iGEM 2020 — Team Bielefeld-CeBiTec

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# Team:Bielefeld-CeBiTec/Notebook

Bielefeld-Cebitec-2020-Notebook

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* [**Home**](https://2020.igem.org/Team%3ABielefeld-CeBiTec)
* [**Overview**](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Description)
* Project ▼
  + [Background](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Background)
  + [Nanobodies & scFv's](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Nanobodies_Project)
  + [Hardware](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Technology_Project)
  + [Implementation](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Implementation)
* Results ▼
  + [Nanobodies & scFv's](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Nanobodies)
  + [Hardware](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Hardware_Results)
  + [Measurement](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Measurement)
  + [Modelling](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Model)
  + [App/Software](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Software)
  + [Contribution](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Contribution)
  + [Engineering](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Engineering)
  + [Proof of concept](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Proof_Of_Concept)
* Lab ▼
  + [Safety](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Safety)
  + [Notebook](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Notebook)
  + [Protocols](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Protocols)
* Parts ▼
  + [Parts](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Parts)
  + [Improve](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Improve)
* Human Practice ▼
  + [Integrated Human Practise](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Human_Practices)
  + [Education &
     Public Engagement](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Education)

  + [Entrepreneurship](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Entrepreneurship)
  + [Collaborations](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Collaborations)
  + [Inclusion](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Inclusion)
* Team ▼
  + [Members](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Team)
  + [Attributions](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Attributions)
  + [CeBiTec](https://2020.igem.org/Team%3ABielefeld-CeBiTec/CeBiTec)
  + [Uni Bielefeld](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Uni_Bielefeld)
  + [Bielefeld](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Bielefeld)
* [**Partner**](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Partner)
* [**Judging Form**](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Judgingform)

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Notebook

## 2020-08-23 - 2020-08-30

##### 2020.08.23

Team: ScFv

Investigators: Niko Klassen & Philipp Kühnel

Superior Experiment: Nanobody grafting

Procedure: Buffer and media prepared

##### 2020.08.24

Team: ScFv

Investigators: Niko Klassen & Philipp Kühnel

Superior Experiment: ScFv grafting

Procedure: Buffer and media prepared

##### 2020.08.25

Team: Nanobodys

Investigators: Stephan Weber

Superior Experiment: Nanobody grafting

Procedure: Streak out of E.coli DH5α on LB-plates

##### 2020.08.26

Team: Nanobodys

Investigators: Stephan Weber

Superior Experiment: Nanobody grafting

Procedure: Phages M13K07 frozen away

Team: ScFv

Investigators: Stephan Weber

Superior Experiment: ScFv grafting

Procedure: Overnight culture of E.coli DH5α in LB-media

Team: ScFv

Investigators: Philipp Kühnel

Superior Experiment: ScFv grafting

Procedure: Plasmid isolation

##### 2020.08.27

Team: Nanobodys

Investigators: Stephan Weber

Superior Experiment: Nanobody grafting

Procedure: Testrun of eror prone PCR of pSB1A3 with Q5 polymerase

Team: Nanobodys

Investigators: Laura Keller & Stephan Weber

Superior Experiment: Nanobody grafting

Procedure: Amplication of Helper Phage M13K07

Team: Nanobodys

Investigators: Philipp Kühnel

Superior Experiment: Nanobody grafting

Procedure: Culture overnight with E.coli ER2738

Team: Nanobodys

Investigators: Philipp Kühnel & Laura Keller

Superior Experiment: Nanobody grafting

Procedure: Culture overnight with E.coli ER2738

Team: ScFv

Investigators: Laura Keller

Superior Experiment: ScFv grafting

Procedure: Preparation of SOC-media and TBS-buffer

##### 2020.08.28

Team: Nanobodys

Investigators: Stephan Weber

Superior Experiment: Nanobody grafting

Procedure: Eroro prone PCR of pSB1A3 with Taq, OneTaq and BioTaq polymerase, buffer prepared

Read More

## 2020-08-31 - 2020-09-06

##### 2020.08.31

Team: Nanobodys

Investigators: Stephan Weber & Neele Kusch

Superior Experiment: Nanobody grafting

Procedure: DPN1-digestion, Plasmid isolation

##### 2020.09.01

Team: Nanobodys

Investigators: Neele Kusch, Laura Keller, Stephan Weber, Marvin Geisler

Superior Experiment: Nanobody grafting

Procedure: Eror prone PCR of pSB1A3 with Taq and OneTaq polymerase

##### 2020.09.02

Team: Nanobodys

Investigators: Stephan Weber, Neele Kusch, Laura Keller

Procedure: PCR cleanup, gibson assembly, Heat shock transformation of E.coli DH5α with pJOE

