Tuning the expression levels of native genes

order the following oligonucleotides:

2 primers annealing in the tetA cassette. These two primers have to each include 50 bp overhangs. The primer annealing upstream of tetA should contain homology to the 50 first basepairs of the gene of interest. The primer annealing downstream of tetA should contain homology to the promoter region of the gene of interest.

For introducing a TIR library while keeping the native promoter:

1 degenerated oligonucleotide. The primer needs to contain the randomization of the 6 nucleotides upstream of the start codon and also change the two codons downstream of the start to all synonymous codons. This primer needs to harbor 50 bp homology to the promoter region and 50 bp downstream of the randomized region.

For introducing a new promoter:

For short promoters, the promoter sequence can be included in the oligonucleotide that contains 50 bp homology to the regions up- and downstream of tetA.

Promoter constructs over 100 bp need to amplified via PCR. Create 2 primers, that bind in the promoter constructs and each contain 50 bp homology to the regions up- and downstream of tetA. Make sure to purify the PCR product over an agarose gel.

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 either 2µL of a 100micromolar (µM) oligonucleotide or 200ng of a gel-purified PCR products.

Recover cells for 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. In case of the TIR library, select colonies and assay the effect on the gene of interest.