Information) | NEB |
| [OneTaq](https://international.neb.com/products/m0480-onetaq-dna-polymerase#Product%20Information) | NEB |
| [Phusion® High-Fidelity DNA Polymerase](https://international.neb.com/products/m0530-phusion-high-fidelity-dna-polymerase#Product%20Information) | NEB |
| [Q5 High-Fidelity DNA Polymerase](https://international.neb.com/products/m0491-q5-high-fidelity-dna-polymerase) | NEB |
| [Taq DNA Polymerase](https://international.neb.com/products/pcr-qpcr-and-amplification-technologies/taq-dna-polymerases/taq-dna-polymerases) | NEB |

## **Others**

| **Others** | **Manufacturer** |
| --- | --- |
| [Klenow Fragment](https://www.thermofisher.com/order/catalog/product/EP0054#/EP0054) | Thermo Scientific |
| [Klenow Fragment](https://international.neb.com/products/m0212-klenow-fragment-3-5-exo#Product%20Information) | NEB |
| [T4 Ligase](https://international.neb.com/products/m0202-t4-dna-ligase#Product%20Information) | NEB |

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:Bielefeld-CeBiTec/Results

[![](https://static.igem.org/mediawiki/2019/2/27/T--Bielefeld-CeBiTec--Test_Logo.png)](https://2019.igem.org/Team%3ABielefeld-CeBiTec)

Menu ≡
[ ]

* Project ▾
  [Project](https://2019.igem.org/Team%3ABielefeld-CeBiTec/OverviewProject)
  [ ]
  + [Overview](https://2019.igem.org/Team%3ABielefeld-CeBiTec/OverviewProject)
  + [Description](https://2019.igem.org/Team%3ABielefeld-CeBiTec/Description)
  + [Background](https://2019.igem.org/Team%3ABielefeld-CeBiTec/Background)
  + [Troygenic Design](https://2019.igem.org/Team%3ABielefeld-CeBiTec/Design)
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  [ ]
  + [Overview](https://2019.igem.org/Team%3ABielefeld-CeBiTec/Results)
  + [Troygenic Assembly](https://2019.igem.org/Team%3ABielefeld-CeBiTec/Construction)
  + [Endocytosis](https://2019.igem.org/Team%3ABielefeld-CeBiTec/Endocytosis)
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Progress Indicator Animation

Results Overview

![](https://static.igem.org/mediawiki/2019/8/8c/T--Bielefeld-CeBiTec--header_stifte.jpg)

![](https://static.igem.org/mediawiki/2019/6/60/T--Bielefeld-CeBiTec--overview.png)

**Summary**
Here you can find the results of all experimental aspects of our project, starting with the Troygenic assembly,
endocytosis and CeDIS. These parts of the project include the cloning of several plasmids. Thereupon, (the) model, measurement, and hardware, each of which greatly contributed to the respective aspects of the construction of the Troygenics, are presented. Finally, we demonstrate our results and achievements, and present an outlook on further research possibilities.

