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:

    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:

    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:

    Promoter library

    Investigators: Levin Joe Klages

    Superior experiment: promoter library

    Procedure:

    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:

    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:

    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