Part:BBa_J61032:Experience

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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 λ_max 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 (λ_Ex), 445 nm (λ_Em)	50 mM in DMSO	50 μM	-	~ 5 minutes

Kinetics
To characterize the enzyme they conducted fluorometric assays to obtain K_m values. To this end bacterial cells were grown until in exponential growth phase. Upon reaching this, gene expression was induced by AHL (see [http://http://2013.igem.org/Team:ETH_Zurich/Infoproc ETHZ system 2013]). After another 4-5 h of growth, cells were harvested and lysed, the cell free extract (CFX) used for the fluorometric assay. The properly diluted CFX was measured on a 96 well plate in triplicates per substrate concentration. A plate reader took measurements at λ_Ex 365 nm and λ_Em 445 nm. The obtained data was evaluated and finally fitted to Michaelis-Menten-Kinetics with SigmaPlot™. See the resulting graph below.

Figure 6. Michaelis-Menten-Kinetics of PhoA cell lysate from E.Coli overexpressing PhoA: plots velocity versus substrate concentration (2.5 μL, 5 μL, 10 μL, 30 μL, 60 μL, 120 μL, 240 μL) in 20 mM Tris buffer of pH 8. A kinetic value for K_m obtained by fitting the raw data to standard the Michaelis Menten equation; K_m = 105.9 ± 5.3 μM. All assays were carried out in triplicates, results are presented as means.

The experimental procedure was as following:

Prepare buffers
- Lysis buffer: 10 mg/ml Lysozyme, 20 mM Tris buffer, pH 8
- Reaction buffer: 20 mM Tris buffer, pH 8
- NOTE: For other enzymes than the ones we tested (Aes,GusA,NagZ,PhoA) you might need different buffers
Cell culture
- Inoculate bacteria in 20 mL of LB with antibiotics
- Let grow at 37°C shaking(200 rpm) to an OD₆₀₀ of 0.6
- Induce enzyme expression (100nM AHL in our case)
- Let grow at 37°C shaking(200 rpm) for 4-5h
Cell lysis
- Transfer to 50 mL Falcon™ tube
- Spin down at 4°C for 5 min with 4 rcf
- Resuspend in lysis buffer, 1 μL/mg pellet
- Transfer to eppendorf tubes
- Incubate at room temperature for 10 min at 220 rpm
- Spin down at 4°C for 10 min with max. speed
- Transfer the supernatant to new tubes, discard pellets
- Cell free extract can be stored at -20°C or continue processing
Dilution
- The following values were provided by Johannes Haerle
  - Aes: Dilute CFX 1:100 in reaction buffer
  - GusA: Dilute CFX 1:100 in reaction buffer
  - NagZ: Use pure
  - PhoA: Dilute CFX 1:10 in reaction buffer
Hydrolysis reaction
- Perform this measurement in a 96 well plate or similar
- Perform 3 replicates for each substrate concentration
- Present 41.6 μL reaction buffer in each well
- Add 8 μL diluted CFX (the further dilution ocurring here is intended)
- Add 30.4 μL of corresponding substrate
- Detection of fluorescence in suitable plate reader (λ_Ex 365 nm, λ_Em 445 nm)

[http://2012.igem.org/Team:Technion Team Technion (2012)] have made an assay using this part. The BioBrick they used is BBa_K784000 consisting of T7 RNAP promoter, phoA and T7 terminator.

We have used the part inside a BL21 strain of E.coli which has an endogenouse PET system.
In the presence of IPTG inducer, T7 RNAP is expressed and activates the part promoter which expresses the Alkaline Phosphatase gene (phoA).
The graph below presents the results of the experiment described. The absorbance at 420nm for ‎each of the different concentrations of the inducer in a range of 0.5 μΜ to 1000 μΜ on a ‎logarithmic scale. The absorbance was calculated via different dilutions of the samples: dilutions ‎by 0.5, by 0.25, and by 0.1. The error bars represent the processing of the data collected. The line at ‎the bottom of the graph represents the basal level according to the control result- no IPTG ‎induction.‎

300X300px

As can be seen, the graph shows a clear positive tendency- the higher the concentration the higher ‎the absorption, as expected. Starting from a concentration of 40 μM and above, there are only ‎small deviations from the absorption value of 1.2, probably due to the fact that saturation has ‎been achieved. ‎

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