iGEM 2018 — Team Bielefeld-CeBiTec

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# Team:Bielefeld-CeBiTec/Notebook

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* [Home](https://2018.igem.org/Team%3ABielefeld-CeBiTec)
* [Project](#!)
  + [Motivation](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Motivation)
  + [Overview](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Description)
  + [Toxicity](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Toxicity_Theory)
  + [Accumulation](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Accumulation)
  + [Silencing](https://2018.igem.org/Team%3ABielefeld-CeBiTec/siRNA)
  + [Nanoparticles](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Ferritin)
* [Modeling](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Model)
* [Results](#!)
  + [Results Overview](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Results)
  + [Toxicity Results](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Toxicity_Results)
  + [Accumulation Results](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Accumulation_Results)
  + [Silencing Results](https://2018.igem.org/Team%3ABielefeld-CeBiTec/siRNA_Results)
  + [Nanoparticles Results](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Ferritin_Results)
  + [InterLab](https://2018.igem.org/Team%3ABielefeld-CeBiTec/InterLab)
  + [Demonstrate](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Demonstrate)
* [Parts](#!)
  + [Parts Overview](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Parts)
  + [Basic Parts](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Basic_Part)
  + [Composite Parts](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Composite_Part)
  + [Parts Collection](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Part_Collection)
  + [Parts Improvement](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Improve)
* [Human Practices](#!)
  + [Human Practices](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Human_Practices)
  + [Outreach](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Public_Engagement)
* [Awards](#!)
  + [Hardware](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Hardware)
  + [Software](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Software)
  + [Measurement](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Measurement)
  + [Product Design](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Design)
* [Notebook](#!)
  + [Notebook](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Notebook)
  + [Experiments](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Experiments)
* [Team](#!)
  + [Members](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Team)
  + [Bielefeld](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Bielefeld)
  + [CeBiTec](https://2018.igem.org/Team%3ABielefeld-CeBiTec/CeBiTec)
  + [Bielefeld University](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Bielefeld-University)
  + [Collaborations](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Collaborations)
  + [Attributions](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Attributions)
* [Partners](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Partners)
* [Judging Form ⇗](https://igem.org/2018_Judging_Form?team=Bielefeld-CeBiTec)

![](https://static.igem.org/mediawiki/2018/5/5f/T--Bielefeld-CeBiTec--notebook.png)

Notebook

### 2018-03-26 - 2018-04-01

### Backbone preparation for BioBricks

**Investigators:** Vanessa Kraemer

**Superior experiment:** HUHF

**Procedure:**

- Plasmid isolation pSB1C3
- Restriction digest with EcoRI and PstI

### 2018-04-16 - 2018-04-22

### Preexperiment toxicity *E.coli*

**Investigators:** Erika Schneider

**Superior experiment:** Toxicity tests

**Procedure:**

- Precultures

* 3x 30 mL LB + chemical competen *E.coli* K12 incubation at 37 °C

### Preexperiment toxicity *E.coli*

**Investigators:** Erika Schneider

**Superior experiment:** Toxicity tests

**Procedure:**

- Measurement of the ODs of the precultures

* Culture 1: 4,21

+ Culture 2: 4,11
+ Culture 3: 4,23

* Inoculation of 3 culures (OD: 0.1, 30 mL LB)

- OD measurement after 2.5 h

* Culture 1: 1.33

+ Culture 2: 1.14
+ Culture 3: 1.08

* OD measurement after 7 h

+ Culture 1: 2.44
+ Culture 2: 3.26
+ Culture 3: 3.13

### Preexperiment toxicity *E.coli*

**Investigators:** Erika Schneider

**Superior experiment:** Toxicity tests

**Procedure:**

- Preparations of stock solutions for CuSO4, CuCl2 and AgNO3
- Inoculation of *E. coli* cultures with 1 mM to 8 mM metal salts

### 2018-04-23 - 2018-04-29

### HmtA

**Investigators:** Erika Schneider

**Superior experiment:** Uptake systems

**Procedure:**

- LB agar plate with *P.brassicacerum*
- Colony PCR on *hmtA* gene
- Amplification failed

### 2018-05-14 - 2018-05-20

### Transformation BioBrick N11

**Investigators:** Levin Joe Klages

**Superior experiment:** Promoter library

**Procedure:**

- Transformation of BioBrick N11 from the registry in *E. coli*

### Transformation BioBrick N12

**Investigators:** Levin Joe Klages

**Superior experiment:** Promoter library

**Procedure:**

- Transformation of BioBrick N11 from the registry in *E. coli*

### 2018-05-21 - 2018-05-27

### Gel extraction Ferritin

**Investigators:** Levin Joe Klages

**Superior experiment:** Ferritin cloning

**Procedure:**

- Gel extraction of the ferritin bands

### Backbone preparation for ferritin cloning

**Investigators:** Levin Joe Klages

**Superior experiment:** Ferritin cloning

**Procedure:**

- Restriction digest of pSB1C3 for linearization of the backbone
- Gel extraction Ferritin

### Ferritin cloning

**Investigators:** Levin Joe Klages

**Superior experiment:** Ferritin cloning

**Procedure:**

- PCR on different ferritin types

### 2018-06-04 - 2018-06-10

### HmtA

**Investigators:** Erika Schneider

**Superior experiment:** Uptake systems

**Procedure:**

- LB agar plate with *P.brassicacerum*
- Colony PCR on *hmtA* gene with *P.aeroginosa* DNA

### 2018-06-25 - 2018-07-01

### OprC cloning

**Investigators:** Jakob Zubek

**Superior experiment:** Importers

**Procedure:**

- Preculture *Pseudomonas syricaris* in LB overnight culture

### OprC cloning

**Investigators:** Jakob Zubek

**Superior experiment:** Importers

**Procedure:**

- single cell separation by plating on agar plates from the overnight culture from 06/27

### OprC cloning

**Investigators:** Jakob Zubek

**Superior experiment:** Importers

**Procedure:**

- DNA isolation of 3 single cells and 16S sequencing

### 2018-07-02 - 2018-07-08

### OprC cloning

**Investigators:** Jakob Zubek

**Superior experiment:** Importers

**Procedure:**

- Q5 PCR on the *P. syricaris* DNA

### OprC cloning

**Investigators:** Jakob Zubek

**Superior experiment:** Importers

**Procedure:**

- Gel purification of the OprC bands

### OprC cloning

**Investigators:** Jakob Zubek

**Superior experiment:** Importers

**Procedure:**

- Backbone (pSB1C3) linearization by restriction digest

### OprC cloning

**Investigators:** Jakob Zubek

**Superior experiment:** Importers

**Procedure:**

- Gibson assembly of the OprC fragment and the linearized backbone
- Transfromation into chemocopetent *E. coli* DH5alpha