##### 2020.09.03

Team: Nanobodys

Investigators: Stephan Weber & Neele Kusch

Superior Experiment: Nanobody grafting

Procedure: Digestion of pJOE with BAM2 and DPN1, heat shock transformation of E.coli DH5α with pJOE

##### 2020.09.04

Team: Nanobodys

Investigators: Stephan Weber & Neele Kusch

Superior Experiment: Nanobody grafting

Procedure: Colony PCR with Q5 polymerase

Read More

## 2020-09-07 - 2020-09-13

##### 2020.09.07

Team: ScFv

Investigators: Philipp Kühnel & Laura Keller

Superior Experiment: ScFv grafting

Procedure: PCR of ScFv with Q5 polymerase, PCR cleanup, gibson assembly, heat shock

 transformation of E.coli DH5α with pTXB1

##### 2020.09.08

Team: Nanobodys

Investigators: Stephan Weber, Neele Kusch, Niko Klassen

Superior Experiment: Nanobody grafting

Procedure: Eror prone PCR of estradiol and progesteron nanobody with OneTaq polymerase

Team: ScFv

Investigators: Philipp Kühnel, Laura Keller, Tim Kettler

Superior Experiment: ScFv grafting

Procedure: Heat shock transformation of E.coli DH5α with pTXB1, overnight culturen of E.coliDH5α

##### 2020.09.09

Team: Nanobodys

Investigators: Stephan Weber & Neele Kusch

Superior Experiment: Nanobody grafting

Procedure: Gel electrophoresis of eror prone PCR, PCR cleanup

Team: Nanobodys

Investigators: Marvin Geisler

Superior Experiment: Nanobody grafting

Procedure: Preparation of antibiotics

Team: ScFv

Investigators: Philipp Kühnel & Laura Keller

Superior Experiment: ScFv grafting

Procedure: Production of competent E.coli DH5α, overnight culture of E.coli DH5α

##### 2020.09.010

Team: Nanobodys

Investigators: Niko Klassen & Tim Kettler

Superior Experiment: Nanobody grafting
Procedure: Eror prone PCR of progesteron nanobody with OneTag polymerase

Team: Nanobodys

Investigators: Stephan Weber & Neele Kusch

Superior Experiment: Nanobody grafting

Procedure: Produktion of electro-competent E.coli ER2738

##### 2020.09.11

Team: Nanobodys

Investigators: Stephan Weber & Neele Kusch

Superior Experiment: Nanobody grafting

Procedure: Grandient PCR of estradiol and progesteron nanobody with OneTaq polymerase

Team: ScFv

Investigators: Philipp Kühnel & Laura Keller

Superior Experiment: ScFv grafting

Procedure: Plasmid isolation , PCR of ScFv with Taq polymerase, PCR cleanup, gibson assembly, heat shock tranformation of E.coli DH5α with pTXB1

##### 2020.09.12

Team: ScFv

Investigators: Philipp Kühnel & Laura Keller

Superior Experiment: ScFv grafting

Procedure: Overnight culture with E.coli DH5α, streak out on LB-plates

Read More

## 2020-09-14 - 2020-09-20

##### 2020.09.14

Team: ScFv

Investigators: Philipp Kühnel, Laura Keller, Tim kettler

Superior Experiment: ScFv grafting

Procedure: Culture with E.coli DH5α, streak out on LB-plates with ampicilin, Plasmid isolation

Team: Nanobodys

Investigators: Stephan Weber & Neele Kusch

Superior Experiment: Nanobody grafting

Procedure: Eror prone PCR of progesteron nanobody with OneTag polymerase

##### 2020.09.15

Team: Nanobodys

Investigators: Stephan Weber & Neele Kusch

Superior Experiment: Nanobody grafting

Procedure: Grandient PCR of progesteron nanobody with OneTaq, eror prone PCR with progesteron nanobody

Team: ScFv

Investigators: Tim Kettler

Superior Experiment: ScFv grafting

Procedure: Plasmid isolation , overnight culture with E.coli DH5α

##### 2020.09.16

Team: Nanobodys

Investigators: Stephan Weber

Superior Experiment: Nanobody grafting

Procedure: Eror prone PCR of progesteron nanobody with OneTag polymerase

##### 2020.09.17

Team: Nanobodys

Investigators: Stephan Weber & Neele Kusch

Superior Experiment: Nanobody grafting

Procedure: Grandient PCR of progesteron nanobody with OneTaq polymerase, PCR of progesteron nanobody with Phusion polymerase and PCR of stradiol nanobody with OneTaq polymerease

