Ampicillin Survival Assay 1

We used this protocol to verify if E. coli transformed with the beta-lactamase gene was able to express the enzyme and survive in media with ampicillin.

Reagents and Materials

• LB Broth
• Ampicillin (100mg/mL)
• Chloramphenicol (30mg/mL)
• Falcon tubes
• E. coli transformed with pLacI resistant to chloramphenicol but not ampicillin
• E. coli transformed with RFP resistant to chloramphenicol but not ampicillin
• E. coli transformed with beta-latacmase (pLacI+beta-lactamase+His) resistant to chloramphenicol

Protocol

1. Add 5mL of LB Broth to 9 Falcon tubes and 3µL of chloramphenicol to 6 Falcon tubes only
2. In 3 of the tubes (2 of them with chloramphenicol and 1 with LB Broth only), add colonies of E. coli transformed with pLacI
3. In 3 of the tubes, add colonies of E. coli transformed with RFP
4. In 3 of the tubes, add colonies of E. coli transformed with beta-latacmase (pLacI+beta-lactamase+His)
5. Incubate the tubes overnight in a shaker at 37°C
6. Spin down the tubes and resuspend cells in 5mL of fresh LB Broth
7. Add ampicillin to the tubes that had no antibiotic on it and to one of the tubes that had chloramphenicol on it. After that, for each group you will have one with Amp only, one with Chlor only and a third one with Amp + Chlor
8. Measure the OD at 600nm in a spectrophotometer in different time points
9. Incubate the tubes for 1h at 37°C
10. For the positive control, inoculate a tube that doesn’t have protein on it with a colony of your bacteria resistant to both antibiotics
11. Add colonies of your bacteria resistant to chloramphenicol but not ampicillin to the other tubes
12. Grow the cells overnight