### OprC cloning

**Investigators:** Jakob Zubek

**Superior experiment:** Importers

**Procedure:**

- No clones recived
- Repetition of the Q5 PCR for the OprC fragment

### OprC cloning

**Investigators:** Jakob Zubek

**Superior experiment:** Importers

**Procedure:**

- Gibson assembly of the OprC fragment and the linearized backbone
- Transfromation into chemocopetent *E. coli* DH5alpha

### 2018-07-09 - 2018-07-15

### OprC cloning

**Investigators:** Jakob Zubek

**Superior experiment:** Importers

**Procedure:**

- Colony PCR on clones from 07/08

### OprC cloning

**Investigators:** Jakob Zubek

**Superior experiment:** Importers

**Procedure:**

- Precultures of positive clones from 07/10

### OprC cloning

**Investigators:** Jakob Zubek

**Superior experiment:** Importers

**Procedure:**

- Plasmid isolation of the precultures from 07/11

### 2018-07-16 - 2018-07-22

### pTet cloning

**Investigators:** Vanessa Kraemer

**Superior experiment:** HUHF

**Procedure:**

- Plasmid isolation pTet from the registry

### OprC expression

**Investigators:** Jakob Zubek

**Superior experiment:** Importers

**Procedure:**

- Restriction digest of the OprC BioBrick with XbaI and PstI

### OprC expression

**Investigators:** Jakob Zubek

**Superior experiment:** Importers

**Procedure:**

- Ligation of the restriction fragment of OprC and pBad

### OprC expression

**Investigators:** Jakob Zubek

**Superior experiment:** Importers

**Procedure:**

- Colony PCR on recived clones from 07/20

### OprC expression

**Investigators:** Jakob Zubek

**Superior experiment:** Importers

**Procedure:**

- Preculture inoculation from the positive clones carrying OprC under control of pBad

### OprC expression

**Investigators:** Jakob Zubek

**Superior experiment:** Importers

**Procedure:**

- Plasmid isolation and sent plasmid to sequencing from the precultures from 07/21

### 2018-07-23 - 2018-07-29

### HmtA

**Investigators:** Erika Schneider

**Superior experiment:** Uptake systems

**Procedure:**

- Linearization of pSB1C3 by restriction digest using EcoRI and SpeI
- Restriction of PCR fragment from HmtA with EcoRI and SpeI
- Ligation and transformation of both fragments by chemical transformation in *E.coli* DH5alpha

### HmtA

**Investigators:** Erika Schneider

**Superior experiment:** Uptake systems

**Procedure:**

- Colony PCR on clones

### HmtA expression

**Investigators:** Erika Schneider

**Superior experiment:** Uptake systems

**Procedure:**

- Restriction of HmtA BioBrick with EcoRI and PstI
- Restriction of T7 and RBS BioBrick with EcoRI and XbaI
- Gel purification of fragments

### Transfromation of different promoters from the library

**Investigators:** Levin Joe Klages

**Superior experiment:** promoter library

**Procedure:**

- Transfromation of different promoters from the library in *E. coli* DH5alpha

### HmtA expression

**Investigators:** Erika Schneider

**Superior experiment:** Uptake systems

**Procedure:**

- Ligation of the fragments from 7/25
- Transformation of the ligation into chemically competent *E.coli* DH5alpha

### Promoter library backbone preparation

**Investigators:** Levin Joe Klages

**Superior experiment:** promoter library

**Procedure:**

- PCR on pSB1C3 and additional fragments for the assembly of the backbone

### Importer growth experiments

**Investigators:** Jakob Zubek

**Superior experiment:** Importers

**Procedure:**

- Preparation of copper salt solutions for importer growth experiments

### HmtA expression

**Investigators:** Erika Schneider

**Superior experiment:** Uptake systems

**Procedure:**

- Colony PCR on the clones recived from the [BioBrick Assembly](https://static.igem.org/mediawiki/2018/d/d1/T--Bielefeld-CeBiTec--Standard_BioBrick_Assembly_LK.pdf) at 7/27, no positive clones

### Importer growth experiments

**Investigators:** Jakob Zubek

**Superior experiment:** Importers

**Procedure:**

- Inocuation of precultures from the importers OprC/HmtA/CopC/D for importer growth experiments

### 2018-07-30 - 2018-08-05

### Importer growth experiments

**Investigators:** Jakob Zubek

**Superior experiment:** Importers

**Procedure:**

- Inoculation of the cultures containing different copper concentrations for the growth experiment (24 well plate, 37 °C, 400 rpm)

### HmtA expression

**Investigators:** Erika Schneider

**Superior experiment:** Uptake systems

**Procedure:**

- Gibson assemby to introduce the T7 promoter in HmtA BioBrick

### HmtA expression

**Investigators:** Erika Schneider

**Superior experiment:** Uptake systems

**Procedure:**

- Repetition of the [BioBrick Assembly](https://static.igem.org/mediawiki/2018/d/d1/T--Bielefeld-CeBiTec--Standard_BioBrick_Assembly_LK.pdf) from 07/28

### Promoter library backbone preparation

**Investigators:** Levin Joe Klages

**Superior experiment:** promoter library

**Procedure:**

- Gelextraction of the PCR Fragments for the assembly of the romoter library backbone

### HmtA expression

**Investigators:** Erika Schneider

**Superior experiment:** Uptake systems

**Procedure:**

- Colony PCR on the clones recived from the [BioBrick Assembly](https://static.igem.org/mediawiki/2018/d/d1/T--Bielefeld-CeBiTec--Standard_BioBrick_Assembly_LK.pdf) from 08/02, no positive clones

### Promoter library backbone preparation

**Investigators:** Levin Joe Klages

**Superior experiment:** promoter library

**Procedure:**

- Gibson assembly and trafo of the gelextracted fragments and trafo into chemocompetent *E. coli* DH5alpha

