Protocol preparation of bacterial cells

We collect bacteria in exponential phase. (OD_600nm between 1 and 2). For our experiments, 400mL of washed bacteria at an OD_600nm of 0.1 (1 × 10⁸ cfu/ml) are required for the lower receiver of each of the LSBAs.

For 1 LSBAs, inoculate 100mL of liquid MOKA medium with appropriate antibiotics (here Rifampicin 50 µg/ml) using Xcc and grow at 28°C under agitation at 200 rpm.

Centrifuge the overnight growth for 10 minutes at 6,000 rpm and wash twice with 1millimolar (mM) MgCl₂.

Centrifuge the overnight growth for 6000rpm and wash twice with 1millimolar (mM)MgCl₂(1/2).

Centrifuge the overnight growth for 6000rpm and wash twice with 1millimolar (mM)MgCl₂(2/2).

Resuspend the final pellet in 5mL of 1millimolar (mM) MgCl₂ and measure the OD_600nm.

Add 1mL of bacterial suspension at a concentration of 4 x 10¹⁰ cells/ml to a syringe with a 25G needle.

We collect bacteria in exponential phase. (OD_600nmbetween 3 and 4). For our experiments, 400 ml of washed bacteria at an OD_600nmof 0.1 (1 × 10⁸cfu/ml) are required for the lower receiver of each of the LSBAs.

For 1 LSBAs, inoculate 100mLof liquid LB medium with appropriate antibiotics (here Rifampicin 50 µg/ml) using So and grow at 28°Cunder agitation at 200 rpm.

Centrifuge the overnight growth for 10 minutes at 3,500 rpm and wash twice with LM medium.

Centrifuge the overnight growth for 3500rpm and wash twice with LM medium. (1/2)

Centrifuge the overnight growth for 3500rpm and wash twice with LM medium. (2/2)

Resuspend the final pellet in 5mL LM medium and measure the OD_600nm.

Add 1mLof bacterial suspension at a concentration of 4 x 10¹⁰cells/ml to a syringe with a 25G needle.