Jul 08, 2024
Stop codon reversal assay
- 1UIUC
Protocol Citation: is Sparrow 2024. Stop codon reversal assay. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl82j39l2w/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 21, 2024
Last Modified: July 08, 2024
Protocol Integer ID: 100204
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Abstract
This protocol is used to characterize viral RNA polymerase based directed evolution tools via fluctuation analysis to calculate the mutation rate. It is specific to the stop codon reversal of the aadA gene, which we use to characterize the mutational power of the tool.
DNA verification
DNA verification
Ensure your constructs are sequenced correctly using Plasmidsaurus, paying particular attention to your stop codon and other mutations in promoter or CDS sequences.
Transformation
Transformation
Transform your plasmid(s) into E. coli BL21 (DE3) line, selecting for the plasmid backbone (kan) using the high efficiency transformation protocol
Thaw a tube of BL21 Competent E. coli cells on ice for 10 minutes. Mix gently and carefully pipette 50 µl of cells into a transformation tube on ice.
Add 1 µl of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
Place the mixture on ice for 30 minutes. Do not mix.
Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
Place on ice for 5 minutes. Do not mix.
Pipette 950 µl of room temperature SOC into the mixture..
Place at 37°C for 45 minutes. Shake vigorously (250 rpm) or rotate.
Spin down cells and remove ~80% of supernatant. Resuspend in remaining supernatant.
Warm selection plates to 37°C.
Mix the cells thoroughly by flicking the tube and inverting.
Plate 50-200 uL on appropriate selections
Grow on a plate overnight at 37C
Outgrowth
Outgrowth
Pick n colonies into separate 15 mL tubes containing 3 mL of LB kan and grow to saturation. This is passage 0 (P0)
Aim for a minimum of 5 biological replicates in each experiment
I typically do passages at 8 AM and 5 PM
Passaging
Passaging
At the end of P0, dilute 1:1000 the culture in fresh LB kan supplemented with filter sterilized arabinose (final concentration 0.2%). This is now passage 1 (P1). Grow to saturation until it is time to do the next passage. This can be repeated any number of times based on your experiment.
For the specR stop codon assay, do up to P8.
Challenge
Challenge
For the challenge part of the assay, create serial dilutions at 10^-2 , 10^-4 , and 10^-6 of each of your strains and replicates.
Spot 5-10 uL of the serial dilutions of each strain and replicate unto 2 different plates:
- Non-selective plate (LB agar kan, selects for plasmid backbone)
- Selective plate (LB agar spec, selects for reversal of stop codon)
Incubate overnight at 37C
Count colonies:
r = cfu / mL in selective plates (mutants/reversants)
N = cfu / mL in non-selective plates
Make sure to account for the dilution factor when calculating cfu/mL.
Suppressor frequency is calculated as r/N and plotted.
Sequencing
Sequencing
For each strain of interest, you can pick 3 random colonies at different cycles, grow in LB kan and prep, then send for sequencing to see mutation frequency downstream and upstream. This needs to be calculated by hand based on generation time estimates.