### Promoter library backbone preparation

**Investigators:** Levin Joe Klages

**Superior experiment:** promoter library

**Procedure:**

- No clones recived, repetition 08/02

### 2018-08-06 - 2018-08-12

### HuHF cloning

**Investigators:** Erika Schneider

**Superior experiment:** Uptake systems

**Procedure:**

- Blunt end ligation of the gBlock 2 and gBlock 3 in pJet

### Promoter library backbone preparation

**Investigators:** Levin Joe Klages

**Superior experiment:** promoter library

**Procedure:**

- Repetition 08/03

### HuHF cloning

**Investigators:** Erika Schneider

**Superior experiment:** Uptake systems

**Procedure:**

- Repetition of the [blunt end ligation](https://static.igem.org/mediawiki/2018/b/b3/T--Bielefeld-CeBiTec--Blunt_end_ligation_LK.pdf) form 08/06

### Promoter library backbone preparation

**Investigators:** Levin Joe Klages

**Superior experiment:** promoter library

**Procedure:**

- colony PCR on clones from 08/06

### Promoter library backbone preparation

**Investigators:** Levin Joe Klages

**Superior experiment:** promoter library

**Procedure:**

- Inoculation of LB precultures from positive clones of the colony PCR from 08/07

### ROS reduction basic parts

**Investigators:** Johannes Ruhnau

**Superior experiment:** ROS reduction

**Procedure:**

- Gene synthesis ordering for basic parts AhpF, ahpC, btuE, Gor, katE

### Promoter library backbone preparation

**Investigators:** Levin Joe Klages

**Superior experiment:** promoter library

**Procedure:**

- Plasmid isolation of the precultures from 08/10
- Linearization of the backbone by restriction

### ROS reduction basic parts

**Investigators:** Johannes Ruhnau

**Superior experiment:** ROS reduction

**Procedure:**

- Primer ordering for the cloning of the basic parts for AhpF, ahpC, btuE, Gor, katE

### 2018-08-13 - 2018-08-19

### Promoter library

**Investigators:** Levin Joe Klages

**Superior experiment:** promoter library

**Procedure:**

- Primer dimerization of the promoter RBS primers

### Promoter library

**Investigators:** Levin Joe Klages

**Superior experiment:** promoter library

**Procedure:**

- Gibson assembly with the linearized backbone and the primer dimers

### Promoter library

**Investigators:** Levin Joe Klages

**Superior experiment:** promoter library

**Procedure:**

- No colonys recived from 08/14
- Repetition primer dimerization

### Promoter library

**Investigators:** Levin Joe Klages

**Superior experiment:** promoter library

**Procedure:**

- Gibson assembly with linearized backbone and primer dimers

### Promoter library

**Investigators:** Levin Joe Klages

**Superior experiment:** promoter library

**Procedure:**

- Colony PCRs of the colonies recived from the [Gibson Assembly](https://static.igem.org/mediawiki/2018/2/26/T--Bielefeld-CeBiTec--Gibson_Assembly_LK.pdf) from 08/17

### Promoter library

**Investigators:** Levin Joe Klages

**Superior experiment:** promoter library

**Procedure:**

- Colony PCRs of the colonies recived from the [Gibson Assembly](https://static.igem.org/mediawiki/2018/2/26/T--Bielefeld-CeBiTec--Gibson_Assembly_LK.pdf) from 08/17

### 2018-08-20 - 2018-08-26

### Promoter library

**Investigators:** Levin Joe Klages

**Superior experiment:** promoter library

**Procedure:**

- Inoculation of positive clones from the promoter library

### ROS reduction basic parts

**Investigators:** Johannes Ruhnau

**Superior experiment:** ROS reduction

**Procedure:**

- PCR on the basic parts AhpF, ahpC, btuE, Gor, katE
- Linearization of the backbone pSB1C3

### Promoter library

**Investigators:** Levin Joe Klages

**Superior experiment:** promoter library

**Procedure:**

- Plasmid isolation of the precultures from 08/20

### ROS reduction basic parts

**Investigators:** Johannes Ruhnau

**Superior experiment:** ROS reduction

**Procedure:**

- Repetition of PCR on the basic parts btuE, Gor, katE
- Linearization of the backbone pSB1C3

### ROS reduction basic parts

**Investigators:** Johannes Ruhnau

**Superior experiment:** ROS reduction

**Procedure:**

- Gel purification of the PCR fragments AhpF, AhpC, BtuE, Gor, KatE

### ROS reduction basic parts

**Investigators:** Johannes Ruhnau

**Superior experiment:** ROS reduction

**Procedure:**

- Gibson assembly of the PCR Fragments for AhpF, AhpC, BtuE, Gor, KatE in pSB1C3

### Promoter library

**Investigators:** Levin Joe Klages

**Superior experiment:** promoter library

**Procedure:**

- Send plasmid to sequencing

### ROS reduction basic parts

**Investigators:** Johannes Ruhnau

**Superior experiment:** ROS reduction

**Procedure:**

- No clones recived
- Repitition of the PCRs on AhpF, AhpC, BtuE, Gor, KatE
- Linearization of pSB1C3 by restriction digest

### ROS reduction basic parts

**Investigators:** Johannes Ruhnau

**Superior experiment:** ROS reduction

**Procedure:**

- Gel purification of the PCR fragments AhpF, AhpC, BtuE, Gor, KatE

### 2018-08-27 - 2018-09-02

### ROS reduction basic parts

**Investigators:** Johannes Ruhnau

**Superior experiment:** ROS reduction

**Procedure:**

- Gibson assembly of the PCR Fragments for AhpF, AhpC, BtuE, Gor, KatE in pSB1C3

### ROS reduction basic parts

**Investigators:** Johannes Ruhnau

**Superior experiment:** ROS reduction

**Procedure:**

- No clones recived
- Repitition of the PCRs on AhpF, AhpC, BtuE, Gor, KatE
- Linearization of pSB1C3 by restriction digest

### ROS reduction basic parts

**Investigators:** Johannes Ruhnau

**Superior experiment:** ROS reduction

**Procedure:**

- Gel purification of the PCR fragments AhpF, AhpC, BtuE, Gor, KatE

### ROS reduction basic parts

**Investigators:** Johannes Ruhnau

**Superior experiment:** ROS reduction

**Procedure:**

- Gibson assembly of the PCR Fragments for AhpF, AhpC, BtuE, Gor, KatE in pSB1C3

### 2018-09-03 - 2018-09-09

### ROS reduction basic parts

**Investigators:** Johannes Ruhnau

**Superior experiment:** ROS reduction

**Procedure:**

- Colony PCR on the clones recived from the [Gibson Assembly](https://static.igem.org/mediawiki/2018/2/26/T--Bielefeld-CeBiTec--Gibson_Assembly_LK.pdf)s from AhpF, AhpC, BtuE, Gor, KatE in pSB1C3

