Our bugs can grow. Oh joy.

Enzyme activity of PHB depolymerase (phaz1)

It can be seen from the 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 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 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

The above graph shows that greater esterase activity occurs when the phaz1 cell lysate is in the reaction mixture. The graph shows that there is also esterase activity occuring in the Empty Vector and Substrate alone reaction mixtures, but this is due to the imidazole present in the Tris-HCl buffer, which acts as a general base catalysis. (6)

Purified enzyme assay

Since the phaz1 BBa_K1149010 construct contains a His tag, the protein could be purified by [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.

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 protocol and the 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.

bdh2 improves growth on 3HB

We have previously observed that MG1655 cells can survive in minimal media that contains 3HB as sole carbon source. This is evidence for that some uptake mechanism and a metabolic pathway is active at a low level in the cells. We hypothesised that the 3HB dehydrogenase could improve growth since it can convert 3HB into acetoacetate, a common metabolite in E.coli.

In order to test this, we run a growth experiment with various 3HB concentrations where the growth of cells containing bdh2 (BBa_K1149050) and Empty vector was recorded on a 96 well plate. We have calculated the growth rates from this data and plot it on the graph below.

3HB sole carbon source growth in minimal media with phaCAB and EV. MG1655 phaCAB were grown in supplemented and minimal M9 media. Relative to the empty vector control, there was no significant difference in growth as t-test gave p = 0.8072 > 0.05. However, an ANOVA of the data gave (F 3,8 = 6.589, p < 0.0149), thus the null hypothesis, there is no difference between M9M and M9S must be rejected. Indeed if we look closer, we see that both M9M EV and M9S EV are significantly different from another (p = 0.045), as are M9M phaCAB and M9S phaCAB (p = 0.0284). Data points show final time point after 6h growth for each concentration. Growth was at 37°C with shaking over 6h. Error bars are SEM, n=4. Figure made by Imperial College London 2013 iGEM.

3HB sole carbon source growth in M9S with P3HB synthesis pathway genes. The 3 phaCAB promoter constructs are represented with native phaCAB, constitutive phaCAB and hybrid phaCAB. In addition bdh2 was tested to see whether its presence increased growth for the cells. This was completed with 3 3HB concentrations - 100 μM, 10 mM and 100 mM. ANOVA analysis shows that there is no significant difference between any of the constructs (F 4,10 = 0.1875, p < 0.9396). Data points show final time point after 6h growth for each concentration. Growth was at 37°C with shaking over 6h. Error bars are SEM, n=4. Figure made by Imperial College London 2013 iGEM.

Bdh2 with no pelB secretion tag growth. Bdh2 MG1655 cells were grown in M9S media to gauge growth, the growth does not differ from the control as p = 0.5543. Data points show final time point after 6h growth for each concentration. Growth was at 37°C with shaking over 6h. Error bars are SEM, n=4. Figure made by Imperial College London 2013 iGEM.

Bdh2 with no pelB secretion tag fluorescence. MG1655 cells with bdh2 were grown in M9S and then tested for fluorescence by measuring their GFP output. There is significant fluorescence as p < 0.0001. Data points show final time point after 6h growth for each concentration. Growth was at 37°C with shaking over 6h. Error bars are SEM, n=4. Figure made by Imperial College London 2013 iGEM.

Conclusion:We can conclude that there is a slight increase in the growth rate of bdh2 containing cells at 10 000 uM 3HB. This suggests that bdh2 is able to function as expected and produces acetoacetate which is used by the cell`s central metabolic pathways for growth. At 100uM or 100 000 uM, we did not observe any differences in growth. This could be be because the rate limiting step at low concentrations could be the uptake of 3HB rather than it`s conversion to acetoacetate. We hope that we could observe an increase in growth if we added the putative permease we designed to the system. At higher level, there is a drop in growth rates in both bdh2 and control cells, probably because of toxicity issues. In addition, fluorescence is highly elevated in bdh2 compared to empty vector control.

Proteinase K is a hydrolytic enzyme and active

Proteinase K is expressed in MG1655 cells transformed with BBa_K1149008

Proteinase K degrades PLA cup

Below come all the fabulous SEM images we shall hopefully have: