Team:Bielefeld-CeBiTec/Notebook

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 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 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 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 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 from 08/17

Promoter library

Investigators: Levin Joe Klages

Superior experiment: promoter library

Procedure:

Colony PCRs of the colonies recived from the Gibson Assembly 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 Assemblys 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 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 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 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 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