Team:Marburg/Labbook/Minicells
15/07/07
Preparation of ONCs
Two times 5 mL LB were inoculated with TB43 and TB43 HU-mCherry and incubated at 37 °C and 200 rpm.
15/07/08
Preparation of Glycerol Stocks
Glycerol stocks of TB43 and TB43 HU-mCherry were prepared according to the glycerol stock generation protocol.
Preparation of electrocompetent Cells
Electrocompetent cells of TB43 and TB43 HU-mCherry were prepared according to the electrocompetent E.coli cells protocol.
Preparation of over-night culture (ONC)
Two times 5 mL LB were inoculated with TB43 and TB43 HU-mCherry and incubated at 37 °C and 200 rpm.
15/07/09
Preparation of electrocompetent Cells
Electrocompetent cells of TB43 and TB43 HU-mCherry were prepared according to the electrocompetent E.coli cells protocol.
15/07/10
Preparation of day culture
20 mL LB were inoculated with TB43 and TB43 HU-mCherry for flow cytometry and grown to an optical density (OD) of 0.5. No minicells could be detected.
15/07/11
Transformation
A GFP plasmid with Chloramphenicol resistance gene was transformed into TB43 and TB43 HU-mCherry via electroporation according to the Transformation into electrocompetent cells protocol.
15/07/13
Preparation of ONC
50 mL LB were inoculated with TB43 GFP and TB43 HU-mCherry GFP and incubated at 37 °C and 200 rpm.
15/07/15
Preparation of Minicells
TB43 GFP and TB43 HU-mCherry GFP were treated according to the Preparation of Mini Cells protocol. Samples were taken and minicells could be detected via flow cytometry but only with very low yields.
15/07/27
Preparation of ONC
5 mL LB were inoculated with TB43, TB43 HU-mCherry, TB43 GFP and TB43 HU-mCherry GFP and incubated at 37 °C and 200 rpm.
15/07/28
Preparation of chemocompetent Cells
Chemocompetent cells of TB43, TB43 GFP, TB43 HU-mCherry and TB43 HU-mCherry GFP were prepared according to the competent E.coli cells (RbCl) protocol.
15/08/21
Preparation of ONC
5 mL LB were inoculated with TB43, TB43 HU-mCherry, TB43 GFP and TB43 HU-mCherry GFP and incubated at 37 °C and 200 rpm.
Preparation of M9 Minimal Medium
Two different M9 minimal media were prepared. Both contained 5x M9 salts, 2 mM MgSO4, 0.1 mM CaCl2 and ddH2O, one additionally 0.4% glucose. The media were sterile filtered.
15/08/24
Plasmid Preparation
Cultures containing a plasmid which encodes enzymes for the biosynthesis of β-carotene (Car) were treated according to the Transformation into chemocompetent (RbCl) cells protocol.
15/08/25
Transformation
Carotinoid plasmids were transformed into chemocompetent TB43 and TB43 HU-mCherry according to Transformation into chemocompetent (RbCl) cells protocol.
Plasmid Preparation
Cultures containing a plasmid which encodes enzymes for the biosynthesis of violacein were treated according to the Macherey-Nagel Plasmid DNA Purification protocol.
15/08/29
Preparation of ONC
Four times 5 mL LB was inoculated with TB43 Car and incubated at 37 °C and 200 rpm.
15/08/30
Plasmid Preparation
ONC were treated according to the Macherey-Nagel Plasmid DNA Purification protocol.
15/09/03
Preparation of ONC
5 mL LB were inoculated with TB43, TB43 GFP, TB43 HU-mCherry, TB43 HU-mCherry GFP and TB43 Car and incubated at 37 °C and 200 rpm.
15/09/04
Preparation of ONC
5 mL M9 minimal medium were inoculated with TB43, TB43 GFP, TB43 HU-mCherry, TB43 HU-mCherry GFP and TB43 Car and incubated at 37 °C and 200 rpm.
15/09/05
Growth Curve
TB43, TB43 HU-mCherry, TB43 GFP and TB43 HU-mCherry GFP were cultivated in LB and M9 minimal medium and grown to an OD of 0.5. No minicells could be detected by flow cytometry.
