Ampicillin Survival Assay 2

We used this protocol to verify if, after protein purification, β-lactamase was functional when linked to one of our TALEs.

Reagents and Materials

• LB Broth
• Ampicillin (100mg/mL)
• Chloramphenicol (30mg/mL)
• Falcon tubes
• Bacteria resistant to chloramphenicol but not ampicillin
• Bacteria resistant to both ampicillin and chloramphenicol
• Purified β-lactamase protein

Protocol

1. To each Falcon tube, add 5mL of LB Broth, 5µL of ampicillin and 3µL of chloramphenicol
2. Add your purified proteins in multiple tubes with different amounts of proteins; for construct we added the following: 20µg, 15µg, 10µg, 5µg, 1µg, 0.1µg and 0.01µg
3. Incubate the tubes for 1h at 37°C
4. For the positive control, inoculate a tube that does not have protein on it with a colony of your bacteria resistant to both antibiotics
5. Add colonies of your bacteria resistant to chloramphenicol but not ampicillin to the other tubes
6. Grow the cells overnight