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• Defining Objectives
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Protocols

Safety

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

SUMMARY: A method of measuring the expression of the lacZ gene when fused to the pBAD promoter. The genes are present in invitrogen TOP10 cells which are exposed to chlorophenol red-B-d-galactopyranoside (CPRG) and arabinose. 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:

• LB broth
• CPRG
• L-Arabinose
• PBS (phosphate buffer saline)

PROTOCOL:

Section 1. Preparing a 3mM CPRG solution that was 2% arabinose (%w/v).*

1. 10mg of L-arabinose was dissolved in 2mL of nanopure water.
2. 80uL of this 5 g/L solution was then added to a sterile tube.
3. 0.047g of CPRG was also added to this tube and 19.920uL of nanopure water was also added bringing the final volume to 20mL.

Section 2. Preparing the bacteria.

1. The E. coli strains carrying the construct were cultured overnight in 5mL LB broth/Kan.
2. The next morning 200uL of the overnight culture was sub cultured in 19 mL of LB/Kan until it reached log phase (~5 hours), which was assessed by an OD value of ~0.6 at 595nm.

Section 3.

1. Once the culture was at log phase 2mL samples were taken (10 samples for 10 time points) in 2mL tubes and pelleted, the supernatant was discarded.
2. To these, 1.9 mL of the solution prepared above (2% arabinose, 3mM CPRG) was added, and the cells were resuspended.
3. One of the treatement 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 2% arabinose 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.

*A buffer should have been used in making this solution as that would have kept the pH constant throughout the whole experiment, minimizing the risk that changing pH might have influenced the color of the solution and therefore the OD readings.

The above procedure was adapted from: Shin HJ, Park HH, Lim WK, 2005. Freeze-dried recombinant bacteria for on-site detection of phenolic compounds by color change. J Biotechnol. 2005 Sep 22;119(1):36-43.