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.

We can degrade the PHB made by our own bacteria

In the below experiments we show that: The phaZ1 PHB depolimerase enzyme that we expressed and purified from E.coli is functional. The white and grey "stuff" that we purified in various experiments is Poly-Hydroxy-Butyrate bioplastc. The grey sample was purified from waste, therefore we made PHB bioplastic from waste. We can close the loop and degrade our own bioplastic.

The plastic samples were incubated with phaZ1 enzyme overnight and the 3HB was detected from the samples using a 3HB colorimetric assay kit. Please see the [http://2013.igem.org/Team:Imperial_College/Protocols#3HB_assay protocol] and the [http://2013.igem.org/Team:Imperial_College/3HB_assay data] for details.

The amount of 3HB monomers freed from the polymer is more in case of PhZ1 treated samples. There is less 3HB produced from the samples that were purified from bacteria than from the pure polymer. This could be because I put less amount in the assay, which I did because there was less available. I put about a 0.1g of grey and white and 0.5 grams of pure PHB.

We do expect to see some background 3HB in the untreated sample since it was in the shaking incubator at 37°C in the Buffer solution. PHB is a bioplastic and degrades spontaneously as well as enzymatically. However, the spontaneous degradation occurs slowly. We have made the process significantly more efficient and we are working on models to predict if this would be industrially scalable.
It is possible to work out how much PHB can be broken down by 1 gram of phaZ1 in an hour: amount of 3HB monomers freed from polymer in average: 153.8 mM in 800uL molecular weight of 3HB: 126.09 amount of phaZ1 enzyme: 0.230 mg/ml, I used 10 uL per reaction time was overnight for about 15 hours.

Growth Assays

Our Phaz1 construct was tested for induction by addition of Xylose.

Phaz1 growth assay. MG1655 E. coli were grown for 6h with 0% and 2% Xylose to induce Phaz1 BBa_K1149010. A two-tailed t-test, with p = 0.4181 shows that there is no significant difference between induction and no induction with regards to growth inhibition, as the value exceeds the critical value of 0.05. Growth was at 37°C in LB with shaking. Error bars are SEM, n=4. Figure made by Imperial College London 2013 iGEM.

Conclusion: There is no growth inhibition caused by induction in Phaz1.

References:

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 99
Illegal BglII site found at 981
Illegal XhoI site found at 1261
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 301
Illegal NgoMIV site found at 776
Illegal NgoMIV site found at 827
Illegal NgoMIV site found at 1021
Illegal NgoMIV site found at 1229
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI site found at 405
Illegal SapI site found at 999