M. tuberculosis Whole cell lysates plus culture filtrate

Harvest 100 mL of culture in Sauton’s grown with no detergent using the following steps:

1. Grow 40 mL cultures first in 7AG (7H9 + ADS + glycerol) tween and antibiotics to saturation.

2. Add 5 mL are into 60 mL 7AG + 0.05% tyloxapol and grow until an OD of 2

3. Wash cultures into Sauton’s + 0.05% tyloxapol and inoculate 100 mL of Sauton’s + 0.05% tyloxapol at an OD of 0.125 with the washed cells.

4. Grow for 5-6 days until an OD of 2 – wash in Sauton’s + 0.02% tyloxapol and resuspend in Sauton’s with no detergent.

5. Innoculate 100 mL of Sauton’s (no detergent or antibiotics) at an OD of 0.125 and grow for 3 days.

a. Without detergent cells are too clumpy to properly OD, but following the protocol above should give you a consistent number of cells. If you are worried about a growth phenotype with a particular mutant, you could grow a smaller side culture containing detergent to OD. Want an OD around 0.5 while harvesting.

b. Pellet cells – keep the cells to make a whole cell lysate prep and double filter sterilize the 100 mL of supernatant to concentration for culture filtrate.

For our proteins, there was no induction step.

M. smegmatis Whole cell lysates plus culture filtrate

Harvest 100 mL of culture in 7H9 grown with no detergent using the following steps:

1. Grow 20 mL culture first in 7H9 + glucose/glycerol with tween and antibiotics to saturation

2. Innoculate 100 mL 7H9 + glucose/glycerol (with no detergent) at an OD of 0.03

a. Without detergent cells are too clumpy to properly OD. Include a small culture with tween to monitor OD

3. Grow overnight to OD of 0.4-0.8.

For our proteins, there was no induction step.