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Protocols

Safety

OBJECTIVE: To verify whether the IPTG inducible Reporter system works and to characterize it.

SUMMARY:

A method of measuring the expression of the lacZ gene when fused to the lacI promoter. The genes are present in invitrogen TOP10 cells which are exposed to chlorophenol red-β-d-galactopyranoside (CPRG) and IPTG buffered to a pH of 7. In such conditions the cells produce chlorophenol red which absorbs 595nm light, thus providing a way to measure the amount of product secreted by the cells.

REAGENTS:

• Need to Prepare a 13mL of a 3mM CPRG, 1mM IPTG solution
• LB broth

PROTOCOL:

Section 1. Preparing the bacteria.

1. The E. coli strains carrying the plasmid with the part 1732901 were cultured overnight in 5mL LB broth with the appropriate antibiotic.
2. The next morning 200uL of the overnight culture was sub cultured in 13 mL of LB with antibiotic until it reached log phase (~5 hours), which was assessed by an OD value of ~0.6 at 595nm.

Section 2. Measuring the concentration of CPR formed.

1. Once the culture was at log phase 1mL samples were taken (the number of samples corresponded to the number of time points) in 1.5mL tubes and pelleted, the supernatant was discarded.
2. To these, 1 mL of the solution prepared above (1mM IPTG, 3mM CPRG) was added, and the cells were resuspended.
3. One of the treatment tubes was immediately centrifuged again for 3 min (see step 4) and an OD reading (595nm) was taken of a 200uL sample of the supernatant.
4. A 200uL sample of the solution of IPTG and 3mM CPRG was used to “blank” the Victor plate reader, while the tube was being centrifuged.
5. The remaining tubes were then incubated in a shaker at 37 degrees until the time when they were required for taking OD readings, at which point they were pelleted and readings were taken using the supernatant.
6. Make a standard curve for chlorophenol red OD readings using concentrations from 0.5 mM to 5 mM in PBS buffer to keep the pH at 7.




The above procedure was adapted from: Shin, H.J., Park, H.H., & Lim, W.K. 2005. Freeze-dried recombinant bacteria for on-site detection of phenolic compounds by color change. Journal of Biotechnology, 119(1), 36-43.