### ROS reduction basic parts

**Investigators:** Johannes Ruhnau

**Superior experiment:** ROS reduction

**Procedure:**

- Precultures of the clones in LB, overnight:

* AhpF: clone 13
* AhpC: clone 7
* BtuE: clone 1
* Gor: cloen 29
* KatE: clone 12

### ROS reduction basic parts

**Investigators:** Johannes Ruhnau

**Superior experiment:** ROS reduction

**Procedure:**

- Plasmid isolation of the precultures from 09/04
- Plasmids send to sequencing

### HmtA expression

**Investigators:** Erika Schneider

**Superior experiment:** Uptake systems

**Procedure:**

- Colony PCR on clones from the Ligation on 08/02
- Inoculation of precultures with the clones 13 and 19

### HuHF cloning

**Investigators:** Erika Schneider

**Superior experiment:** Uptake systems

**Procedure:**

- Colny PCR on the HuHF fragments cloned into pJet
- Inoculation of precultures of positive clones

### 2018-09-10 - 2018-09-16

### HuHF cloning, HmtA expression

**Investigators:** Erika Schneider

**Superior experiment:** Uptake systems

**Procedure:**

- Plasmid isolation from the vernight cultures

### Promoter library

**Investigators:** Levin Joe Klages

**Superior experiment:** promoter library

**Procedure:**

- Additional primers dimerized

### Terminator cloning

**Investigators:** Erika Schneider

**Superior experiment:** Uptake systems

**Procedure:**

- Transformation of a double terminator from the registry

### Promoter library

**Investigators:** Levin Joe Klages

**Superior experiment:** promoter library

**Procedure:**

- Gibson assembly with linearized backbone and primer dimers

### HmtA cloning

**Investigators:** Erika Schneider

**Superior experiment:** Uptake systems

**Procedure:**

- Repitition of the plasmid isolation of the HmtA T7 BioBrick

### Promoter library

**Investigators:** Levin Joe Klages

**Superior experiment:** promoter library

**Procedure:**

- Colony PCRs of the colonies recived from the [Gibson Assembly](https://static.igem.org/mediawiki/2018/2/26/T--Bielefeld-CeBiTec--Gibson_Assembly_LK.pdf) from 09/12

### Importer growth experiments

**Investigators:** Jakob Zubek

**Superior experiment:** Importers

**Procedure:**

- Preparation of copper salt solutions for importer growth experiments

### Promoter library

**Investigators:** Levin Joe Klages

**Superior experiment:** promoter library

**Procedure:**

- Colony PCRs of the colonies recived from the [Gibson Assembly](https://static.igem.org/mediawiki/2018/2/26/T--Bielefeld-CeBiTec--Gibson_Assembly_LK.pdf) from 09/13

### Importer growth experiments

**Investigators:** Jakob Zubek

**Superior experiment:** Importers

**Procedure:**

- Inocuation of precultures from the importers OprC/HmtA/CopC/D for importer growth experiments

### HmtA cloning

**Investigators:** Erika Schneider

**Superior experiment:** Uptake systems

**Procedure:**

- Plasmid isolation BBa\_K823009 and BBa\_K823012
- Restriction BBa\_K823017 with EcoRI and XbaI and HmtA T7 with EcoRI and SpeI

### Promoter library

**Investigators:** Levin Joe Klages

**Superior experiment:** promoter library

**Procedure:**

- Inoculation of positive clones from the promoter library

### Importer growth experiments

**Investigators:** Jakob Zubek

**Superior experiment:** Importers

**Procedure:**

- Inoculation of the cultures containing different copper concentrations for the growth experiment (24 well plate, 37 °C, 400 rpm)

### ROS reduction composite parts

**Investigators:** Johannes Ruhnau

**Superior experiment:** ROS reduction

**Procedure:**

### HmtA cloning

**Investigators:** Erika Schneider

**Superior experiment:** Uptake systems

**Procedure:**

- Ligation of the restriction fragments from 09/15
- Transfromation of the ligation into chemocompetent *E. coli*

### ROS reduction composite parts

**Investigators:** Johannes Ruhnau

**Superior experiment:** ROS reduction

**Procedure:**

- Restriction digest of the fragments from the basic parts AhpF, AhpC, BtuE, Gor, KatE with PstI and XbaI
- Restriction digest of pBad with SpeI and PstI

### 2018-09-17 - 2018-09-23

### HmtA cloning

**Investigators:** Erika Schneider

**Superior experiment:** Uptake systems

**Procedure:**

- Ligation K823009 + RBS (B0034)
- Trafo in *E. coli* DH5alpha

### RNAi improved vector cloning

**Investigators:** Antonin Lenzen Antonio

**Superior experiment:** RNAi improved

**Procedure:**

- Transfromation of Promoter, RBS and marker from the registry into chemocompetent *E. coli* DH5alpha

### CopC CopD cloning

**Investigators:** Erika Schneider

**Superior experiment:** Uptake systems

**Procedure:**

- PCR on CopC/CopD gene synthesis

### HUHF expression

**Investigators:** Vanessa Kraemer

**Superior experiment:** HUHF

**Procedure:**

- Inoculation of a LB preculture containing HUHF

### Promoter library

**Investigators:** Levin Joe Klages

**Superior experiment:** promoter library

**Procedure:**

- Plasmid isolation of the precultures from 09/15

### RNAi vector cloning

**Investigators:** Antonin Lenzen Antonio

**Superior experiment:** RNAi imroved

**Procedure:**

- Precultures from clones recived from the transfromation from 09/17

### ROS reduction composite parts

**Investigators:** Johannes Ruhnau

**Superior experiment:** ROS reduction

**Procedure:**

- Gel purification of the restriction fragments

### CopC CopD cloning

**Investigators:** Erika Schneider

**Superior experiment:** Uptake systems

**Procedure:**

- Linearization of pSB1C3 by restriction
- Ligation of CopC/CopD into pSB1C3

### CoAP cloning

**Investigators:** Erika Schneider

**Superior experiment:** Uptake systems

**Procedure:**

- Plasmid isolation of CopAP
- PCR on sfGFP fpr localisation of CopAP

### Promoter library

**Investigators:** Levin Joe Klages

**Superior experiment:** promoter library

**Procedure:**

- Preculture of all promoter library samples (3 replicates) in 24 well plates, 1 mL, LB, 400 rpm

