Contents 1 Resourceful Waste 1.1 Overview 1.1.1 Petrochemical Plastic degradation 1.1.1.1 Pathway of PUR hydrolysis: 1.1.2 Bioplastic Synthesis 1.2 Specification 1.3 Modelling 1.3.1 Degradation models: The enzyme cocktail 1.3.2 Biosynthetic models: The path to bioplastic 1.4 Design 1.4.1 Polyurethane Degradation 1.4.2 Gene expression 1.4.3 Secretion 1.4.3.1 Alternative strategies for protein secretion in E.coli: 1.4.3.2 Extracellular Secretion in E.coli: 1.4.3.3 Outer Membrane Anchors and Periplasmic Expression in E.coli: 1.4.4 PUR degradation constructs: 1.4.5 Tolerance to waste 1.4.6 PHB synthesis Design 1.4.7 Assembly: Adding better promoter and RBS to phaCAB operon 1.4.7.1 Partial sequence of the constitutive phaCAB construct ([http://parts.igem.org/Part:BBa_K1149052 BBa_K1149052]): 1.4.7.2 Partial sequence of the hybrid phaCAB construct ([http://parts.igem.org/Part:BBa_K1149051 BBa_K1149051]): 1.5 Results 1.6 Protocols 1.6.1 LB-Agar Plates containing Nile Red stain 1.6.2 Nile Red Culture staining 1.6.3 Fluorescent Microscopy 1.6.4 Extracting PHB 1.6.5 Relationship between OD and dry biomass 1.7 Safety 1.7.1 Dimethyl Sulfoxide(DMSO)

Resourceful Waste

The mixed waste produced from materials recovery facilities is currently disposed of by incineration, causing harm to the environment and health. It also wastes the resources present, many of which are non-renewable. We have developed a system to produce the valuable bioplastic P3HB, from this waste. To utilise the plastics in the mixed waste we have expanded the range of plastic degradation parts in the registry, expressing Polyurethane esterases which break down polyurethane to produce the platform chemical ethylene glycol, which can be filtered off and sold. Once the oil based plastics have been removed, E.coli transformed with the phaCAB operon from Ralstonia eutropha will produce P3HB from the remaining waste material. In this way, the mixed can be used to produce commodities.

Overview

Petrochemical Plastic degradation

It is known from previous research that some lignin degrading microbial enzymes to be so tough that they can even deal with more difficult polymers: plastics. Some previous iGEM teams have exploited this and worked on enzymatic degradation of various kinds of plastic. We would like to build on this work and extend the plastic degradation capabilities of the synthetic biology community by improving PUR degradation in particular as this has not been successfully achieved before. We have identified 5 PUR-esterase enzymes from the literature that are capable of catalyzing the below reaction.

Pathway of PUR hydrolysis:

We have synthesised all of the genes in the below table and are testing them for expression in E.coli, secretion, activity and PUR degradetion capabilities. Our ultimate design is to be able to control the relative levels of different enzymes in waste degrading bio-reactor in order to adjust it to the composition of waste. Therefore we designed the expression constructs accordingly. Our models predict the degradation rate of PUR at the bioreactor scale.

enzyme	source organism	biobrick	reference
EstCS2	uncultured unknown bacterium (GU256649.1)	[http://parts.igem.org/Part:BBa_K1149002 BBa_K1149002]	[http://www.microbialcellfactories.com/content/10/1/41 Kang et.al 2011]
pueA	Pseudomonas chlororaphis	[http://parts.igem.org/Part:BBa_K1149003 BBa_K1149003]	[http://onlinelibrary.wiley.com/doi/10.1111/j.1574-6968.2000.tb09056.x/pdf Stern et al., 2000]
pueB	Pseudomonas chlororaphis	[http://parts.igem.org/Part:BBa_K1149004 BBa_K1149004]	[http://www.sciencedirect.com/science/article/pii/S0964830501000427 Howard et al., 2001]
pudA	Comamonas acidovorans	[http://parts.igem.org/Part:BBa_K1149005 BBa_K1149005]	[http://www.sciencedirect.com/science/article/pii/S0964830598000663 Allen et al. 1999]
pulA	Pseudomonas fluorescens	[http://parts.igem.org/Part:BBa_K1149006 BBa_K1149006]	[http://www.sciencedirect.com/science/article/pii/S0964830598000687 Vega et al., 1999]

Bioplastic Synthesis

Poly-3-hydroxybutyrate(P3HB) is a bioplastic which is naturally produced inside bacteria such as Ralstonia eutropha, where it accumulates as globules inside the cell. In its native bacteria it is produced as an energy store(1) but it is as a plastic that it interests researchers and industrialists.

