TITLE: Gene knockout strategy
AUTHORS: Carolyn N Bayer, Ana Gabriela Veiga Sepulchro, Maja Rennig, Morten Norholm

[DESCRIPTION]
This protocol collection describes how to use our optimised tetAOPT dual selection marker in E. coli K12 and Nissle. This dual selection marker can be used for positive selection based on tetracycline resistance and counterselection based on NiCl2 sensitivity. tetA can be used to engineer all stages of the central dogma of molecular biology. On the DNA-level tetAOPTcan be used to create scarless knockouts across the E. coli genome with an efficiency above 90%, whereas recombinant gene integrations can be achieved with approximately 50% efficiency. On the expression level, tetAOPT enables advanced genome engineering of both gene translation and transcription.

[STEPS]
SECTION: Ordering of oligonucleotides
1. order the following oligonucleotides:

SECTION: Ordering of oligonucleotides
1.1. 2 primers annealing in the tetA cassette including 50 bp overhangs that correspond to the regions up-and downstream of the locus that will be removed. 
SECTION: Ordering of oligonucleotides
1.2. 1 oligonucleotide (100bp) with the same 50 bp homology as the first primer set. This primer needs to anneal to the lagging strand. Use modest.biosustain.dtu.dk to create a sample "MAGE" oligonucleotide. Select your locus of interest in the dropdown menu "gene" and delete "A" in position 1. This will generate a MAGE oligonucleotide that will delete the A of the start codon in your gene that you want to delete. Align this oligonucleotide in your sequence software to see which strand it aligns to. This strand represents the lagging strand. Now create your custom oligonucleotide that aligns to the same strand as the MAGE sample oligonucleotide. 
SECTION: preculture and PCR - day 1
2. Setup a preculture of the strain with pSIM19 (recombineering plasmid) in LB medium supplemented with Spectinomycin0.05 mg/mL and incubate at   overnight. From now on the strain has to be kept at 30 °C to maintain pSIM19 inside the cells. 

SECTION: Recombineering: tetA integration - day 2-4
4. 
Prepare:
SECTION: Recombineering: tetA integration - day 2-4
4.1. Cold sterile water
SECTION: Recombineering: tetA integration - day 2-4
4.2. Cold Glycerol 15 % volume

SECTION: Recombineering: tetA integration - day 2-4
4.3. Pre-chilled centrifuge and tabletop centrifuge at 4 °C

SECTION: Recombineering: tetA integration - day 2-4
4.4. LB agar supplemented with 0.05 mg/mL tetracycline

SECTION: Recombineering: tetA integration - day 2-4
5. Inoculate 50 mL LB-Medium supplemented with Spectinomycin (0.05 mg/mL) with 500 µL of the preculture from step 

SECTION: Recombineering: tetA integration - day 2-4
6. Induce expression by transferring the culture to a shaking water bath at  

SECTION: Recombineering: tetA integration - day 2-4
5.1. Incubate at  until cultures reached an OD600 of 0.5

SECTION: Recombineering: tetA integration - day 2-4
7. Transfer culture to prechilled 50 mL falcon tubes and put on ice for 15 min

SECTION: Recombineering: tetA integration - day 2-4
8. Spin the culture down at 4000 x g, 15 min, 4 °C and discard the supernatant

SECTION: Recombineering: tetA integration - day 2-4
9. Add 1 mL of ice cold water, resuspend and transfer to a 1.5 mL tube

SECTION: Recombineering: tetA integration - day 2-4
10. Spin at 11000 x g, 30 s, 4 °C  in a tabletop centrifuge

SECTION: Recombineering: tetA integration - day 2-4
11. Wash pellet twice with 1 mL ice cold water   

SECTION: Recombineering: tetA integration - day 2-4
12. Resuspend the pellet in 600 µL cold glycerol (15 % volume)

SECTION: Recombineering: tetA integration - day 2-4
13. Unused cells can be stored at-80 °C

 

SECTION: Recombineering: tetA integration - day 2-4
14. Electroporate50 µL of cells with 200 ngof purified PCR product from step 3 
SECTION: preculture and PCR - day 1
3. Prepare a PCR product of the tetA casette using a proof-reading polymerase and purify it.
SECTION: Recombineering: tetA integration - day 2-4
15. Recover cells  in a tabletop shaker using SOC medium. 

SECTION: Recombineering: tetA integration - day 2-4
16. plate cells on LB agar supplemented with 0.05 mg/mL tetracycline. Cell might need up to 2 days to grow.

SECTION: Recombineering: tetA removal - day 4
17. Select a colony from the LB tetracycline plate and start a preculture in LB medium supplemented with Spectinomycin0.05 mg/mL. Incubate at   overnight.
SECTION: Recombineering: tetA removal - day 4
18. prepare cells following steps 5-13
SECTION: Recombineering: tetA removal - day 4
20. Recover cells at  . Afterwards, transfer the cells into 5 mL LB medium supplemented with Spectinomycin

SECTION: Recombineering: tetA removal - day 4
21. Incubate at  overnight
Important! Cells need to lose tetA transporter in the membrane to get resistant to NiCl2
SECTION: Plating - day 5-7
22. Wash 1 mL of the recovered cells twice with sterile water. Centrifuge at 11000 rpm, 30 s, 20 °C

SECTION: Plating - day 5-7
23. Make a dilution series and plate 100 µL of the 1:10 - 1:1000 dilution on M9 agar supplemented with 50 micromolar (µM) NiCl2

SECTION: Recombineering: tetA integration - day 2-4
4.5. M9 agar supplemented with 50 micromolar (µM) NiCl2

SECTION: Plating - day 5-7
24. incubate the plates at 30 °C for 2880 min to 4320 min

 

SECTION: Screening - day 8
25. Screen for positive colonies by colony PCR to identify the correct recombinants. Restreak correct colony on LB agar. 
SECTION: Recombineering: tetA removal - day 4
19. Electroporate 50 µL of the prepared cells with 2 µL of a 100 micromolar (µM) oligonucleotide