May 22, 2024
Version 4
HTTM : Transposon mutagenesis V.4
Peer-reviewed method
- Antoine Champie1,
- Amélie De Grandmaison1,
- Sebastien Rodrigue1
- 1Université de Sherbrooke
External link: https://doi.org/10.1371/journal.pone.0283990
Protocol Citation: Antoine Champie, Amélie De Grandmaison, Sebastien Rodrigue 2024. HTTM : Transposon mutagenesis. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgq72n3vk5/v4Version created by Antoine Champie
Manuscript citation:
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: October 03, 2023
Last Modified: May 22, 2024
Protocol Integer ID: 100188
Keywords: HDTM, TnSeq, HTTM
Abstract
Part of the HTTM protocol dedicated to the transposon mutagenesis of targets cells.
The last step in this version contains a supplemental video with extra context and tips, as part of the protocols.io Spotlight series, featuring conversations with protocol authors.
Attachments
HDTM _ Protocol.pdf
444KB
Image Attribution
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Before start
Per plate refers to the number of 96 well plates of target cells that need to be processed.
Day 1
Day 1
3m
(1-A) Make a 15 mL LB (Diaminopimelic acid [Dap], Ampicillin [Amp], Spectinomycin [Spec]) pre-culture ( 2 mL per plate minimum) of the donor strain eAC494 and incubate with agitation at 37 °C overnight.
(1-B) Prepare the 96 deep-well plates for conjugation :
- Preheat the deep-well plates at 60 °C in a sterile incubator for 00:10:00
- Prepare 50 mL of LB-Agar for each plate and keep it above 70 °C
10m
Using a multichannel pipette transfer 300 µL of molten LB-Agar in each well of the deep-well plates, paying attention not to create bubbles by keeping the tips on the side of the wells and not dispensing all the liquid.
Let dry in a biological hood for 3 days or until well dried but not cracked. (Optional : can be placed on a heating mat set at 30 °C to shorten the drying time to 2 days).
Day 2
Day 2
(2-A) Prepare a 500 mL LB (Dap, Amp, Spec) culture of the donor strain per plate by making a 1/250 dilution of the pre-culture and incubate overnight at 37 °C with 180 rpm .
(2-B) Fill the deep-well plates with chosen medium (1.5 mL per well) and inoculate each well with the recipient strains. Incubate overnight at 37 °C with 180 rpm .
Day 3
Day 3
20m
(3-A) Pellet the donor strain by centrifugation 6000 x g, 00:10:00 and discard the liquid.
10m
(3-B) Resuspend the pellet in 10 mL LB per plate.
(3-C) Dispense 100-150 µL (total volume) donor culture into each recipient well.
(3-D) Pellet the cells by centrifugation 3270 x g, 00:10:00 and remove the supernatant with the Aspir-8 + 50 µL guide.
10m
If not using the Aspir-8 + 50 µL guide, remove all supernatant and add 50 µL of LB to each well.
(3-E) Resuspend by agitating on a shaker 900 rpm, 00:10:00 and do a quick spin to recover all the cells at the bottom of the plate.
(3-F) Take 50-100 µL (total volume) from the resupended cells and deposit them on the dried agar at the bottom of the prepared deep-well plate. Let dry 01:00:00 at 30 °C in a biological hood and cover with a gas permeable plate seal.
1h
(3-G) Incubate the deep-well plates 02:00:00 at 37 °C for conjugation.
2h
(3-H) Add 400 µL of selection media to each well and resuspend by agitating on a shaker at 900 rpm, 00:10:00 and do a quick spin to recover all the cells at the bottom of the plate.
(3-I) Transfert 400 µL (total volume) of the resuspended cells to a new deep-well filled with 1500 µL of selection media (with antibiotics to select for newly obtained mutants). Cover with a gas permeable plate seal and incubate at 37 °C with 180 rpm Overnight .
(3-J)/(3-K) (Optional) Using 20 µL of the conjugation mix make serial dilutions and spot on selective plates to estimate the number of mutants obtained per well.
Selection markers :
- Donor strain : Dap, Amp, Spec
- Recipient : Target-dependant
- Transposon mutants : Target-dependant + Spec
Days 4 to 7
Days 4 to 7
Make a passage from the previous plate to a new deep-well plate filled with selective medium.
The volume of the passage (optimized to pass 3 millions mutants in E.coli) varies from day to day :
- 200 µL of day 4 (4-A)
- 100 µL on day 5 (5-A), 6 (6-A) and 7 (7-A)
(7-B) (Optional) In order to have a backup in case of an issue during DNA extraction, make a glycerol stock: pellet cells of the culture after passage 3270 x g and remove supernatant, add 75 µL of 50 % glycerol solution and resuspend by agitating 900 rpm 00:10:00 . Store it at-80 °C .
10m
Day 8
Day 8
10m
(8-A)/(8-B) Pellet cells by centrifugation 3270 x g, 00:10:00 and remove the supernatant. Aspir-8 can be used to accelerate this step. Cells are ready for DNA extraction and can be stored at -80 °C until ready to process.
10m
Spotlight video
Spotlight video