|  |  |
| --- | --- |
| [![](https://static.igem.org/mediawiki/2019/e/e8/T--Bielefeld-CeBiTec--IconAssembly.png)](https://2019.igem.org/Team%3ABielefeld-CeBiTec/Construction) | [Troygenic Assembly](https://2019.igem.org/Team%3ABielefeld-CeBiTec/Construction)  Pivotal for our project is the construction of our Troygenics. To achieve this, we designed two plasmids and co-transformed them into *E. coli* which than produce Troygenics. Afterwards, we demonstrated the correct assembly with different methods, including atomic force microscopy, ddPCR and nanopore sequencing. |
| [![](https://static.igem.org/mediawiki/2019/0/0f/T--Bielefeld-CeBiTec--Ikon_Endocytosis.png)](https://2019.igem.org/Team%3ABielefeld-CeBiTec/Endocytosis) | [Endocytosis](https://2019.igem.org/Team%3ABielefeld-CeBiTec/Endocytosis)  One of the key issues we had to face was to get our Troygenic selectively internalized into the target cell. We were able to prove specific endocytosis of several cell specific ligands by fusing them to the fluorescence reporter mCherry and observing the uptake via fluorescence measurements. |
| [![](https://static.igem.org/mediawiki/2019/9/93/T--Bielefeld-CeBiTec--Ikon_CeDIS.png)](https://2019.igem.org/Team%3ABielefeld-CeBiTec/CeDIS) | [CeDIS](https://2019.igem.org/Team%3ABielefeld-CeBiTec/CeDIS)  Our varieties of Cell Death Inducing System (CeDIS) consist of different Cas13a systems in combination with guide RNAs. The CeDIS results in collateral RNA cleavage and leads to cell death. We have demonstrated the functionality of Lbu and Lsh with growth experiments. |
| [![](https://static.igem.org/mediawiki/2019/f/fc/T--Bielefeld-CeBiTec--Ikon_Model.png)](https://2019.igem.org/Team%3ABielefeld-CeBiTec/Model) | [Model](https://2019.igem.org/Team%3ABielefeld-CeBiTec/Model)  Using our model, we identified the optimal split points for split antibiotic resistances by implementing structural- and 3D-modeling. We verified this model in wet lab experiments of a split-chloramphenicol resistance.Furthermore, we calculated the adequate number of gRNAs for lab experiments, using a mathematical modeling approach including growth curve modeling. |
| [![](https://static.igem.org/mediawiki/2019/c/c1/T--Bielefeld-CeBiTec--Ikon_Hardware.png)](https://2019.igem.org/Team%3ABielefeld-CeBiTec/Hardware) | [Hardware](https://2019.igem.org/Team%3ABielefeld-CeBiTec/Hardware)  To get a deeper insight into the behavior of our model organisms, we designed microfluidic chips, which we produced using 3D-printing. On these chips we cultivated *S. cerevisiae* and *A. niger*. We also designed and built a low-cost microfluidic laboratory starter kit to make microfluidics easily accessible for other teams. |
| [![](https://static.igem.org/mediawiki/2019/a/a1/T--Bielefeld-CeBiTec--Ikon_Measurement.png)](https://2019.igem.org/Team%3ABielefeld-CeBiTec/Measurement) | [Measurement](https://2019.igem.org/Team%3ABielefeld-CeBiTec/Measurement)  To enable the generation of scientifically sound data, it is necessary to standardize all measurements. In the context of life sciences, it is of uttermost importance to achieve this for fluorescence data. After the iGEM measurement committee had established fluorescein as a reference for all GFP measurements, we conferred the same principle to mCherry measurements. We introduced Texas Red as a reference fluorophore for several mCherry applications. |
| [![](https://static.igem.org/mediawiki/2019/9/91/T--Bielefeld-CeBiTec--Ikon_Demonstrate.png)](https://2019.igem.org/Team%3ABielefeld-CeBiTec/Demonstrate) | [Demonstrate](https://2019.igem.org/Team%3ABielefeld-CeBiTec/Demonstrate)  We demonstrated that every part of our Troygenic workes as designed. Firstly, we showed the correct assembly of the Troygenic. Thereafter, we showed receptor induced endocytosis of specific ligands for *S. cerevisiae* and *A. niger*. Moreover, we demonstrated that our Cas13a-based CeDIS is working. Finally, we have successfully assembled a Troygenic which was able to transform *S. cerevisiae*. |
| [![](https://static.igem.org/mediawiki/2019/c/c7/T--Bielefeld-CeBiTec--Ikon_Outlook.png)](https://2019.igem.org/Team%3ABielefeld-CeBiTec/Outlook) | [Outlook](https://2019.igem.org/Team%3ABielefeld-CeBiTec/Outlook)  On this page, we describe the possible approaches to optimize the effectivity of our Troygenics and the potentials of customizing our system for various applications in the future. |