We decided that targeting only two different antibiotic resistance will be enough for a proof of concept. We give up AmpR because otherwise they might be experimental issues with AmpR that is also on phagemid litmus28i.
Week 05: 1st - 7th July
Monday 01st July
Drug Screening
Media was made, Strains sD001-sD004 were received, inoculated, and put into stock and catalog.
4 strains were received from Jake:
E. coli: BL21 (DE3) ko20 ΔcysI, Δfpr, ΔydbK
E. coli: NEBTurbo zmSIR Chloramphenicol
E. coli: NEBTurbo zmFNR Spectinomycin
E. coli: NEBTurbo soFD, zmSIR Chloramphenicol
Media and Glycerol stock were prepared:
Media Preparation
3 500ml bottles of LB broth and LB agar were prepared by standard methods. 12.5g/500ml powder/water for broth and 20g/500ml powder/water for agar. Bottles were autoclaved. Additionally 1 flask of 500ml of LB broth was made in the same fashion.
Glycerol Stocks
Single colonies from agar plates were picked and used to innoculate 5ml LB broth overnight. 750ml of overnight culture was added to 250ml of 60% glycerol in a cryotube. Two sets of Glycerol stocks were used for each of sD001, sD002, sD003, sD004; one set was frozen at -20ºC and the other set was frozen at -80ºC.
Phage Sensor
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Trojan Horse
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TB-ception
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Human Practices
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Modeling
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Tuesday 02st July
Drug Screening
Plasmids were extracted from strains sD002, sD003, sD004
Plasmid Extraction
Plasmids pD004 (pCDF.ew12 zmFNR Spectinomycin), pD005 (pACYC.ew13 soFD, zmSIR Chloramphenicol), pD006 (pACYC.ew17 zmSIR Chloramphenicol) were extracted from NEBTurbo cells using a Thermo Scientific GeneJet Plasmid mini prep kit as described in the protocol (available on google drive). Lacking Resuspention solution we used some from a different mini prep upstairs. Plasmids were eluted in 100ul of nanopure water and frozen at -20ºC. Plasmids were included in catalog.
Phage Sensor
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Trojan Horse
Glycerol stock was made and cells were transformed.
Glycerol stock
From overnight culture of MG1655-6300 O/N : T001
Centrifuge 4000rpm, 10 minutes,
Take out liquid
Resuspend cells in 1mL glycerol, 2mL LB
Separate in two cryotubes, one for the -80°C, one for the -20°C
Electroporation
Making MG1655-6300 competent using Electroporation protocol
Test of the competent cells (negative
Transforming with pCOLADuet-1
Transforming with pACYCDuet-1
Plating 3 different quantity of cells (20ul, 50uL, 100uL) respectively on Cm and Kan plates (dilution 1000x for the antibiotics)
TB-ception
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Wednesday 03st July
Drug Screening
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Competent Cells : BL21 (DE3) dCysI dFpr dydbk
We grew 5ml of sD001 cells overnight from a single colony.
This 5ml was used to innoculate 500ml of LB broth.
Broth was incubated for 1.5h and optical density was read at 0.62 OD600.
Cells were alloquoted into 10 falcon tubes (50ml each) and centrifuged at 4000xg for 20 minutes at 4C.
Supernatant was removed and cells were resuspended in 200ml of Buffer 1 (4x 50ml) and left on ice for 20 minutes.
Cells were centrifuged again for 20 minutes at 4000xg.
Supernatant was removed and cells were resuspended in 12ml Buffer 2.
Cells were alloquoted at 500ul into microcentifuge tubes and frozen at -80C.
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