### RNAi vector cloning

**Investigators:** Antonin Lenzen Antonio

**Superior experiment:** RNAi

**Procedure:**

- Plasmid isolation from the precultures from 08/18

### ROS reduction composite parts

**Investigators:** Johannes Ruhnau

**Superior experiment:** ROS reduction

**Procedure:**

- Ligation of the fragments from 09/19

### Promoter library

**Investigators:** Levin Joe Klages

**Superior experiment:** promoter library

**Procedure:**

- Inoculation of all promoter library samples (3 replicates) in 24 well plates, OD 0.1, 1 mL, LB, 400 rpm

### RNAi vector cloning

**Investigators:** Antonin Lenzen Antonio

**Superior experiment:** RNAi

**Procedure:**

- PCR on the fragments needed for the RNA vectors including:

* Backbone
* Promoter
* Reporter

### ROS reduction composite parts

**Investigators:** Johannes Ruhnau

**Superior experiment:** ROS reduction

**Procedure:**

- Colony PCR on the clones recived from the ligations from AhpF, AhpC, BtuE, Gor, KatE with pBad

### CoAP cloning

**Investigators:** Erika Schneider

**Superior experiment:** Uptake systems

**Procedure:**

- Gibson for sfGFP labeling of CopAP

### Promoter library

**Investigators:** Levin Joe Klages

**Superior experiment:** promoter library

**Procedure:**

- Fluorescence measurements of the promoter library

### Promoter library

**Investigators:** Levin Joe Klages

**Superior experiment:** promoter library

**Procedure:**

- Repitition of the promoter library fluorescence measurement experiment

### RNAi vector cloning

**Investigators:** Antonin Lenzen Antonio

**Superior experiment:** RNAi

**Procedure:**

- Gelpurification of the fragments from the PCR from 08/07

### OprC cloning

**Investigators:** Erika Schneider

**Superior experiment:** Uptake systems

**Procedure:**

- PCR purification of Jakobs PCR on OprC

### ROS reduction composite parts

**Investigators:** Johannes Ruhnau

**Superior experiment:** ROS reduction

**Procedure:**

- Precultures of the clones in LB, overnight:

* AhpF: clone 1
* AhpC: clone 5
* BtuE: clone 1
* Gor: cloen 4
* KatE: clone 11

### OprC localization

**Investigators:** Erika Schneider

**Superior experiment:** Uptake systems

**Procedure:**

- PCRs on fragments for HmtA lokalization
- Plasmid isolation of araBad for expression plasmids

### RNAi vector cloning

**Investigators:** Antonin Lenzen Antonio

**Superior experiment:** RNAi improved

**Procedure:**

- Gibson assembly of the fragments for the RNAi vector
- Transformation into chemocompeten *E. coli*

### ROS reduction composite parts

**Investigators:** Johannes Ruhnau

**Superior experiment:** ROS reduction

**Procedure:**

- Plasmid isolation of the precultures from 09/22
- Plasmids send to sequencing

### 2018-09-24 - 2018-09-30

### OprC localization

**Investigators:** Erika Schneider

**Superior experiment:** Uptake systems

**Procedure:**

- PCRs on fragments for the localizations of the different importers
- Gelextraction of the bands

### RNAi vector cloning

**Investigators:** Antonin Lenzen Antonio

**Superior experiment:** RNAi improved

**Procedure:**

- No clones recived from the [Gibson Assembly](https://static.igem.org/mediawiki/2018/2/26/T--Bielefeld-CeBiTec--Gibson_Assembly_LK.pdf) at 08/09
- Repetition of the PCR from 08/08

### RNAi vector cloning

**Investigators:** Antonin Lenzen Antonio

**Superior experiment:** RNAi improved

**Procedure:**

- Gelpurification of the PCR fragments from 09/25
- Gibson assembly of the fragments
- Transformation into chemocompetent *E. coli* cells

### Calgary ferritin

**Investigators:** Erika Schneider

**Superior experiment:** Uptake systems

**Procedure:**

- Blunt end cloning of the calgary ferritin (ordered as gene synthesis) into pSB1C3

### RNAi vector cloning

**Investigators:** Antonin Lenzen Antonio

**Superior experiment:** RNAi improved

**Procedure:**

- Colony PCR on clones from the transfromation at 08/11

### RNAi vector cloning

**Investigators:** Antonin Lenzen Antonio

**Superior experiment:** RNAi improved

**Procedure:**

- Precultures of positive clones from the colony PCR from 08/13

### ROS reduction growth experiments

**Investigators:** Johannes Ruhnau

**Superior experiment:** ROS reduction

**Procedure:**

- Precultures from the composite parts of AhpF, AhpC, BtuE, Gor, KatE with pBad

### RNAi vector cloning

**Investigators:** Antonin Lenzen Antonio

**Superior experiment:** RNAi improved

**Procedure:**

- Plasmid isolation of the precultures of 08/14

### ROS reduction growth experiments

**Investigators:** Johannes Ruhnau

**Superior experiment:** ROS reduction

**Procedure:**

- Inoculation of the parts AhpF, AhpC, BtuE, Gor, KatE with pBad in Copper rich LB to an OD of 0.1, 37 °C and 400 rpm in 24 well plates

### RNAi vector cloning

**Investigators:** Antonin Lenzen Antonio

**Superior experiment:** RNAi improved

**Procedure:**

- Transfromation of Promoter, RBS and marker from the registry into chemocompetent *E. coli* DH5alpha

### RNAi vector cloning

**Investigators:** Antonin Lenzen Antonio

**Superior experiment:** RNAi improved

**Procedure:**

- Precultures from clones recived from the transfromation from 08/01

### 2018-10-01 - 2018-10-07

### RNAi vector cloning

**Investigators:** Antonin Lenzen Antonio

**Superior experiment:** RNAi improved

**Procedure:**

- Plasmid isolation from the precultures from 08/02

### Growth experiments importers expression

**Investigators:** Erika Schneider

**Superior experiment:** Uptake systems

**Procedure:**

- Precultures for growth experiment on OprC, CopD and HmtA

### RNAi vector cloning

**Investigators:** Antonin Lenzen Antonio

**Superior experiment:** RNAi improved

**Procedure:**

- PCR on the fragments needed for the RNA vectors including:

* Backbone
* Promoter
* Reporter

### RNAi vector cloning

**Investigators:** Antonin Lenzen Antonio

**Superior experiment:** RNAi improved

**Procedure:**

- Gelpurification of the fragments from the PCR from 08/07

### ROS reduction growth experiments 2

**Investigators:** Johannes Ruhnau

**Superior experiment:** ROS reduction

**Procedure:**

- Precultures from the composite parts of AhpF, AhpC, BtuE, Gor, KatE with pBad

### ROS reduction growth experiments 2

**Investigators:** Johannes Ruhnau

**Superior experiment:** ROS reduction

**Procedure:**

- Inoculation of the parts AhpF, AhpC, BtuE, Gor, KatE with pBad in Copper rich LB to an OD of 0.1, 37 °C and 400 rpm in 24 well plates

