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Applications of BBa_J61032

[http://2013.igem.org/Team:ETH_Zurich ETH Zurich 2013] used PhoA in their project as a reporter enzyme.

Colorimetric and fluorometric response

Figure 2. Liquid culture from the triple knockout E.Coli strain overexpressing PhoA or PhoA-His respectively after reacting with pNPP. The negative control contains liquid culture without pNPP added.

Figure 3. Liquid culture from the triple knockout E.Coli strain overexpressing PhoA after reacting with BCIP. The negative control contains liquid culture without BCIP added.

To test the functionality of the enzyme, liquid culture of the ΔaesΔgusAΔnagZ Escherichia coli strain overexpressing PhoA was incubated with pNPP (Figure 2). Another suitable chromogenic substrate for detection of PhoA is BCIP (Figure 3).

Figure 1. Enzymatic reaction of PhoA with pNPP (1) or BCIP (2).

Cell lysate for the assay described below was tested for active enzyme in the same way, but with the fluorescent substrate 4-MU-phosphate. The picture in Figure 5 was taken with a common single lens reflex camera mounted on a dark hood at λ_Ex 365 nm.

Figure 4. Enzymatic reaction of PhoA with 4-MU-phosphate.

Figure 5. Cell lysate from the ΔaesΔgusAΔnagZ E scherichia coli strain overexpressing PhoA after reacting with 4-MU-phosphate.

Hydrolase	Substrate	Absorption λmax or Excitation/Emission	Stock solution	Liquid culture: end concentration	Colonies: 1.5 μl of substrate solution	Response time
PhoA	4-Nitrophenoyl-phosphate (pNPP)	Yellow, 405 nm	0.5 M in DEA	50 mM	0.5 M	~ 1 minute
	5-Bromo-4-Chloro-3-indolyl phosphate (BCIP)	Blue, 615 nm	0.1 M in H2O	1 mM	50 mM	~ 30 minutes
	4-MU-phosphate	Blue (fluorescent), 372 nm (λEx), 445 nm (λEm)	50 mM in DMSO	50 μM	-	~ 5 minutes

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