Polylactic acid (PLA) Degradation Module

Introduction

The efficiency of Polylactic acid is important for the performance of MAPLE. Therefore, we chose a strong enzyme Proteinase K which is suggested by many literature as an efficient PLA degrading enzyme. We performed several assays in order to determine the kinetic properties of Proteinase K. The PLA degradation model was then built based on the experimental results.

Objective and Design

1. With the defined kinetic properties of Proteinase K. The model can predict the time needed to degrade a certain amount of PLA. The information is important as it estimates the efficiency of the MAPLE system when degrading the large amount of plastic.Therefore, further improvements can be made if the system is not efficient.

2. The model also involves the gene expression model of the Proteinase K with an inducible promoter. Therefore, gene expression can be regulated by adjusting the inducer concentration. The overall plastic degradation can be regulated by the gene expression.

3. The secretion model is also contained in the model which assumes the efficiency of enzyme secretion to the culture.

Specifications of the model

Polylactic acid (PLA) degradation involves proteinase K:

The overall PLA degradation model is shown as below:

There are two compartments which represents cells and the culture from left to right. The "cell" compartment contains the gene expression module whereas the "culture" compartment contains the degradation module. The "secretion" block that connects two compartments is the secretion module.

Parameters and assumptions

Gene expression module of Proteinase K

1.Derivation of the maximal expression rate,β

• Average molecular weight (Mw) of a base pair = 660g/mol[http://www.geneinfinity.org/sp/sp_dnaprop.html][http://www.lifetechnologies.com/uk/en/home/references/ambion-tech-support/rna-tools-and-calculators/dna-and-rna-molecular-weights-and-conversions.html]
• Average mass of a base pair = 660g/mol x 1.66x10^-24 = 1.1x10^-21g
• Volume of an E.coli cell = 1µm³[http://kirschner.med.harvard.edu/files/bionumbers/fundamentalBioNumbersHandout.pdf] = 1x10^-15L
  • ∴Mass concentration =
  • ∴Molar concentration of 1 base pair in the volume of E.coli = = 1.66x10^-6 mM
• BioBrick assembly plasmid pSB1C3 is a high copy number plasmid (100-300 copies per cell)[http://parts.igem.org/Part:pSB1C3?title=Part:pSB1C3]
  • assume 200 copies per cell
• ∴ concentration of the gene per cell = N x 200 x 1.66x10^-6mM, where N = number of base pairs
  • ∴ concentration of the gene proteinase K (N = 1194) in the volume of an E.coli cell is = 0.40mM
• Transcription rate in E.coli = 80bp/s[http://kirschner.med.harvard.edu/files/bionumbers/fundamentalBioNumbersHandout.pdf] = 80 x 1.66x10^-6mM/s = 80 x 1.66x10^-6 x 60mM/min = 7.97x10^-3mM/min
• ∴ Rate of mRNA_Proteinase K production under the control of pBAD = 7.97x10^-3 ÷ 0.40 = 0.020/min

• Average molecular weight(Mw) of an amino acid(aa)= 110g/mol[http://www.genscript.com/conversion.html][http://www.promega.com/~/media/Files/Resources/Technical%20References/Amino%20Acid%20Abbreviations%20and%20Molecular%20Weights.pdf]
• Average mass of an amino acid = 110g/mol x 1.66x10^-24=1.83x10^-22g/L
  • ∴Mass concentration of one aa in the volume of an E.coli = = 1.83x10^-6g/L
  • ∴Molar concentration of one aa = = 1.66x10^-5mM
• Translation rate = 20aa/s = (20 x 1.66x10^-5 x 60)mM/min = 0.020mM/min
• Proteinase K comprises of 398aa[http://www.uniprot.org/uniprot/Q2PEN2&format=html]
  • ∴concentration of Proteinase K's aa in the volume of an E.coli = 1.66x10^-5mM x 398 = 6.61x10^-3mM
• ∴ Rate of protein production = 0.020 ÷ 4.25x10^-3 = 3.0/min

Degradation

The reaction equation of the PLA degradation is:

[Polylactic acid]+[Proteinase K]= 660 [lactic acid] + [Proteinase K]

Assumptions:

We assumed 1 mole of polylactic acid can produce 660 moles of lactic acid.