### Growth experiments importers expression

**Investigators:** Erika Schneider

**Superior experiment:** Uptake systems

**Procedure:**

- Growth experiment on OprC, CopD and HmtA

### RNAi vector cloning

**Investigators:** Antonin Lenzen Antonio

**Superior experiment:** RNAi

**Procedure:**

- Gibson assembly of the fragments for the RNAi vector
- Transformation into chemocompeten *E. coli*

### RNAi vector cloning

**Investigators:** Antonin Lenzen Antonio

**Superior experiment:** RNAi

**Procedure:**

- No clones recived from the [Gibson Assembly](https://static.igem.org/mediawiki/2018/2/26/T--Bielefeld-CeBiTec--Gibson_Assembly_LK.pdf) at 08/09
- Repetition of the PCR from 08/08

### Cloning CopC/D and HmtA

**Investigators:** Erika Schneider

**Superior experiment:** Uptake systems

**Procedure:**

- PCR on the Fragments for expression plasmids of CopC/D and HmtA

### RNAi vector cloning

**Investigators:** Antonin Lenzen Antonio

**Superior experiment:** RNAi improved

**Procedure:**

- Gelpurification of the PCR fragments from 08/10
- Gibson assembly of the fragments
- Transformation into chemocompetent *E. coli* cells

### RNAi vector cloning

**Investigators:** Antonin Lenzen Antonio

**Superior experiment:** RNAi improved

**Procedure:**

- Colony PCR on clones from the transfromation at 08/11

### Cloning CopC/D and HmtA

**Investigators:** Erika Schneider

**Superior experiment:** Uptake systems

**Procedure:**

- Gibson assembly to clone CopC/D and HmtA in pSB1C3

### RNAi vector cloning

**Investigators:** Antonin Lenzen Antonio

**Superior experiment:** RNAi improved

**Procedure:**

- Precultures of positive clones from the colony PCR from 08/13

### 2018-10-08 - 2018-10-14

### RNAi vector cloning

**Investigators:** Antonin Lenzen Antonio

**Superior experiment:** RNAi improved

**Procedure:**

- Plasmid isolation of the precultures of 08/14

### RNAi testing

**Investigators:** Antonin Lenzen Antonio

**Superior experiment:** RNAi testing

**Procedure:**

- RNAi golden gate assembly

### RNAi testing

**Investigators:** Antonin Lenzen Antonio

**Superior experiment:** RNAi testing

**Procedure:**

- RNAi golden gate evaluation

![](https://static.igem.org/mediawiki/2018/7/7e/T--Bielefeld-CeBiTec--top_arrow_white_vk.png)


[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:Bielefeld-CeBiTec/Experiments

[![](https://static.igem.org/mediawiki/2018/1/1b/T--Bielefeld-CeBiTec--nanofactory_navbar_vk.png)](https://2018.igem.org/Team%3ABielefeld-CeBiTec)

* [Home](https://2018.igem.org/Team%3ABielefeld-CeBiTec)
* [Project](#!)
  + [Motivation](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Motivation)
  + [Overview](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Description)
  + [Toxicity](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Toxicity_Theory)
  + [Accumulation](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Accumulation)
  + [Silencing](https://2018.igem.org/Team%3ABielefeld-CeBiTec/siRNA)
  + [Nanoparticles](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Ferritin)
* [Modeling](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Model)
* [Results](#!)
  + [Results Overview](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Results)
  + [Toxicity Results](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Toxicity_Results)
  + [Accumulation Results](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Accumulation_Results)
  + [Silencing Results](https://2018.igem.org/Team%3ABielefeld-CeBiTec/siRNA_Results)
  + [Nanoparticles Results](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Ferritin_Results)
  + [InterLab](https://2018.igem.org/Team%3ABielefeld-CeBiTec/InterLab)
  + [Demonstrate](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Demonstrate)
* [Parts](#!)
  + [Parts Overview](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Parts)
  + [Basic Parts](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Basic_Part)
  + [Composite Parts](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Composite_Part)
  + [Parts Collection](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Part_Collection)
  + [Parts Improvement](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Improve)
* [Human Practices](#!)
  + [Human Practices](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Human_Practices)
  + [Outreach](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Public_Engagement)
* [Awards](#!)
  + [Hardware](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Hardware)
  + [Software](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Software)
  + [Measurement](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Measurement)
  + [Product Design](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Design)
* [Notebook](#!)
  + [Notebook](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Notebook)
  + [Experiments](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Experiments)
* [Team](#!)
  + [Members](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Team)
  + [Bielefeld](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Bielefeld)
  + [CeBiTec](https://2018.igem.org/Team%3ABielefeld-CeBiTec/CeBiTec)
  + [Bielefeld University](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Bielefeld-University)
  + [Collaborations](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Collaborations)
  + [Attributions](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Attributions)
* [Partners](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Partners)
* [Judging Form ⇗](https://igem.org/2018_Judging_Form?team=Bielefeld-CeBiTec)

![](https://static.igem.org/mediawiki/2018/e/e8/T--Bielefeld-CeBiTec--plates_vk.png)