##### 2020.09.18

Team: Nanobodys

Investigators: Niko Klassen & Marvin Geisler

Superior Experiment: Nanobody grafting

Procedure: Gel electrophoresis

Read More

## 2020-09-21 - 2020-09-27

##### 2020.09.21

Team: ScFv

Investigators: Philipp Kühnel & Laura Keller

Superior Experiment: ScFv grafting

Procedure: Plasmid isolation, heat shock tranformation of E.coli DH5α with pTXB1

Team: Nanobodys

Investigators: Stephan Weber & Neele Kusch

Superior Experiment: Nanobody grafting

Procedure: Eror prone PCR of estradiol nanobody with OneTaq polymerase

##### 2020.09.22

Team: ScFv

Investigators: Philipp Kühnel & Laura Keller

Superior Experiment: ScFv grafting

Procedure: Culture with E.coli DH5α, preparatio of LB-plates with ampicillin

Team: Nanobodys

Investigators: Stephan Weber & Neele Kusch

Superior Experiment: Nanobody grafting

Procedure: Eror prone PCR of estradiol nanobody with OneTaq polymerase

##### 2020.09.23

Team: Improve a Part

Investigators: Melina Müller

Superior Experiment: FbFP caracterization

Procedure: PCR of pJOE and FbFP with Phusion polymerase, PCR cleanup, gibson assembly

Team: ScFv

Investigators: Philipp Kühnel & Laura Keller

Superior Experiment: ScFv grafting

Procedure: Plasmid isolation, PCR of pTXB1 with Taq polymerase

Team: Improve a Part

Investigators: Tim Kettler

Superior Experiment: FbFP caracterization

Procedure: Gel electrophoresis

Team: Nanobodys

Investigators: Stephan Weber & Neele Kusch

Superior Experiment: Nanobody grafting

Procedure: Eror prone PCR of progesteron and estradiol nanobody with Taq polymerase

##### 2020.09.24

Team: Improve a Part

Investigators: Melina Müller & Tim Kettler

Superior Experiment: FbFP caracterization

Procedure: Heat shock tranformation of E.coli DH5α with pJOE

Team: Nanobodys

Investigators: Stephan Weber & Neele Kusch

Superior Experiment: Nanobody grafting

Procedure: Eror prone PCR of progesteron and estradiol nanobody with Taq polymerase

Team: ScFv

Investigators: Tim Kettler

Superior Experiment: ScFv grafting

Procedure: Overnight Culture with E.coli DH5α

##### 2020.09.25

Team: Improve a Part

Investigators: Melina Müller & Tim Kettler

Superior Experiment: FbFP caracterization

Procedure: Colony PCR of FbFP with Phusion polymerase

Team: ScFv

Investigators: Philipp Kühnel & Laura Keller

Superior Experiment: ScFv grafting

Procedure: PCR of FbFP with Phusion polymerase and gel-extraction, Plasmid isolation

##### 2020.09.27

Team: ScFv

Investigators: Melina Müller

Superior Experiment: ScFv grafting

Procedure: Overnight Culture with E.coli DH5α

Team: Improve a Part

Investigators: Tim Kettler

Superior Experiment: FbFP caracterization

Procedure: Overnight Culture with E.coli DH5α

Read More

## 2020-09-28 - 2020-10-04

##### 2020.09.28

Team: Improve a Part

Investigators: Angelika Urban & Tim Kettler

Superior Experiment: FbFP caracterization

Procedure: Overnight Culture with E.coli DH5α

Team: Nanobodys

Investigators: Neele Kusch, Niko Klassen, Stephan Weber, Marvin Geisler

Superior Experiment: Nanobody grafting

Procedure: Sequencing of estradiol and progesteron nanobody, preparation of media and buffer

##### 2020.09.29

Team: Improve a Part

Investigators: Angelika Urban, Tim Kettler, Melina Müller

Superior Experiment: FbFP caracterization

Procedure: Culture with E.coli DH5α, Plasmid isolation, PCR of FbAP with Phusion, PCR cleanup, heat shock transformation of E.coli DH5α with pJOE

Team: ScFv

Investigators: Philipp Kühnel & Laura Keller

Superior Experiment: ScFv grafting

Procedure:PCR with phusion and gel-extraction, gibson assembly, heat shock transformation of E.coli DH5α with pTXB1, Overnight Culture with E.coli DH5α

