5. Add 380 μL of electrocompetent/recombinogenic M. tuberculosis cells containing pKM461 to the mixture, pipet back and
forth 2–3 times to mix the cells with the Orbit oligo and
integrating plasmid, and transfer the mixture to a 0.2 cm
cuvette (BioRad).
6. Place cuvette into the electroporation chamber and apply a
pulse at 2.5 kV, 1000 Ω, 25 μF.
7. Remove the cuvette from the chamber, recover the cells with a
sterile 1 mL disposable transfer pipet, and transfer the cells to a
30 mL inkwell bottle containing 5 mL of 7H9-OADCTween 80.
8. Shake the cultures at 37
C overnight.
9. Collect the cells by centrifugation at 3500 Â g for 10 min and
remove the supernatant, leaving behind ~200 μL of media to
resuspend the cells.
10. Spread the entire suspension on a 7H10 plate containing the
appropriate drug (either hygromycin or zeocin, depending on
the drug selection marker on the integrating plasmid).
11. As a control, also plate a culture where only the integrating
plasmid is added to the cells (no oligo control).
Fig. 3 Examples of the ORBIT oligo sequences used for deletion of 4 target genes in M. tuberculosis: Rv0989c
(right replicore, counter-clockwise); Rv1321 (right replicore, clockwise); Rv3211 (left replicore, clockwise);
Rv3544c (left replicore, counter-clockwise). The attP sequences are highlighted in blue; the core sequences
are highlighted in red
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