Monday 27/08/2018

Cells were refreshed 1:200 in 2ml of M9 minimal media plus antibiotics plus anhydrotetracycline and grown at 37C with shaking at 220rpm for 2 hours. Cells were then added (1uL in 199uL ddH2O) to black-sided clear-bottom 96-well plates and incubated in a TECAN infinite F500 fluorescent plate reader at 37C with shaking. Optical density (OD600nm) and fluorescence (480/20nm excitation 530/25 emission) were measured in 15 minute intervals with 3 biological repeats and 3 technical repeats. All measurements were blanked against M9 media plus antibiotics plus ATC (100ng/ml), then against MG1655Z1 cells containing pSB1C3 and pSB1AK8 plasmid backbones so that they have no basal fluorescence.

Test plasmid constructs: Measure GFP levels in plate reader in M9 minimal media with spectinomycin (17μg/ml); Positive control - Cells with loss of the Z1 cassette to constitutively express sfGFP; Negative control - Cells, non-induced; Inducible - ATC (anhydrotetracycline) (moisture-, light-, and air-sensitive!) at 100ng/ml.

Fluorescence protocol

Grow cells overnight to early stationary phase at 37°C at 220rpm overnight in M9 minimal media with appropriate antibiotics (spec + chlor/kan) [and ATC for induced test cells at 100ng/ml]. Meanwhile, prepare negative control cells by transforming pSB1C3 and pSB1AK8 into MG1655Z1 cells.

Refresh cells 1:200 in 2ml M9 minimal media plus antibiotics [plus ATC for induced cells], and grow at 37°C at 220rpm for 2 hours. Add cells to black-sided clear-bottom 96-well plate (3 replicates of each; induced cells, non-induced cells, positive controls, negative controls, blanks)

Incubate plates in plate reader at 37°C with shaking (Standard Protocol - OD = 600nm, fluorescence = 480/20nm excitation, 530/25nm emission) in 15 minute intervals (3 repeats, and 3 replicates - 1 std deviation in error bars) blanked against M9 media with antibiotics and ATC as necessary, and then against MG1655Z1 cells containing empty plasmids (no igRNA/trigger/dCas9/sfGFP components to generate basal fluorescence levels).

Experiment: 3 replicates of each, 15 minute intervals, 37C, 80 cycles (20 hours)
OD = 600nm, Fluor for sfGFP = 480/20nm excitation, 530/25nm emission.

Induced (In) (E. coli MG1655Z1 cells with PAJ379 and PAJ390 + chlor + kan +spectinomycin + 100ng/ml ATC in M9)

Non-induced (NIn) (E. coli MG1655Z1 cells with PAJ379 and PAJ390 + chlor + kan +spectinomycin, no ATC in M9)
Positive control (Pos) (E. coli MG1655Z1 LacI-SpR cells with PAJ379 and PAJ390 + chlor + kan in M9)
Negative control (Neg) (E. coli MG1655Z1 cells with pSB1C3 and pSB1AK8 + chlor + kan in M9)
379 sfGFP (379) (E. coli MG1655Z1 cells with PAJ379 encoding 3A switch sgRNA and sfGFP + kan)
390 dCas9 (390) (E. coli MG1655Z1 cells with PAJ390 encoding the sRNA [3A trigger] and dCas9 + chlor)
ATC only (ATC) (0.1ug/ml ATC + chlor + kan + spectinomycin in M9)
Blank (Blnk) (M9 + chlor + kan + spectinomycin, no cells)

Team:Warwick/Notebook

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