Team: Nanobodys

Investigators: Stephan Weber & Neele Kusch

Superior Experiment: Nanobody grafting

Procedure:Phage display with M18K07, produktion of elektrokompetent E.coli ER2738, elctroshock transformation of E.coli ER2738 with PZMB0062

##### 2020.10.01

Team: Nanobodys

Investigators: Stephan Weber & Neele Kusch

Superior Experiment: Nanobody grafting

Procedure:Digestion of PZMB0062 with Sfi1

Team: ScFv

Investigators: Philipp Kühnel & Laura Keller

Superior Experiment: ScFv grafting

Procedure:PCR of ScFv with Q5 polymerase, PCR cleanup, gibson assembly, heat shock transformation of E.coli DH5α with pTXB1

Team: Improve a Part

Investigators: Tim Kettler

Superior Experiment: FbFP caracterization

Procedure:Overnight Culture with E.coli DH5α, colony PCR with Phusion polymerase

##### 2020.10.02

Team: ScFv

Investigators: Philipp Kühnel & Laura Keller

Superior Experiment: ScFv grafting

Procedure:PCR with phusion and gel-extraction, colony PCR with Taq polymerase, Plasmid isolation

Team: Improve a Part

Investigators: Melina Müller & Tim Kettler

Superior Experiment: FbFP caracterization

Procedure:Plasmid isolation, colony PCR with Taq polymerase

Team: Nanobodys

Investigators: Stephan Weber & Marvin Geilser

Superior Experiment: Nanobody grafting

Procedure:Preparation of antibiotics, electroshock transformation of E.coli ER2738 with PZMB0062

Read More

## 2020-10-05 - 2020-10-11

##### 2020.10.05

Team: Improve a Part

Investigators: Melina Müller & Tim Kettler

Superior Experiment: FbFP caracterization

Procedure:PCR of FbFP with Phusion polymerase, PCR cleanup, gibson assembly, heat shock transformation of E.coli DH5α with pJOE, overnight culture with E.coli DH5α

Team: Nanobodys

Investigators: Stephan Weber & Marvin Geilser

Superior Experiment: Nanobody grafting

Procedure:Elctroshock transformation of E.coli ER2738 with PZMB0062, preparation of LB-plates

Team: ScFv

Investigators: Philipp Kühnel & Laura Keller

Superior Experiment: ScFv grafting

Procedure:Gibson assembly, AQUA cloning, PCR of ScFv with Phusion polymerase, PCR cleanup, heat shock transformation of E.coli DH5α

##### 2020.10.06

Team: ScFv

Investigators: Philipp Kühnel & Laura Keller

Superior Experiment: ScFv grafting

Procedure:Colony PCR with Taq polymerase, plasmid isolation

Team: Improve a Part

Investigators: Angelika Urban & Tim Kettler

Superior Experiment: FbFP caracterization

Procedure:Plasmid isolation, colony PCR with Taq polymerase

Team: Nanobodys

Investigators:Stephan Weber, Neele Kusch, Marvin Geisler

Superior Experiment: Nanobody grafting

Procedure:Eror prone PCR of progesteron and estradiol nanobody with Taq polymerase, PCR cleanup, preparation of LB-plates

##### 2020.10.7

Team: Nanobodys

Investigators:Stephan Weber & Neele Kusch

Superior Experiment: Nanobody grafting

Procedure:Digestion of PZMB0062 with Sfi1, gel extraction

Team: Improve a Part

Investigators:Melina Müller

Superior Experiment: FbFP caracterization

Procedure:PCR of FbFP with Phusion polymerase, PCR cleanup, gibson assembly, heat shock transformation of E.coli DH5α with pJOE, overnight culture with E.coli DH5α

Team: ScFv

Investigators:Philipp Kühnel & Laura Keller

Superior Experiment: ScFv grafting

Procedure:PCR of ScFv with Phusion polymerase, PCR cleanup

##### 2020.10.8

Team: Improve a Part

Investigators:Melina Müller

Superior Experiment: FbFP caracterization

Procedure:Overnight Culture with E.coli DH5α, colony PCR with Taq polymerase

Team: Nanobodys

Investigators:Stephan Weber, Neele Kusch, Niko Klassen

Superior Experiment: Nanobody grafting

Procedure:PCR of estradiol and progesteron nanobody, PCR cleanup

Team: ScFv

Investigators:Laura Keller

Superior Experiment: ScFv grafting

Procedure:Plasmid isolation, PCR of ScFv with Taq polymerase

##### 2020.10.9

Team: ScFv

Investigators:Philipp Kühnel

Superior Experiment: ScFv grafting

Procedure:Colony PCR with Taq polymerase, plasmid isolation

Team: Nanobodys

Investigators:Stephan Weber & Neele Kusch

Superior Experiment: Nanobody grafting

Procedure:Digestion of PZMB0062 with Sfi1, gel extraction, PCR of estradiol und progesteron nanobody with Phusion polymerase, eror prone PCR of estradiol and progesteron with Taq polymerase, PCR cleanup, ligation

