Gene knockout strategy

order the following oligonucleotides:

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.

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.

Setup a preculture of the strain with pSIM19 (recombineering plasmid) in LB medium supplemented with Spectinomycin0.05mg/mL and incubate at 250rpm overnight. From now on the strain has to be kept at 30°C to maintain pSIM19 inside the cells.

Prepare a PCR product of the tetA casette using a proof-reading polymerase and purify it.

Prepare:

Cold sterile water

Cold Glycerol 15% volume

Pre-chilled centrifuge and tabletop centrifuge at 4°C

LB agar supplemented with 0.05mg/mL tetracycline

M9 agar supplemented with 50micromolar (µM) NiCl₂

Inoculate 50mL LB-Medium supplemented with Spectinomycin (0.05mg/mL) with 500µL of the preculture from step

Incubate at 250rpmuntil cultures reached an OD600 of 0.5

Induce expression by transferring the culture to a shaking water bath at 150rpm

Transfer culture to prechilled 50mL falcon tubes and put on ice for 0h 15m 0s

Spin the culture down at 4000x g,4°C and discard the supernatant

Add 1mL of ice cold water, resuspend and transfer to a 1.5mL tube

Spin at 11000x g,4°C in a tabletop centrifuge

Wash pellet twice with 1mL ice cold water

Resuspend the pellet in 600µL cold glycerol (15% volume)

Unused cells can be stored at-80°C

Electroporate50µL of cells with 200ngof purified PCR product from step 3

Recover cells800rpm in a tabletop shaker using SOC medium.

plate cells on LB agar supplemented with 0.05mg/mL tetracycline. Cell might need up to 2 days to grow.

Select a colony from the LB tetracycline plate and start a preculture in LB medium supplemented with Spectinomycin0.05mg/mL. Incubate at 250rpm overnight.

prepare cells following steps 5-13

Electroporate 50µL of the prepared cells with 2µL of a 100micromolar (µM) oligonucleotide

Recover cells at 800rpm. Afterwards, transfer the cells into 5mL LB medium supplemented with Spectinomycin

Incubate at 250rpmovernight

Important! Cells need to lose tetA transporter in the membrane to get resistant to NiCl₂

Wash 1mL of the recovered cells twice with sterile water. Centrifuge at 11000rpm,20°C

Make a dilution series and plate 100µL of the 1:10 - 1:1000 dilution on M9 agar supplemented with 50micromolar (µM) NiCl₂

incubate the plates at 30°C for 48h 0m 0s to 72h 0m 0s

Screen for positive colonies by colony PCR to identify the correct recombinants. Restreak correct colony on LB agar.