TITLE: HTTM : Transposon mutagenesis 
AUTHORS: Antoine Champie, Amélie De Grandmaison

[DESCRIPTION]
Part one of the HTTM protocol. A low-cost and high-throughput Tn-seq protocol. 
This part cover the transposon mutagenesis and selection steps. 

[BEFORE_START]
Per plate refers to the number of 96 well plates of target cells that need to be processed. 

[STEPS]
SECTION: Day 1
1. (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. 

SECTION: Day 1
2. (1-B) Prepare the 96 deep-well plates for conjugation : 

SECTION: Day 1
2.1. Preheat the deep-well plates at 60 °C in a sterile incubator for 10 min
Prepare 50 mLof LB-Agar for each plate and keep it above 70 °C



SECTION: Day 1
2.2. Using a multichannel pipette transfer 300 µLof 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. 

SECTION: Day 1
2.3. 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).

SECTION: Day 2
3. (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.  

SECTION: Day 2
4. (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 at37 °C.

SECTION: Day 3
5. (3-A) Pellet the donor strain by centrifugation 6000 x g, 10 min  and discard the liquid.


SECTION: Day 3
6. (3-B) Resuspend the pellet in 10 mLLB per plate. 

SECTION: Day 3
7. (3-C) Dispense 100 µLof concentrated donor culture into each recipient well. 

SECTION: Day 3
8. (3-D) Pellet cells by centrifugation 4000 x g, 10 min  and remove the supernatant with the Aspir-8 + 50 µL guide. 
SECTION: Day 3
8.1. If not using the Aspir-8 + 50 µL guide, remove all supernatant and add 50 µL of LB to each well.
SECTION: Day 3
9. (3-E) Resuspend by agitating on a shaker   and do a quick spin to recover all the cells at the bottom of the plate. 

SECTION: Day 3
10. (3-F) Take 50 µLfrom the resupended cells and deposit them on the dried agar at the bottom of the prepared deep-well plate. Let dry 60 min in a biological hood and cover with a gas permeable plate seal. 

SECTION: Day 3
11. (3-G) Incubate the deep-well plates 120 min at 37 °C for conjugation.

SECTION: Day 3
12. (3-H) Add 400 µLof selection media to each well and resuspend by agitating on a shaker at   and do a quick spin to recover all the cells at the bottom of the plate. 

SECTION: Day 3
13. (3-I) Transfer 250 µL 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 with agitation at 37 °C overnight. 

SECTION: Day 3
14. (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
SECTION: Days 4 to 7
15. 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 million mutants in E.coli) varies from day to day : 
200 µLof day 4 (4-A)
100 µL on day 5 (5-A), 6 (6-A) and 7 (7-A) 


SECTION: Days 4 to 7
16. (7-B) (Optional) In order to have a backup in case of an issue during DNA extraction, make a glycerol stock using 150 µLof the culture after the passage, and store it at-80 °C .

SECTION: Day 8
17. (8-A)/(8-B) Pellet cells by centrifugation 4000 x g, 10 min  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 °Cuntil ready to process.