Team: Improve a Part

Investigators:Tim Kettler

Superior Experiment:FbFP caracterization

Procedure:Gel electrophoresis

##### 2020.10.10

Team: Nanobodys

Investigators:Stephan Weber

Superior Experiment:Nanobody grafting

Procedure:Electroshock transformation of E.coli ER2738 with PZMB0062

##### 2020.10.11

Team: ScFv

Investigators:Philipp Kühnel

Superior Experiment:ScFv grafting

Procedure:Overnight colony with E.coli DH5α

Read More

## 2020-10-12 - 2020-10-18

##### 2020.10.12

Team: ScFv

Investigators:Philipp Kühnel & Laura Keller

Superior Experiment:ScFv grafting

Procedure:Plasmis isolation

Team: Nanobodys

Investigators:Stephan Weber & Neele Kusch

Superior Experiment:Nanobody grafting

Procedure:Electroshock transformation of E.coli ER2738 with PZMB0062, preparation of LB-plates

Team: Improve a Part

Investigators:Tim Kettler & Melina Müller

Superior Experiment:FbFP caracterization

Procedure:PCR of FbFP with Phusion polymerase, overnight culture with E.coli DH5α

##### 2020.10.13

Team: Improve a Part

Investigators:Tim Kettler & Melina Müller

Superior Experiment:FbFP caracterization

Procedure:PCR of FbFP with Phusion polmerase, PCR cleanup, gibson assembly, heat shock transformation of E.coli DH5α with pJOE, Plasmid isolation

Team: ScFv

Investigators:Philipp Kühnel & Laura Keller

Superior Experiment:ScFv grafting

Procedure:Overnigt culture with E.coli DH5α

Team: Nanobodys

Investigators:Stephan Weber & Neele Kusch

Superior Experiment:Nanobody grafting

Procedure:Electroshock transformation of E.coli ER2738, production of electrocompetent E.coli ER2738

##### 2020.10.14

Team: Nanobodys

Investigators:Stephan Weber & Neele Kusch

Superior Experiment:Nanobody grafting

Procedure:Digestion of pZMB0062 with Sti1, PCR cleanup, ligation

Team: Improve a Part

Investigators:Tim Kettler & Melina Müller

Superior Experiment:FbFP caracterization

Procedure:Colony PCR with Taq polymerase

##### 2020.10.15

Team: Improve a Part

Investigators:Tim Kettler

Superior Experiment:FbFP caracterization

Procedure:Colony PCR with Taq polymerase, overnight culture of E.coli DH5α

Team: Nanobodys

Investigators:Stephan Weber & Neele Kusch

Superior Experiment:Nanobody grafting

Procedure:Electroshock transformation of E.coli ER2738, Digestion of pZMB0062 with Sti1, ligation

##### 2020.10.16

Team: Improve a Part

Investigators:Tim Kettler

Superior Experiment:FbFP caracterization

Procedure:Plasmid isolation

Team: Nanobodys

Investigators:Stephan Weber & Neele Kusch

Superior Experiment:Nanobody grafting

Procedure:Electroshock transformation of E.coli ER2738

##### 2020.10.18

Team: ScFv

Investigators:Philipp Kühnel & Laura Keller

Superior Experiment:ScFv grafting

Procedure:PCR of ScFv with Phusion polymerase, PCR cleanup, DPN1 digestion, gibsen assembly, heat shock transformation of E.coli DH5α with pTXB1

Read More

## 2020-10-19 - 2020-10-25

##### 2020.10.19

Team: Nanobodys

Investigators:Stephan Weber & Neele Kusch

Superior Experiment:Nanobody grafting

Procedure:Culture of E.coli ER2738

Team: ScFv

Investigators:Philipp Kühnel & Laura Keller

Superior Experiment:ScFv grafting

Procedure:PCR of ScFv with Phusion polymerase, PCR cleanup, DPN1 digestion of pTXB1, gibsen assembly, heat shock transformation of E.coli DH5α with pTXB1

Team: Improve a Part

Investigators:Tim Kettler

Superior Experiment:FbFP caracterization

Procedure:Heat shock transformation of E.coli Bl21D3 with pJOE

##### 2020.10.20

Team: ScFv

Investigators:Philipp Kühnel & Laura Keller

Superior Experiment:FbFP caracterization

Procedure:PCR of ScFv with Phusion polymerase, PCR cleanup, digestion of pTXB1 with Spe1 and Nde1, ligation, heat shock transformation of E.coli DH5α with pTXB1