We have made P3HB in E.coli, transferring three genes, naturally found in Ralstonia eutropha into E.coli MG1655. These encode the three enzymes necessary for P3HB production; polyhydroxyalkanoate synthase(phaC), 3-ketothiolase(phaA) and acetoacetyl coenzyme A reductase(phaB). These are encoded by the phaCAB operon. We have altered the expression of these three genes to maximise the production of P3HB as high yields are required for it to be economically viable.

http://www.igem.org/wiki/images/f/f3/Reaction_phaC.jpg

Specification

1. The bacteria should survive and grow in mixed waste

In order for the bacteria to produce bioplastic from the mixed waste they first need to be able to use it as a carbon source

2. The bacteria should secrete a functional polyurethane esterase

To recover the resources from polyurethane in the mixed waste, the enzymes should secrete in an active form.

3. Our Bacteria should be able to tolerate mixed waste degradation products, such as Ethylene Glycol

The products of Polyurethane degradation are toxic and so any bacteria growing with them must be able to survive to a concentration which will allow economical production of ethylene glycol. We have chosen a strain of E.coli-MG1655, which is resistant to ethylene glycol toxicity.

4. The bacteria should produce P3HB

Modelling

Degradation models: The enzyme cocktail

Predictions for the degradation system as a whole with different enzyme combinations and how this informed the design of our constructs. <p>Scaling up to bio-reactor size and how that informed our plans for industrial implementation and MAPLE.

Biosynthetic models: The path to bioplastic

Metabolic models to predict how we can use various monomers for the production of bioplastic. Tell how flexible the system is in terms of being able to use either sugars or 3HB. This informed our design in that it predicts that it is enough to have phaCB when 3HB is the main monomer but the full phaCAB is better when PHB synthesis is from glucose. Therefore, we are constructing both and we are testing them experimentally to see if this matches our predictions. (Bobby, can you make predictions in the units and format of what we would be likely to measure experimentally?)

Other aspects of the metabolic can be found under PHB recycling. (?)(MK)

Synthesis model scaled up and what that means for industrial implementation and MAPLE.

Design

Polyurethane Degradation

Gene expression

construct design: Inducible promoters, His and Flag tags for western, GFP in operon .

later, these enzymes can be combined

Secretion

align="justify">E.coli is commonly used as a chassis in innovative iGEM project that aim to prove the concept and make the case for a novel function in a biologically engineered machine. In our case, we aim to degrade and synthesise plastic and the degradation part of our system needs to be extacellular. There are not only such biotechnological but also many other reasons for the extracellular targeting of a protein: It can be a product with a medical function, like the [http://www.2009.igem.org/Team:METU-Gene/EGF_Transportation EGF hormone in a previous iGEM project] or be important in bioremediation such as in Dundee Team’s toximop system and much more. There are many strategies for secretion. On the following page, you can read about these and find out why we chose the pelB tag for all our degradation enzymes and how we improved the ESTCS2 esterase by changing the phoA outer-membrane anchoring tag in the original Biobrick to pelB. Our story will help you to choose a secretion strategy best suited to your project and also give you guidance with the technical details of using it.

Alternative strategies for protein secretion in E.coli:

N terminal secretion-signal peptides are recognised by the sec pathway . The pathway transports to the periplasm and additional mechanism are usually necessary to further export the protein to the extracellular space. An approach for using this pathway is the addition of an N terminal signal peptide to the target protein which can obtain as high as 90% efficiency in secretion to the extracellular space. Such signals are pelB and phoA tags which are available as biobricks. PelB can get cleaved off by pelB peptidase in the periplasm but phoA will anchor your protein to this localisation.

Fusion partners are endogenous proteins that naturally get secreted in E.coli and can be fused to the target protein. Some such proteins were demonstrated to be a powerful carriers of medically relevant human proteins in E.coli. The yebF and ompF proteins exit the cell via the sec pathway and use additional mechanism for leaving the periplasm where they interact with outer-membrane porins for extracellular secretion. The osmY is available as a Biobrick and we have submitted ompF this year.

The porin proteins such as ompF and ompA, can be used as fusion partners too and anchor proteins to the outer surface of E.coli.

ABC transporters use ATP for transport of specific proteins across the bacterial membrane. The ABC transporter of Erwinia chrysanthemi can be expressed in E.coli and export proteins that contain the LARD1 domain as a C terminal fusion. The system is biobricked in two separate plasmids (transporter, LARD1) with different antibiotic resistance and it is important to use both at the same time.

The [http://parts.igem.org/Protein_domains/Localization page] in the registry that lists localisation tags] is incomplete and contains eukaryotic and prokaryotic localisation signals mixed together. We have therefore put together the tables below for you to help choose an E.coli secretion strategy that is suitable for your project. </p>

Extracellular Secretion in E.coli:

	Description	Biobrick	Team
pelB	type II secretion signal peptide, cleaved off in periplasm	BBa_K208004 BBa_K1149022 BBa_K208004 BBa_J32015	lots eg. Imperial 2013
yebF	large fusion-protein, Sec-pathway and additional mechanism	BBa_K1149001	Imperial 2013
osmY	large fusion-protein, Sec-pathway and additional mechanism	BBa_K892008	Washington 2012
LARD 1	Lipase ABC transporter recognition domain	BBa_K258001	METU-GEne 2009

Outer Membrane Anchors and Periplasmic Expression in E.coli:

	Description	Biobrick	Team
ompF	porin protein that can be fused to a protein, anchors to outer membrane	BBa_K864204	Uppsala 2012
ompA	porin protein that can be fused to a protein, anchors to outer membrane	BBa_K103006	Wasraw 2008
INP	short domain,anchors in periplasm	BBa_K523013 (INP-YFP) BBa_K632002 (INP-Silicatein)	Minnesota 2011 Edinburgh 2011
torA	anchors in periplasm		Dundee 2013
phoA	tag for sec pathway, anchors to OM	BBa_K808028	Darmstadt 2011

Our choice is to use the pelB secretion tag as it has been demonstrated to work in many cases with sometimes as high as 90% transport efficiency. The pelB has been used in iGEM project for many years and is part of 50+ constructs. The UC-Davis team last year used it to secrete LC-Cutinase, a PET plastic degrading enzyme ( BBa_K936013) successfully which is somewhat similar to our plastic degradation enzymes.

Assembly methods and toolkit:

An advantage of the pelB is that it is relatively short (only 66 BP) and we could get our gene synthesised with the tag. A problem with standard biobrick assembly is that the scar site contains a STOP codon and therefore [http://parts.igem.org/Protein_domains alternative strategies are needed]. One of the ways around this is to use Infusion/Gibson assembly which we also had a go with and you can find instructions under our protocols section. We have constructed a biobrick where LARD1 is after a constitutive promoter and RBS in order to facilitate quicker cloning with less assembly steps, [http://parts.igem.org/Part:BBa_K1149021 BBa_K1149021].

Unfortunately, the pelB tag did not prove to be the best strategy for secreting our degradation enzymes in the MG1655 strain. Therefore, we are changing the strategy to use osmY.

References

1. Economou A. Following the leader: bacterial protein export through the Sec pathway. Trends in microbiology 1999;7(8) 315-320.
2. Thanassi DG, Hultgren SJ. Multiple pathways allow protein secretion across the bacterial outer membrane. Current opinion in cell biology 2000;12(4) 420-430.
3. Sletta H, Tondervik A, Hakvag S, Aune TEV, Nedal A, Aune R, et al. The presence of N-terminal secretion signal sequences leads to strong stimulation of the total expression levels of three tested medically important proteins during high-cell-density cultivations of Escherichia coli. Applied and Environmental Microbiology 2007;73(3) 906-912.
4. Qian Z, Xia X, Choi JH, Lee SY. Proteome-based identification of fusion partner for high-level extracellular production of recombinant proteins in Escherichia coli. Biotechnology and bioengineering 2008;101(3) 587-601.
5. Prehna G, Zhang G, Gong X, Duszyk M, Okon M, McIntosh LP, et al. A Protein Export Pathway Involving Escherichia coil Porins. Structure 2012;20(7) 1154-1166.
6. Binet R, Letoffe S, Ghigo JM, Delepelaire P, Wandersman C. Protein secretion by Gram-negative bacterial ABC exporters - A review. Gene 1997;192(1) 7-11.
7. Chung CW, You J, Kim K, Moon Y, Kim H, Ahn JH. Export of recombinant proteins in Escherichia coli using ABC transporter with an attached lipase ABC transporter recognition domain (LARD). Microbial Cell Factories 2009;8 11.
8. Francetic O, Pugsley AP. Towards the identification of type II secretion signals in a nonacylated variant of pullulanase from Klebsiella oxytoca. Journal of Bacteriology 2005;187(20) 7045-7055.
9. Fu LL, Xu ZR, Li WF, Shuai JB, Lu P, hu CXH. Protein secretion pathways in Bacillus subtilis: Implication for optimization of heterologous protein secretion. Biotechnology Advances 2007;25(1) 1-12.
10. Cho HY, Yukawa H, Inui M, Doi RH, Wong SL. Production of minicellulosomes from Clostridium cellulovorans in Bacillus subtilis WB800. Applied and Environmental Microbiology 2004;70(9) 5704-5707.
11. Zhang XZ, Cui ZL, Hong Q, Li SP. High-level expression and secretion of methyl parathion hydrolase in Bacillus subtilis WB800. Applied and Environmental Microbiology 2005;71(7) 4101-4103.

PUR degradation constructs:

Tolerance to waste

We chose the MG1655 E.coli strain as chassis because it constitutively expresses genes that make it resistant to the potentially toxic molecule. The [http://parts.igem.org/Part:BBa_K892010 AldA] and [http://parts.igem.org/Part:BBa_K892009 FucO] genes have an important role in decreasing the toxic effects of Ethylene-glycol by converting it into Glycolaldehyde which is a link to the cell`s central metabloism. We have received these genes from the registry and future work could be to express these in Bacillus or cellulose degrading organisms to make them tolerant as well.

PHB synthesis Design

We will first use the phaCAB biobrick Part:BBa_K934001 P3HB

Assembly: Adding better promoter and RBS to phaCAB operon

We made two new improved constructs by modifying the native operon:

<p> The modellers in the drylab predicted that an increased level of phaB enzyme will increase the amount of PHB produced and therefore we decided to change the native Ralstonia eutropha promoter and RBS to a strong constitutive E.coli promoter (J23104) and RBS (0034) in front of the operon. We have succeeded with the swap and built the new biobrick [http://parts.igem.org/Part:BBa_K1149052 BBa_K1149052] which we refer to as constitutive phaCAB. In parallel, we have also assembled a different configuration of transcription initiation region: the "hybrid". Surprisingly, our results show that the "hybrid" phaCAB construct [http://parts.igem.org/Part:BBa_K1149051 BBa_K1149051] if highly efficient at producing bioplastic. Please read on to discover more.

We tested the promoter and RBS we were going to use by cloning them in front of amilCP blue chromoprotein ([http://parts.igem.org/Part:BBa_K1149020 BBa_K1149020]). It was bright blue and we had some fun playing around with it as part of our communication work "E.coli Art".

In order to change the promoter, we designed a forward primer that binds to the beginning of the first gene in the operon and contains a Xba restriction site: Pha_Fw (cgcttctagagatggctactgggaaaggagccg). We used pha_Fw and the suffix primer G1005 primer pain to amplify the insert out of the backbone. The new promoter and RBS were J23104 and B0034 from the biobrick [http://parts.igem.org/Part:BBa_K608002 BBa_K608002] We digested the pSB1C3 backbone with this biobrick with SpeI and PstI.

Summary diagram of construction steps:

We performed DpnI digest on the gel purified PCR product (insert) in order to get rid of any contamination from the original plasmid. We did Alkaline Phosphatase treatment of the backbone to remove terminal phosphates in order to decrease self-ligation of plasmid. We transformed the ligation into NEB5 cells and we grew overnight cultures from single cell colonies. The next day, we purified the plasmids and digested 200 ng of them with Xba and PstI to screen for the right clones:

Colony #8 had the expected 4.2kB insert and therefore we sent this plasmid for sequencing. After having the sequenced it, we submitted the new part to registry as [http://parts.igem.org/Part:BBa_K1149051 BBa_K1149051]. At the sequencing step, we realised that it has double P+RBS since the native P+RBS stayed there in between the new P+RBS and the operon. Anyhow, this hybrid configuration should increase the amount of protein products from the genes in the operon and we are in the process characterizing and testing our new construct. We have identified a clone (from colony #6) that contains the new constitutive promoter and submitted it as [http://parts.igem.org/Part:BBa_K1149052 BBa_K1149052] biobrick. We are testing both the hybrid and normal constitutive constructs for PHB bioplastic production.

Partial sequence of the constitutive phaCAB construct ([http://parts.igem.org/Part:BBa_K1149052 BBa_K1149052]):

Partial sequence of the hybrid phaCAB construct ([http://parts.igem.org/Part:BBa_K1149051 BBa_K1149051]):

Results

E.coli MG1655 containing the PhaCAB construct accumulates the bioplastic PHB inside its cells as illustrated in the figure below. A control strain with a plasmid containing the BBa_J23104 promoter and BBa_B0034 ribosome binding site and the strain containing PhaCAB were grown on plates with Nile Red stain in the media. Nile Red binds to the membranes surrounding the PHB and fluoresces.

Figure x. More intense fluorescence is seen for J and S which contain phaCAB and phaCB respectively. These operons allow them to produce the bioplastic PHB. PHB is stained by Nile Red which fluoresces when the plate is imaged at 473nm.

The improved biobrick:

Protocols

LB-Agar Plates containing Nile Red stain

Nile Red final concentration in media should be 0.5µg/ml

1. After autoclaving 300ml of LB-Agar, add 600µl Nile Red solution(0.25mg/ml DMSO)
2. Add 150µl chloramphenicol
3. Pour plates in a clean fume cupboard

Nile Red Culture staining

Nile Red final concentration should be 20µg/ml media

Add 320µl to 4 ml overnight culture and incubate for 30mins at room temp.

Fluorescent Microscopy

Agar pads are first made

Extracting PHB

• Centrifuge settings: 4000RPM, 10mins.
• Scale as appropriate.
• After each centrifuge step the supernatant should be poured off.
• This should provide PHB with 99% purity and a high molecular weight.

1. Centrifuge 50ml culture and resuspend in PBS.
2. Resuspend pellet in 5ml Triton X-100(1% v/v in PBS) for 30mins at room temp.
3. Centrifuge, resuspend in 5ml PBS.
4. Centrifuge, add 5ml sodium hyperchlorite solution and incubate at 30˚C for 1 hour.
5. Centrifuge, wash with 5ml distilled water and 70% EtOH several times.
6. Allow powder to dry.

Relationship between OD and dry biomass

1. Grow 3 large cultures in 3% glucose 1L/300ml LB - inoculate with 1ml OD 0.1 and grow for 24h.
2. Calculate OD of original culture and dilute until there is a range of 0.2 - 1.0 OD(try to have large volumes for low OD).
3. Weigh centrifuge containers.
4. Transfer solutions to these containers, recording what volume of each OD is transferred. .
5. Centrifuge these solutions down and dry the pellet out overnight in a 50°C oven.
6. Dry cell mass = (container mass + dry cell mass)/container mass
7. Plot OD against dry cell mass.

Measure OD of culture. If OD>0.4 then dilute by a dilution factor d. Divide new OD by d to give the corrected OD.

Either create a calibration curve or assume dry biomass is proportional.

1. Weigh each container,then culture PHB producing microbes.
2. Calculate the culture’s optical density(OD).
3. Centrifuge culture and pour off supernatant.
4. Weigh container and pellet.
5. Heat in oven until weight is constant.
6. Re-weigh container and pellet, subtracting container weight to calculate biomass dry weight.

Measure OD of cultures before extracting PHB to estimate dry biomass.

Safety

Dimethyl Sulfoxide(DMSO)

It has known carcinogenic effects and can permeate your skin, carrying other chemicals with it. Handle in a fume cupboard, wearing nitrile gloves and goggles.

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