The molecular weight of a single polylactic acid monomer is 90 g/mol [http://www.chemspider.com/Chemical-Structure.592.html]whereas the molecular weight of the solid polylactic acid is around 59500 g/mol.[http://www.sciencedirect.com/science/article/pii/S0014305711003582]Therefore, the short-chain polylactic acid consists approximately 660 monomers. 660 molecules of lactic acid will be produced by degrading one chain of polymer.

We also assumed a simple Michaelis-Menten mechanism for proteinase K

The parameters for the kinetic equations are:

Derivation:Turnover number (Kcat) = Vmax*Mw/[proteinase K] = 2348.4

The efficiency of Secretion is assumed to be 90% secretion over 2 hours.[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1251600/] The rate of secretion in the model is therefore:

rate of secretion = 0.9[concentration of Proteinase K]/120 (mM/mins)

Simulations and Results

Summary

Our chassis can grow and survive in reasonable concentrations of P(3HB) and 3HB. To increase tolerance to 3HB we have re-designed our system to include expression of Bdh2 (BBa_K1149050, 3HB dehydrogenase), which breaks down 3HB to acetoacetate. Acetoacetate toxicity was examined and these data are being utilised along with our metabolic models to determine the effects of differing levels of 3HB and acetoacetate on bioplastic (P3HB) production.

MG1655 transformed with either native phaCAB or empty vector (control) plasmid were grown for 6h in LB containing 3-HB or P-3HB to assess any potential toxicities associated with these bioplasrics.

3-hydroxybutyrate (3HB)

Toxicity of 3-hydroxybutyrate in LB with phaCAB and EV expression. 3HB was tested at 5 concentrations for toxicity - 1 μM, 100 μM, 1 mM, 10 mM and 237 mM. Between 10 mM and 237 mM, there is a clear toxicity effect observed. 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.

Poly(3-hydroxybutyrate) P(3HB)

Toxicity of Poly(3-hydroxybutyrate) in LB with phaCAB and EV expression. P3HB toxicity was only tested at one concentration in LB - 31 μg/L, it was also tested at 31 mg/L however the resulting OD was over 1, so this data was not considered accurate. Growth was at 37°C with shaking over 6h. Error bars are SEM, n=4. Figure made by Imperial College London 2013 iGEM.

Conclusion:3HB is toxic above 10mM and P(3HB) had no effect on growth at 31 μg/L. These data can be utilised to determine the maximum level of 3HB which should be present in our final bioreactor design. We have shared these data with our modelling team in order to determine maximum P(3HB) production at or below 10mM (3HB). In light of these data we have modified our design to include the expression of bdh2 (BBa_K1149050)</b>, which is a 3HB dehydrogenase that can convert 3HB into acetoacetate, a common metabolite in E.coli. Expression of bdh2 should overcome the toxicity limit of 3HB, since E. coli can metabolise acetoacetate</b>To this end we tested whether an increase in acetoacetate would present any toxicities.

Acetoacetate

Toxicity of acetoacetate in LB with phaCAB and EV expression. Acetoacetate was also tested for toxicity at 4 concentrations - 1 μM, 100 μM, 1mM and 287 mM. In phaCAB and EV this manifested itself between 1 mM and 287 mM. 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:Acetoacetate toxicity lies somewhere between 1mM and 287 mM.

Enzyme activity of PHB depolymerase (phaz1)

It can be seen from the Western Blot results that phaz1 [http://parts.igem.org/Part:BBa_K1149010 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 [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

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. [http://pubs.acs.org/doi/pdf/10.1021/ja00874a035 (6)]

Purified enzyme assay

Since the phaz1 [http://parts.igem.org/Part:BBa_K1149010 BBa_K1149010] construct contains a His tag, the protein could be purified by 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

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 kinetic activtiy

According to the literature, protease K breaks down poly L-lactic acid as well as fibrous proteins, such as silk fibroin. In the active site of proteinase K is a catalytic triad containing serine, histidine and aspartic acid. L-lactic acid is analogous to L-alanine. Poly L-lactic acid, therefore, can bind to and be catalysed by the catalytic triad, as it mimics silk fibroin which is rich with L-alanine residues. Proteinase K cannot catalyse the breakdown of poly D-lactic acid, because the catalytic triad will not accommodate it.

Substrate analogue, N-succinyl Ala-Ala-Pro-Leu p-nitroanilide (AAPL p-NA) was used to measure the kinetic activity of proteinase K. Cleavage of AAPL p-NA releases 4-nitroaniline (4-NA), which can be recorded by an increase in absorbance at wavelength 405nm.

Molecular structure of N-succinyl Ala-Ala-Pro-Leu p-nitroanilide. Image by [http://www.sigmaaldrich.com/catalog/product/sigma/s8511?lang=en&region=GB Sigma]

The extinction coefficient of 4-nitroaniline is 9,500 M-1s-1 at wavelength 405 nm, according to [http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Product_Information_Sheet/2/m4765pis.Par.0001.File.tmp/m4765pis.pdf Sigma]. Using the Beer-Lambert law, the concentrations of 4-NA were calculated and plotted against time.

The absorbance change recorded during the catalysis of different concentrations of AAPLp-NA by Proteinase K. Figure by Imperial College London 2013 iGEM

The concentration of 4-NA produced from different starting concnetrations of AAPLp-NA. Figure by Imperial College London 2013 iGEM

The initial velocities were calculated as the gradient between 0 and 30 seconds, when time = 0 seconds. These were plotted against the concentration AAPLp-NA and [http://www.graphpad.com/ graphpad] was used to estimate the Michaelis-Menten fit. It determined the Vmax as 46.31 µM/s and the Km as 19.28 µM.

Michaelis-Menten plot for proteinase K activity cleaving AAPLp-NA at 37ºC and pH 7.4. Figure by Imperial College London 2013 iGEM

Proteinase K is a hydrolytic enzyme and active

Proteinase K is expressed in MG1655 cells transformed with BBa_K1149008

Proteinase K degrades PLA cup

Chemical reagents

Some of the reagents we used were slightly toxic to human. In order to reduce the risks of using toxic reagents, we always wear gloves and labcoats. For some toxic reagents, we performed in fume cupboard. Click on the reagents to see the MSDS.

1. [http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=GB&language=en&productNumber=363502&brand=ALDRICH&PageToGoToURL=http%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog%2Fproduct%2Faldrich%2F363502%3Flang%3Den Poly(R)-3-hydroxybutyric acid].

2. [http://www.sigmaaldrich.com/MSDS/MSDS/PleaseWaitMSDSPage.do?language=&country=GB&brand=SIAL&productNumber=P2308&PageToGoToURL=http://www.sigmaaldrich.com/catalog/product/sial/p2308?lang=en&region=GB Proteinase K].

3. [http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=GB&language=en&productNumber=54965&brand=ALDRICH&PageToGoToURL=http%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog%2Fproduct%2Faldrich%2F54965%3Flang%3Den Sodium 3-hydroxybutyrate].

4. [http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=GB&language=en&productNumber=A3256&brand=SIGMA&PageToGoToURL=http%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog%2Fproduct%2Fsigma%2Fa3256%3Flang%3Den Arabinose].

5. [http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=GB&language=en&productNumber=A8509&brand=ALDRICH&PageToGoToURL=http%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog%2Fproduct%2Faldrich%2Fa8509%3Flang%3Den Acetoacetate].

Papers Referenced

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3. Kim S, Dale BE. Energy and Greenhouse Gas Profiles of Polyhydroxybutyrates Derived from Corn Grain: A Life Cycle Perspective. Environ Sci Technol 2008 OCT 15;42(20):7690-7695.
4. Jendrossek D, Handrick R. Microbial degradation of polyhydroxyalkanoates. Annu Rev Microbiol 2002;56:403-432.
5. Philip S, Keshavarz T, Roy I. Polyhydroxyalkanoates: biodegradable polymers with a range of applications. Journal of Chemical Technology and Biotechnology 2007 MAR;82(3):233-247.
6. Thomas C. Bruice , Thomas H. Fife , John J. Bruno , Patricia. BenkovicHydroxyl Group (V)1 and Imidazole (X)2 Catalysis. The General Base Catalysis of Ester Hydrolysis by Imidazole and the Influence of a Neighboring Hydroxyl Group. J. Am. Chem. Soc., 1962, 84 (15), pp 3012–3018