Team: Improve a Part

Investigators:Tim Kettler

Superior Experiment:FbFP caracterization

Procedure:Overnight culture with E.coli BL21D3

Team: Nanobodys

Investigators:Stephan Weber & Neele Kusch

Superior Experiment:Nanobody grafting

Procedure:Preparation of phages and immunotubes

##### 2020.10.21

Team: Improve a Part

Investigators:Tim Kettler

Superior Experiment:FbFP caracterization

Procedure:Culture with E.coli BL21D3

Team: Improve a Part

Investigators:Tim Kettler, Melina Müller, Angelika Urban

Superior Experiment:FbFP caracterization

Procedure:Fluorescens messurment with tecan-reader

Team: Nanobodys

Investigators:Stephan Weber & Neele Kusch

Superior Experiment:Nanobody grafting

Procedure:Digestion of pZMB0062 with Sti1, PCR cleanup, ligation, electroshock transformation of ER2738

Team: ScFv

Investigators:Laura Keller

Superior Experiment:ScFv grafting

Procedure:Colony PCR with Phusion Polymerase, PCR cleanup, digestion of pTXB1 with Spe1 and Nde1, ligation, heat shock transformation of E.coli DH5α

##### 2020.10.22

Team: ScFv

Investigators:Philipp Kühnel & Laura Keller

Superior Experiment:ScFv grafting

Procedure:PCR od ScFv with Phusion polymerase, colony PCR with Phusion polymerase

Team: Nanobodys

Investigators:Stephan Weber & Neele Kusch

Superior Experiment:Nanobody grafting

Procedure:Culture with E.coli ER2783 with phages

Team: Improve a Part

Investigators:Tim Kettler

Superior Experiment:FbFP caracterization

Procedure:Culture with E.coli BL21D3

##### 2020.10.23

Investigators:Philipp Kühnel & Laura Keller

Superior Experiment:ScFv grafting

Procedure:Plasmid isolation, digestion of pTXB1with Kpn1 and Hind3, digestion of pTXB1with EcoR5 and Hind3

Team: Improve a Part

Investigators:Tim Kettler

Superior Experiment:FbFP caracterization

Procedure:Preparation of buffer, Fluorescens messurment with tecan-reader

Team: Nanobodys

Investigators:Stephan Weber & Neele Kusch

Superior Experiment:Nanobody grafting

Procedure:Colony PCR with Phusion Polymerase

##### 2020.10.24

Team: Nanobodys

Investigators:Stephan Weber & Neele Kusch

Superior Experiment:Nanobody grafting

Procedure:Culture with E.coli ER2783, Preparation of phage ELISA

##### 2020.10.25

Team: Nanobodys

Investigators:Stephan Weber & Neele Kusch

Superior Experiment:Nanobody grafting

Procedure:Phage ELISA

Read More

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Bielefeld-CeBiTec/Engineering

Bielefeld-Cebitec-2020-Engineering

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* [**Home**](https://2020.igem.org/Team%3ABielefeld-CeBiTec)
* [**Overview**](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Description)
* Project ▼
  + [Background](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Background)
  + [Nanobodies & scFv's](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Nanobodies_Project)
  + [Hardware](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Technology_Project)
  + [Implementation](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Implementation)
* Results ▼
  + [Nanobodies & scFv's](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Nanobodies)
  + [Hardware](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Hardware_Results)
  + [Measurement](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Measurement)
  + [Modeling](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Model)
  + [App/Software](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Software)
  + [Contribution](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Contribution)
  + [Engineering](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Engineering)
  + [Proof of concept](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Proof_Of_Concept)
* Lab ▼
  + [Safety](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Safety)
  + [Notebook](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Notebook)
  + [Protocols](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Protocols)
* Parts ▼
  + [Parts](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Parts)
  + [Improve](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Improve)
* Human Practice ▼
  + [Integrated Human Practise](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Human_Practices)
  + [Education &
     Public Engagement](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Education)

  + [Entrepreneurship](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Entrepreneurship)
  + [Collaborations](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Collaborations)
  + [Inclusion](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Inclusion)
* Team ▼
  + [Members](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Team)
  + [Attributions](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Attributions)
  + [CeBiTec](https://2020.igem.org/Team%3ABielefeld-CeBiTec/CeBiTec)
  + [Uni Bielefeld](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Uni_Bielefeld)
  + [Bielefeld](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Bielefeld)
* [**Partner**](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Partner)
* [**Judging Form**](https://2020.igem.org/Team%3ABielefeld-CeBiTec/Judgingform)

