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Team:Imperial College/BioPlastic Recycling: PHB
From 2013.igem.org
Module 2: Plastic Fantastic
Plastic Fantastic is a complete P(3HB) bioplastic recycling platform, where P(3HB) is degraded into monomeric form and then re-polymerised back into de novo P(3HB) for future applications.
Overview
Specifications
Design
Modelling
Assemble
Testing & results
Future work
Overview
We are developing a system by which PHB can be recycled when products made from it come to the end of their life. In in order to do this, we have engineered E.coli to break down P3HB extracellularly to release the monomers of 3-hydroxybutyrate (3-HB). E.coli in a different bioreactor will then use the 3-HB as their carbon source for the reproduction of P3HB. We have thoroughly characterised the phaZ1 PHB depolimerase enzyme and demonstrated that it degrades PHB in various experiments. Our models predict that use of the enzyme on a bioreactor scale will be effective in degrading bioplastic on an industrial or local scale. For the adjustment of the PHB synthesis pathway, we designed a metabolic pathway where a permease will take up 3HB and the bdh2 dehydrogenase enzyme will convert it to acetoacetate, which can be then used for PHB synthesis.
We are also establishing a second bioplastic recycling platform for polylactic acid (PLA)
This aspect of our module is in collaboration with the Yale iGEM team
Specification
Degradation of P(3HB)
Our bacteria should be resist any toxic effects that are associated with P(3HB) or 3HB
Our bacteria should degrade (P3HB)
Synthesis of P(3HB)
Our bacteria should take up and internalise 3HB from the surrounding media
Our bacteria should be able to utilise P(3HB) as a sole carbon source
Design
In order to ensure efficient expression in our Chassis, we ordered E.coli codon optimized versions of our proetin coding genes.
Degradation of P(3HB)
We are using E.coli chasis and we made sure to test for relevant toxicity issues, please see our data. PHB and 3HB are not toxic to the cells under conditions relevant to our bioreactor design.
[http://parts.igem.org/Part:BBa_K1149010 BBa_K1149010]: Extracellular expression of phaZ1, PHB depolymerase enzyme. It is regulated by a strong xylose the inducible promoter [http://parts.igem.org/Part:BBa_I741018 BBa_I741018] and we are using the RBS 0034. For extracellular secretion, we fused pelB secretion tag [http://parts.igem.org/Part:BBa_J32015 BBa_J32015] to the N terminus of the protein.
http://www.igem.org/wiki/images/8/86/Reaction_phaz.jpg
Synthesis of P(3HB) from 3HB
3HB Permease:We designed a biobrick for the expression of a Putative permease identified from the literature [http://www.ncbi.nlm.nih.gov/nuccore/AB330992(AB330992.1)] for 3HB inport. We optimised the sequence for E.coli and planned experiments with the construct. However, the gene synthesis was delayed and we did not get to the stage of characterizing and submitting the part.
[http://parts.igem.org/Part:BBa_K1149050 BBa_K1149050]: Intracellular expression of bdh2, 3HB dehydrogenase enzyme . We have used a strong arabinose inducible promoter [http://parts.igem.org/Part:BBa_K206000 BBa_K206000] in front of the operon for controlled expression of the enzyme since the enzyme is only necessairy for the cell if 3HB is present. We have included superfolder GFP [http://parts.igem.org/Part:BBa_K515005 BBa_K515005] in operon with phaZ1 so that we can monitor gene expression from the promoter via fluorescence measurements. (see our corresponding data under results)
http://www.igem.org/wiki/images/f/f0/Reaction_bdh.jpg
Safety
Bacteria strains
We used E. coli K-12 strains MG1655, NEB 10 beta, NEB 5 alpha and TOP10 (similar to E. coli K-12 DH 10 beta that fall under Risk Group 1 for the experiments, but eventually only MG1655 strain will be used in the bioreactors. This strain is shown rarely survives outside labs, although minor symptoms may show if infected. Meanwhile, the parts we designed are specialised for degrading PUR and they are not expected to harm human health.
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®ion=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].
Bioreactor safety
We have come up with a few strategies to prevent release of our bacteria into the environment. Firstly, the bioreactor containing bacteria will have a kill switch that kills bacteria if the bioreactor is opened when operating. Secondly, we lyse the cells at the end of reactions to harvest our product. This also ensures living bacteria will not be released unexpectedly.
Polylactic acid degradation model
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:
| enzyme | source organism | biobrick | reference |
|---|---|---|---|
| Proteinase K | Engyodontium album | [http://parts.igem.org/Part:BBa_K1149002 BBa_K1149002] | [http://www.ncbi.nlm.nih.gov/nuccore/X14689.1] |
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
| Parameter | Description | Value | Units | Sources | Assumptions |
|---|---|---|---|---|---|
| β | maximum rate of transcription | 0.032 | mM/min | Please see derivation 1 below. | Please see derivation 1 below. |
| K | Activation coefficient | 0.0031 | mM | [http://parts.igem.org/Part:BBa_K206000:Characterization] | Taking the "switch point" as the activation coefficient |
| dmRNA | mRNA degradation rate | 0.10 | 1/min | [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC420366/pdf/07X.pdf] | Taking the value of mRNA half-life in E.coli strain MG1655 as 6.8min. rate = ln2/half-life = ln2/6.8 = 0.10 |
| dprotein | Protein degradation rate | 0.050 | 1/min | [http://jb.asm.org/content/189/23/8746.full] | There is no active degradation pathway and that dilution is the dominant way by which the protein level decreases in a cell. Rate = 1/doubling time, where doubling time = 20min. Assuming steady-state growth in LB broth as presented in paper. |
| k2 | Protein production rate (Proteinase K) | 4.7 | 1/min | Please see derivation 2 below. | Please see derivation 2 below. |
| [Arabinose] | Concentration of arabinose | Initial: 0.008 | mM | ||
| [mRNA] | Concentration of mRNA | - | mM | - | - |
| [Proteinase K] | Concentration of Proteinase K | - | mM | - | - |
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
- ∴Mass concentration =
- 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 BDH2 (N = 768) in the volume of an E.coli cell is = 0.25mM
- 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 ÷ 0.25 = 0.032/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
- ∴Mass concentration of one aa in the volume of an E.coli =
- Translation rate = 20aa/s = (20 x 1.66x10-5 x 60)mM/min = 0.020mM/min
- BDH2 comprises of 256aa[http://www.uniprot.org/uniprot/Q2PEN2&format=html]
- ∴concentration of BDH2's aa in the volume of an E.coli = 1.66x10-5mM x 256 = 4.25x10-3mM
- ∴ Rate of protein production = 0.020 ÷ 4.25x10-3 = 4.7/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:
| Parameter | Description | Value | Units | Sources |
|---|---|---|---|---|
| Km | Michaelis constant | 0.032 | mM | - |
| Vmax | Maximum velocity | 2.472 | mM/min | - |
| Proteinase K | Concentration of the enzyme in assays | 30 | mg/L | - |
| Kcat | turnover number | 2348.4 | 1/min | See derivation below |
| Mw | Molecular weight of proteinase K | 28.5 | KDa | [http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Datasheet/2/p4850dat.Par.0001.File.tmp/p4850dat.pdf] |
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
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Our bugs can grow. Oh joy.
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.
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 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 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.
 |  |
.png) | _fluor.png) |
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:
Papers Referenced
- ANDERSON A, DAWES E. Occurrence, Metabolism, Metabolic Role, and Industrial Uses of Bacterial Polyhydroxyalkanoates. Microbiol Rev 1990 DEC;54(4):450-472.
- Harding KG, Dennis JS, von Blottnitz H, Harrison STL. Environmental analysis of plastic production processes: Comparing petroleum-based polypropylene and polyethylene with biologically-based poly-beta-hydroxybutyric acid using life cycle analysis. J Biotechnol 2007 MAY 31;130(1):57-66.
- 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.
- Jendrossek D, Handrick R. Microbial degradation of polyhydroxyalkanoates. Annu Rev Microbiol 2002;56:403-432.
- 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.
- 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