Preparation of ONC
5 mL LB were inoculated from colonies of the first plate (TB43 and TB43 HU-mCherry) and incubated at 37 °C and 200 rpm.
15/09/06
Growth Curve
The procedure from 15/09/05 was repeated with the same result. The calibration of the side scatter sensor of the flow cytometer appeared to be wrong.
15/09/07
Preparation of ONC
5 mL LB were inoculated with the violacein and β-carotene plasmid containing strains and incubated at 37 °C and 200 rpm.
Plasmid Preparation
The cultures containing the constructs AL11 (GFP & Amp-resistance) and AL13 (RFP & Amp-resistance) were treated according to the Macherey-Nagel Plasmid DNA Purification protocol.
15/09/08
Plasmid Preparation
Cultures with a deletion of the MinC gene were treated according to the Macherey-Nagel Plasmid DNA Purification protocol.
PCR
The backbone pILS was amplified according to the Standard PCR protocol. 1 μL template DNA was used.
Gel Extraction
The PCR product was cleaned according to the modified gel extraction protocol.
Preparation of ONC
5 mL LB were inoculated with all previous minicell producing strains (both, with and without additional plasmids).
15/09/09
Preparation of electrocompetent Cells
ONC from 15/09/08 were treated according to the competent E.coli cells (Electroporation) protocol.
Transformation
All fluorescent protein producing plasmids (both, inducible and constitutive promoters) were transformed into minicell producing strains according to according to the Transformation into electrocompetent cells protocol.
Preparation of ONC
5 mL LB were inoculated with AL11 and AL13 for induction assays.
15/09/10
PCR
The primers iMC1 and iMC2 were used. The product was cleaned up according to the modified gel extraction protocol.
PCR
The primers iMC3 and iILS5 were used. The product was cleaned up according to the modified gel extraction protocol.
CPEC
A construct for the production of a sRNA was built according to the standard CPEC protocol.
Microscopy
The ONC were examined via fluorescence microscopy. Minicells could clearly be detected.
15/09/11
Plasmid Preparation
Cultures containing the constructs pAB.001 and M13K07 were treated according to the Macherey-Nagel Plasmid DNA Purification Kit protocol.
Flow Cytometry
ONC of minicell producing strains were examined via flow cytometry. The calibration of the side scatter sensor was still not right but minicells could be identified by comparison with a negative control.
15/09/12
Growth Curve
TB43 with and without HU-mCherry were cultivated in LB and examined in the flow cytometer and the plate reader.
Preparation of Glycerol Stocks
ONC of minicell-producing strains containing the constructs AL11 and AL13 were treated according to the glycerol stock generation protocol.
Preparation of ONC
5 mL LB+CAM were inoculated with TB43 AL11 & TB43 AL13 for an inducer assay.
PCR
4 PCRs were prepared. The first with primers iMC1, iMC2 and template pAB001, the second with primers iMC3, iILS5 and template pAB001, the third with primers iILS1, iILS2 and template pILS8. The fourth one was an extension PCR with the products of PCR 1 & 2 as templates.
CPEC
A CPEC was performed to build the sRNA encoding plasmids according to the CPEC standard protocol. One reaction used the PCR products 1 & 2 as template, the other one was performed with the product of the extension PCR.
15/09/13
CPEC
Due to a dissatisfying result, the CPEC from 15/09/12 was repeated. Additionally, the construct was built by a Gibson Assembly according to the respective protocol.
Transformation
The CPEC and Gibson Assembly products were dialyzed for 15 min each and transformed into electrocompetent NEB Turbo and chemocompetent DH5α cells.
15/09/14
Preparation of ONC
ONC of TB43 were prepared and then cultivated in 600 mL LB.
Purification of Minicells
To isolate the produced minicells, the TB43 cultures were centrifuged for 15 min at 4 °C and 4,000 rpm. The supernatant was filtered twice over 0.45 μm filters and once over a 0.22 μm filter. The minicells were washed from the filter and rebuffered into glucose-free M9 minimal medium.
Preparation of HPLC samples
Glucose was added to the minicell suspension. In steps of 1 min, samples were taken and quenched with acetonitrile.
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