Experiments

| Protocol | Protocol |
| --- | --- |
| [“Typical” Restriction Digest](https://static.igem.org/mediawiki/2018/5/5e/T--Bielefeld-CeBiTec--%E2%80%9CTypical%E2%80%9D_Restriction_Digest_LK.pdf) | [50x TAE Stock Solution](https://static.igem.org/mediawiki/2018/e/e0/T--Bielefeld-CeBiTec--50x_TAE_Stock_Solution_LK.pdf) |
| [Blunt end ligation](https://static.igem.org/mediawiki/2018/b/b3/T--Bielefeld-CeBiTec--Blunt_end_ligation_LK.pdf) | [Cloning Strategy with Restriction Enzymes](https://static.igem.org/mediawiki/2018/9/94/T--Bielefeld-CeBiTec--Cloning_Strategy_with_Restriction_Enzymes_LK.pdf) |
| [Colony PCR GoTaq® G2 DNA Polymerase](https://static.igem.org/mediawiki/2018/d/d3/T--Bielefeld-CeBiTec--Colony_PCR_GoTaq%C2%AE_G2_DNA_Polymerase_LK.pdf) | [Fast cell lysis for SDS-Page](https://static.igem.org/mediawiki/2018/1/15/T--Bielefeld-CeBiTec--Fast_cell_lysis_for_SDS-Page_LK.pdf) |
| [Generating chemocompetent cells](https://static.igem.org/mediawiki/2018/9/9f/T--Bielefeld-CeBiTec--Generating_chemocompetent_cells_LK.pdf) | [Generating electrocompetent cells](https://static.igem.org/mediawiki/2018/c/c3/T--Bielefeld-CeBiTec--Generating_electrocompetent_cells_LK.pdf) |
| [Genome editing via the CRISPR-Cas9 system](https://static.igem.org/mediawiki/2018/2/26/T--Bielefeld-CeBiTec--Genome_editing_via_the_CRISPR_LK.pdf) | [Gibson Assembly](https://static.igem.org/mediawiki/2018/2/26/T--Bielefeld-CeBiTec--Gibson_Assembly_LK.pdf) |
| [IMPACT-CN](https://static.igem.org/mediawiki/2018/b/bd/T--Bielefeld-CeBiTec--IMPACT-CN_LK.pdf) | [innuPREP Gel Extraction Kit from Analytik Jena](https://static.igem.org/mediawiki/2018/b/b1/T--Bielefeld-CeBiTec--innuPREP_Gel_Extraction_Kit_from_Analytik_Jena_LK.pdf) |
| [Lenox Broth (LB)](https://static.igem.org/mediawiki/2018/1/1c/T--Bielefeld-CeBiTec--Lenox_Broth_%28LB%29_LK.pdf) | [Maldi](https://static.igem.org/mediawiki/2018/a/a3/T--Bielefeld-CeBiTec--MALDI_LK.pdf) |
| [PCR with Phusion High-Fidelity PCR Master Mix with GC Buffer](https://static.igem.org/mediawiki/2018/4/46/T--Bielefeld-CeBiTec--PCR_with_Phusion_High-Fidelity_PCR_Master_Mix_with_GC_Buffer_LK.pdf) | [Plasmid Assembly Protocol with Golden Gate Assembly](https://static.igem.org/mediawiki/2018/6/69/T--Bielefeld-CeBiTec--Plasmid_Assembly_Protocol_with_Golden_Gate_Assembly_LK.pdf) |
| [Produce gold in Ferritin](https://static.igem.org/mediawiki/2018/3/35/T--Bielefeld-CeBiTec--Produce_gold_in_Ferritin_LK.pdf) | [PureYieldTM Plasmid Miniprep System from Promega](https://static.igem.org/mediawiki/2018/1/16/T--Bielefeld-CeBiTec--PureYieldTM_Plasmid_Miniprep_System_from_Promega_LK.pdf) |
| [ssDNA Annealing](https://static.igem.org/mediawiki/2018/f/f7/T--Bielefeld-CeBiTec--ssDNA_Annealing_LK.pdf) | [Standard BioBrick Assembly](https://static.igem.org/mediawiki/2018/d/d1/T--Bielefeld-CeBiTec--Standard_BioBrick_Assembly_LK.pdf) |
| [TAE buffer](https://static.igem.org/mediawiki/2018/b/b3/T--Bielefeld-CeBiTec--TAE_buffer_LK.pdf) | [ThermoFischer Scientific Gel Extraction GenJet](https://static.igem.org/mediawiki/2018/c/cb/T--Bielefeld-CeBiTec--ThermoFischer_Scientific_Gel_Extraction_GenJet_LK.pdf) |
| [Transformation via electroporation](https://static.igem.org/mediawiki/2018/4/41/T--Bielefeld-CeBiTec--Transformation_via_electroporation_LK.pdf) | [Transformation via heat shock](https://static.igem.org/mediawiki/2018/1/18/T--Bielefeld-CeBiTec--Transformation_via_heat_shock_LK.pdf) |
| [Western Blot](https://static.igem.org/mediawiki/2018/d/dd/T--Bielefeld-CeBiTec--Western_Blot_LK.pdf) | [Wizard SV Gel and PCR Clean-Up Promega](https://static.igem.org/mediawiki/2018/0/01/T--Bielefeld-CeBiTec--Wizard_SV_Gel_and_PCR_Clean-Up_Promega_LK.pdf) |
| [ZymoPURETM Plasmid Miniprep Kit from Zymo Research](https://static.igem.org/mediawiki/2018/f/f3/T--Bielefeld-CeBiTec--ZymoPURETM_Plasmid_Miniprep_Kit_from_Zymo_Research_LK.pdf) | [Silver nanoparticle synthesis](https://static.igem.org/mediawiki/2018/f/f9/T--Bielefeld-CeBiTec--Silver_nanoparticle_synthesis_LK.pdf) |
| [Phytochelatin assay](https://static.igem.org/mediawiki/2018/e/ef/T--Bielefeld-CeBiTec--Phytochelatin_assay_LK.pdf) | [ONPG membrane permeability assay](https://static.igem.org/mediawiki/2018/5/51/T--Bielefeld-CeBiTec--ONPG_membrane_permeability_assay_LK.pdf) |
| [Remove Iron of Ferritin](https://static.igem.org/mediawiki/2018/6/65/T--Bielefeld-CeBiTec--Remove_Iron_of_Ferritin_LK.pdff) | [NPN assay](https://static.igem.org/mediawiki/2018/1/14/T--Bielefeld-CeBiTec--NPN_assay_LK.pdf) |
| [Measurement of the phytochelatin synthase assay](https://static.igem.org/mediawiki/2018/6/67/T--Bielefeld-CeBiTec--Measurement_of_the_phytochelatin_synthase_assay_LK.pdf) | [Cultivation for Expression of recombinant proteins](https://static.igem.org/mediawiki/2018/9/98/T--Bielefeld-CeBiTec--Cultivation_for_Expression_of_recombinant_proteins_LK.pdf) |
| [Roti-Nanoquant](https://www.carlroth.com/downloads/ba/de/K/BA_K880_DE.pdf) | [Ferritin Purification](https://static.igem.org/mediawiki/2018/7/7e/T--Bielefeld-CeBiTec--Ferritin_Purification_LK.pdf) |
| [Roti-Nanoquant(for protein concentration estimation)](https://static.igem.org/mediawiki/2018/5/5a/T--Bielefeld-CeBiTec--Roti_Nanoquant_LK.pdf) | [SDS-PAGE](https://static.igem.org/mediawiki/2018/7/73/T--Bielefeld-CeBiTec--SDS_PAGE_LK.pdf) |
| [SDS-PAGE running buffer](https://static.igem.org/mediawiki/2018/e/e8/T--Bielefeld-CeBiTec--SDS_Running_Puffer_LK.pdf) |