-->

![](https://static.igem.org/mediawiki/2020/6/60/T--Bielefeld-CeBiTec--WikiWave.png)

Engineering

# Part 1 – The PCB

If we think about engineering, it is all about imagining a new system, design, build and test our approach, realize improvement potential and start again with redesign. This is called the design-build-test cycle – The fundamental concept of engineering. One example of how this concept is applied in our project, is the construction of our Printed Circuit Board (PCB). The PCB is one of the most important parts of the technical realization. But let’s start with the beginning - the design. From there our design, the build test cycle starts.

# Design

![](https://static.igem.org/mediawiki/2020/1/1c/T--Bielefeld-CeBiTec--PCB-3D.png)

Fig. 1 - 3D Model of the PCB

For the design of our PCB we worked together with the CITEC of the University of Bielefeld to define the technical implementation of our project idea and discussed the practical realization. The design of the board, as well as the schematics, were created by the working group "Cognitronics and Sensor Systems" according to our requieremnts to use the PCB for SAW masurements.
To be able to measure a phase shift at all, it is possible to design various circuits. Therefore, we discussed two possible realizations of the PCB.
One solution was to use an analog mixer. An exclusion criterion is the amplitude dependence of an analog mixer. This is problematic due to the fact that at the time the circuit diagrams were developed, we did not have any knowledge of the amplitude of the frequency passing through the SAW chip. Therefore, we decided to search for another approach.
A second possibility, which was finally chosen, was the use of an XOR-gate. The functionality of the XOR is described in the Project/Hardware section. We attempted to capture the signal curve of the XOR at the contact points to visualize it on the scope. The signal curves of the XOR are shown in Fig. 2 and 3.

![](https://static.igem.org/mediawiki/2020/b/bc/T--Bielefeld-CeBiTec--Scope_XOR_1.png)

Fig. 2 - XOR curve on the scope.

![](https://static.igem.org/mediawiki/2020/5/5a/T--Bielefeld-CeBiTec--Scope_XOR_2.png)

Fig. 3 - XOR curve on the scope.

# Build

With the big support of the CITEC, we were able to realize our idea and build our own PCB.
Since there was no sensing chip, no real phase shift could be evaluated by the antibody binding. So we had to simulate artificial phase shifts via the pin headers directly on the PCB. The areas marked in red in Fig. 4 can be connected in bridges, allowing a total of 16 different time shifts (in ps) to be simulated.

![](https://static.igem.org/mediawiki/2020/9/99/T--Bielefeld-CeBiTec--PCB_TOP_Marked.png)

Fig. 4 - PCB Pinheader Marked.

# Test

In order to cover the entire bandwidth of the DDS, we have generated the frequencies from 50 MHz to 600 MHz in 50 MHz steps for all 16 time shifts. We were able to detect the analog to digital converted voltage values on the serial console of the Arduino IDE. The results can be found in a table available for download below and in figure 5 below.

 [Download Table](https://github.com/Benni42/iGEM-2020--Bielefeld-CeBiTec)

![](https://static.igem.org/mediawiki/2020/9/93/T--Bielefeld-CeBiTec--PlottingShifts.png)

Fig. 5 - Matlab Plot of the derived phase shift and time shift in 2D and 3D illustration.

The 3D plots illustrate the dependence of the time shift in [ps] or the phase shift in [°] and the frequency in [Hz] to the resulting voltages in [V]. At first, it must be mentioned that the time shift was initiated with a specified track extension for a signal, which might cause the procedure to be incorrect. Furthermore, the phase shift is derived based on the time shift, which can also be a source of mistakes.
We can prove by our measurement that our PCB delivers a voltage change at the output in case of a phase shift on the input, which we could detect by reading the analog output. However, it must be clarified that our PCB model is not able to detect a perfectly valid phase shift as well as voltage change.

# Improve

To measure the precision of our board, further tests have to follow. The maximum mistake must be calculated to receive the percentage deviation. Moreover, the DDS generates the frequencies with a certain interference factor as well, which has already been analyzed in the section "Frequency spectra of the DDS".
Looking again at Figure 5, it becomes clear that e.g. there is supposed to be a minimum with a phase shift of 180°. But this point is located at a phase shift of 150°, which results a deviation of approximately 30°.

In conclusion, it can be said that even though there have been a number of systematic measurement deviations, we have developed a functional evaluation board that allows us to determine a phase shift from the analog output voltage value.

