Latest revision as of 22:21, 3 October 2013

Phaz1 expression

Depolymerises poly 3-hydroxybutyrate extracellularly

(image from BRENDA)

Enzyme activity of PHB depolymerase (phaz1)

It can be seen from the [http://2013.igem.org/Team:Imperial_College/Western_Blots Western Blot results] that phaz1 BBa_K1149010 was being expressed. To show that this enzyme has esterase activity, colourimetric assays were performed using the substrate analog para-Nitrophenyl butyrate. When the ester bond in this substrate is cleaved, 4-Nitrophenol is released. This is accompanied by an increase in absorbance at the wavelength 405 nm and a colour change from colourless to yellow. The concentration of 4-Nitrophenol produced could then be calculated with the Beer-Lambert Law, as the extinction coefficient of 4-NP at 405 nm is 18,000 M-1 cm-1. This experiment was performed with both the crude cell lysate and purified PHB depolymerase. Our data shows that this enzyme is definitely active.

para-Nitrophenyl butyrate is cleaved by PHB depolymerase to release 4-Nitrophenol. This is accompanied by an increase in absorbance at 405 nm. Figure adapted from [http://2012.igem.org/Team:TU_Darmstadt/Protocols/pNP_Assay#Enzyme TU Darmstadt 2012 iGEM]

When the substrate para-Nitrophenyl butyrate is added to the reaction mixture of purified PHB depolymerase (phaz1) in phosphate buffer, there is a colour change from colourless to yellow. Figure by Imperial College London iGEM 2013.

Cell lysate assay

After 48 hours of growing and inducing the phaz1 culture, the cells were lysed by probe-sonication, spun down and resuspended in 50mM Tris-HCl buffer. A reaction mixture containing 5 µL of crude phaz1 lysate, 4 µL of para-Nitrophenyl butyrate and 1 mL of 50 mM Tris-HCl pH 7.4 buffer was incubated in the [http://www.eppendorf.com/int/index.php?sitemap=2.1&action=products&contentid=1&catalognode=87236 Eppendorf BioSpectrometer] for 570 seconds, whilst the absorbance at 405 nm was automatically recorded every 30 seconds.

The increase in absorbance that accompanies the cleavage of para-Nitrophenyl butyrate by PHB depolymerase (phaz1). Figure by Imperial College London 2013 iGEM

The concentration of 4-Nitrophenol released by PHB depolymerase (phaz1) activity. Empty Vector and Substrate alone were used as negative controls. Figure by Imperial College London 2013 iGEM

Purified enzyme assay

Since the phaz1 BBa_K1149010 construct contains a His tag, the protein could be purified by [http://2013.igem.org/Team:Imperial_College/Protocols#Protein_purification_by_metal_affinity_chromatography metal affinity chromatography]. The colourimetric assay was carried out, as with the cell lysate. 100 mM potassium phosphate (pH 7.4) buffer was used instead of the Tris-HCl buffer, to eliminate the esterase activity caused by imidazole in the buffer used previously. Every 30 seconds for 30 minutes the absorbance at 405 nm of the reaction mixture was recorded. The reaction mixture contained 1 ml of phosphate buffer, 2.5 µL of purified PHB depolyermase (stock concentration 0.230 mg/ml) and para-Nitrophenyl butyrate to make final concentrations of 50 µM, 100 µM, 150 µM, 200 µM and 250 µM of substrate.

The purified PHB depolyermase (phaz1) is definitely active. The graph shows that more 4-Nitrophenol is produced with greater concentrations of para-Nitrophenyl butyrate. Figure by Imperial College London 2013 iGEM

The above graph shows how the concentration of 4-Nitrophenol produced is greater when the para-Nitrophenyl butyrate substrate concentration is greater. This data shows that we have successfully purified active PHB depolyermase.

SDS PAGE gel shows that PHB depolymerase was purified from the crude cell extract. Figure by Imperial College London 2013 iGEM

Clearing Zone Assay

Clearing zone experiment was conducted to semi-quantitatively test the ability of PhaZ1 to break down P3HB. PhaZ1 cell lysate was tested on a P3HB LB agar plate. The protocol can be found [http://2013.igem.org/Team:Imperial_College/Protocols#Clear_zone_assay here].

PhaZ1 showing P3HB degradation ability by generating a clear zone on a P3HB LB agar plate. A. No difference between empty vector cell lysate and PhaZ1 cell lysate right after they were pipetted into the wells. B. PhaZ1 in the cell lysate started to clear P3HB around the well after 1 day. C. PhaZ1 created a clear zone around the well after 3 days, whereas it was still cloudy around the empty vector cell lysate well.