| Enzyme | Producer |
| --- | --- |

## Enzyme

| [BsaI](https://www.neb.com/products/r0535-bsai) | NEB |
| [DpnI](https://www.neb.com/products/r0176-dpni#Product%20Information) | NEB |
| [EcoRI-HF](https://www.neb.com/products/r3101-ecori-hf#Product%20Information) | NEB |
| [EcoRV](http://www.thermofisher.com/order/catalog/product/ER0301) | Thermo Fisher Scientific |
| [NotI](https://www.neb.com/products/r3189-noti-hf#Product%20Information) | NEB |
| [PstI-HF](https://www.neb.com/products/r3140-psti-hf#Product%20Information) | NEB |
| [SpeI](https://www.neb.com/products/r0133-spei#Product%20Information) | NEB |
| [XbaI](https://www.neb.com/products/r0145-xbai#Product%20Information) | NEB |

---

 References ▾

![](https://static.igem.org/mediawiki/2018/7/7e/T--Bielefeld-CeBiTec--top_arrow_white_vk.png)

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:Bielefeld-CeBiTec/Results

[![](https://static.igem.org/mediawiki/2018/1/1b/T--Bielefeld-CeBiTec--nanofactory_navbar_vk.png)](https://2018.igem.org/Team%3ABielefeld-CeBiTec)

* [Home](https://2018.igem.org/Team%3ABielefeld-CeBiTec)
* [Project](#!)
  + [Motivation](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Motivation)
  + [Overview](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Description)
  + [Toxicity](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Toxicity_Theory)
  + [Accumulation](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Accumulation)
  + [Silencing](https://2018.igem.org/Team%3ABielefeld-CeBiTec/siRNA)
  + [Nanoparticles](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Ferritin)
* [Modeling](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Model)
* [Results](#!)
  + [Results Overview](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Results)
  + [Toxicity Results](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Toxicity_Results)
  + [Accumulation Results](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Accumulation_Results)
  + [Silencing Results](https://2018.igem.org/Team%3ABielefeld-CeBiTec/siRNA_Results)
  + [Nanoparticles Results](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Ferritin_Results)
  + [InterLab](https://2018.igem.org/Team%3ABielefeld-CeBiTec/InterLab)
  + [Demonstrate](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Demonstrate)
* [Parts](#!)
  + [Parts Overview](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Parts)
  + [Basic Parts](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Basic_Part)
  + [Composite Parts](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Composite_Part)
  + [Parts Collection](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Part_Collection)
  + [Parts Improvement](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Improve)
* [Human Practices](#!)
  + [Human Practices](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Human_Practices)
  + [Outreach](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Public_Engagement)
* [Awards](#!)
  + [Hardware](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Hardware)
  + [Software](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Software)
  + [Measurement](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Measurement)
  + [Product Design](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Design)
* [Notebook](#!)
  + [Notebook](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Notebook)
  + [Experiments](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Experiments)
* [Team](#!)
  + [Members](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Team)
  + [Bielefeld](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Bielefeld)
  + [CeBiTec](https://2018.igem.org/Team%3ABielefeld-CeBiTec/CeBiTec)
  + [Bielefeld University](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Bielefeld-University)
  + [Collaborations](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Collaborations)
  + [Attributions](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Attributions)
* [Partners](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Partners)
* [Judging Form ⇗](https://igem.org/2018_Judging_Form?team=Bielefeld-CeBiTec)

![](https://static.igem.org/mediawiki/2018/2/21/T--Bielefeld-CeBiTec--pipetten_vk.png)

Results Overview

This is **nanoFactory** - a combined system to clean up mining drainage and to produce nanoparticles.

![](https://static.igem.org/mediawiki/2018/8/8f/T--Bielefeld-CeBiTec--cg--Overview.png)

## [Dual Use](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Public_Engagement)

We analyzed the awareness of Dual Use and Dual Use Research of Concern issues through a nationnal and an international survey.
Both surveys revealed the lack of unified definitions, insufficient education, and failed science communication as major problems. To improve the situation and to prevent restrictions on free research, we increased awareness through improved
science communication, appealed to the science community, and provided open source material for education of scientists.

## [Modeling](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Model)

Modeling contributed to several project parts. One modeling approach identified lethal metal ion
concentrations and led to the construction of a ROS reducing system to improve the tolerance towards metal ions.

## [Promoter Collection](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Part_Collection)

Since there is a shortage of reliable information about promoter strengths, we tested a promoter and RBS library to identifiy the appropriate combination for our project.
Therefore, we constructed a plasmid backbone, which enables reliable promoter strength measurement through normalization based on a second reporter encoded in the backbone. It has not escaped our notice that this system could be applied by further iGEM teams to characterize any promoter sequence of interest.

## [Toxicity](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Toxicity_Results)

Metal ions have a toxic effect on *Escherichia coli* cells. We identified critical concentrations for our experiments and developed several methods to reduce ROS.

## [Accumulation](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Accumulation_Results)

To increase the nanoparticle yield, we cloned and characterized dedicated metal ions importers. We investigated the specifcity towards their respective ions and the influence on the growth.

## [Silencing](https://2018.igem.org/Team%3ABielefeld-CeBiTec/siRNA_Results)

We designed and assembled vectors for assessment and expression of siRNAs. We used our software to design suitable siRNAs and developed an improved vector set for experimenntal validation.

## [Nanoparticles](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Ferritin_Results)

We were able to enhance iron nanoparticle formation by overexpressing ferritin in *Escherichia coli*. Furthermore, we developed a mutated variant of the human ferritin to produce gold and silver nanoparticles.

## [Reactor](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Hardware)

We designed and printed a customized cross-flow bioreactor to filter huge amounts of mining drainage while accumulating metal ions. Through iterated feedback from our modeling we improved our prototype and developed an improved bioreactor to facilitate application of our system for the cleaning of mining drainage.

## [Proof of Concept](https://2018.igem.org/Team%3ABielefeld-CeBiTec/Demonstrate)

During our project we were able to demonstrate accumulation of metal ions in *Escherichia coli*, while increasing the tolerance towards such ions. We engineered ferritin to enable iron, silver and gold nanoparticle formation. Furthermore, we demonstarted that nanoparticles could be used to print conductive paths.

![](https://static.igem.org/mediawiki/2018/7/7e/T--Bielefeld-CeBiTec--top_arrow_white_vk.png)