# Files to download

To allow this project to be executed by further iGEM teams and possibly expanded, we have uploaded the files for the design of the PCB into our github repository. These files are available to download for free. Furthermore, one can find the source code for the Arduino Mega, which is used to read the PCBs analog output and additionally simulate phase shifts. We also uploaded the table of the results of the measurement as well as the matlab-plot so that the 3D plots can be reconstructed and viewed in three-dimensional. All these files can be used by future iGEM teams to do their own design, build and test cycles to use and improve our PCB prototype for their projects.

# Part 2 – Engineering of FbFPs

Fluorescent proteins are a fundamental building block in synthetic biology research. Therefore, we decided to contribute an improved version of flavin-based fluorescent proteins (FbFPs) to the iGEM community. Due to their small size and their functionality in oxygen-free environment there are a perfect alternative to the commonly used fluorescent protein GFP.

# Design

After a long research for a FbFP candidate, we found the protein SB2 from Pseudomonas putida, which shows a measurable fluorescent activity. So, we designed a testing system to measure the fluorescent activity of the SB2 protein (BBa\_3410007) in contrast to the FbFP protein available in the parts registry of iGEM (BBa\_K3013002). For this purpose, we chose the vector pJoe as our backbone and cloned the testing construct in silico with SnapGene (figure 6).

![](https://static.igem.org/mediawiki/2020/7/76/T--Bielefeld-CeBiTec--IAP_pJoe_parts.png)

Figure 6: Plasmid map of the vector pJoe with the insert SB2

# Build

With the in silico design plan, we were able to start with the construction of our vector. First, we decided to made a gradient PCR to identify the ideal temperature for the primer annealing. We measured the best annealing temperature at 63,2°C (figure 7).

![](https://static.igem.org/mediawiki/2020/6/6b/T--Bielefeld-CeBiTec--IAP_PCRGelbildnew.png)

Figure 7: Gel image for control of the PCR of the SB2 protein, the registry part and the vector pJoe.

With Gibson Assembly we were able to clone the amplified inserts into the pJoe backbone and transformed them in E. coli DH5α. Afterwards, we made a colony PCR to determine the clones carrying the right insert (figure 8) and validated the results with Sanger sequencing.

![](https://static.igem.org/mediawiki/2020/thumb/5/51/T--Bielefeld-CeBiTec--IAPColonyPCRGel.png/1200px-T--Bielefeld-CeBiTec--IAPColonyPCRGel.png)

Figure 8: Colony PCR from SB2, the registry part and protein KOFP-7 in three different gels

The results of both steps prove the successful cloning of the inserts inside the pJoe vector. Finally, to measure the fluorescence, a retransformation from the strain DH5alpha to BL21 (D3) was performed, since pJoe carries a T7 promoter and requires a strain carrying a T7 polymerase for expression.

# Test

For the characterization, the liquid cultures were induced at an OD(600)=0.7 with 0.1mM IPTG and then incubated for 16 hours at 20°C. After 10 hours the protein expression was measured. For this purpose, the emission was recorded via plate reader at an excitation of 450nm and an emission of 495nm.
To measure the fluorescence, the cells had to be washed with PBS buffer beforehand, because the LB medium falsified the measurement. Therefore, the samples were centrifuged, decanted and resuspended in the PBS buffer. The fluorescence of the cells in PBS was then measured in the plate reader (figure 9).

![](https://static.igem.org/mediawiki/2020/4/47/T--Bielefeld-CeBiTec--FluorescenceMeassurement2.png)

Figure 9: Results of fluorescence measurement after 10 to 16 hours after induction with IPTG in the Tecan reader

Figure 9 shows a slightly higher fluorescence for the cloned protein SB2 (Original, BBa\_3410007) in contrast to the FbFP from the part registry (Parts, BBa\_K3013002). Therefore, we were able to design, build and test an improved FbFP for the iGEM community.

## Improve

The results in figure 9 show a slightly higher fluorescence for our designed part. More research on this protein SB2 could consult in potential point mutations to grant a higher stability or a stronger fluorescence signal, which would improve the fluorescence even more. Future iGEM teams could use this part to design better FbFPs to establish this interesting class of fluorescent proteins deeper in iGEM.

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Bielefeld-CeBiTec/Experiments

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Bielefeld-CeBiTec/Results

# Results

You can describe the results of your project and your future plans here.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2014 TU Darmstadt](https://2014.igem.org/Team%3ATU_Darmstadt/Results/Pathway)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Results)
* [2014 Paris Bettencourt](https://2014.igem.org/Team%3AParis_Bettencourt/Results)