Contents 1 Making Mixed Waste Useable 1.1 Overview 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 Gene expression 1.4.2 Secretion 1.4.2.1 Alternative strategies for protein secretion in E.coli: 1.4.2.2 Extracellular Secretion in E.coli: 1.4.2.3 Outer Membrane Anchors and Periplasmic Expression in E.coli: 1.4.3 Plastic degradation 1.4.3.1 Pathway of PUR hydrolysis: 1.4.4 PUR degradation constructs: 1.4.5 Tolerance to waste 1.4.6 PHB synthesis 1.5 Results 1.6 Protocols 1.7 Safety

Making Mixed Waste Useable

Overview

We are utilising the various polymers in mixed waste by flooding the rubbish with a cocktail of degradation enzymes. Next, some of the monomers are taken up by our E.coli to produce bioplastic.

Specification

Our Bacteria should survive and grow in mixed waste

Our Bacteria should be able to express our constructs whilst being cultured with mixed waste

Our Bacteria should be able to degrade mixed waste

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

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

Gene expression

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

later, these enzymes can be combined

Secretion

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.

Plastic degradation

Some previous iGEM teams have worked on degrading various kinds of plastic before. 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:

<p> polyurethane polyisocyanate ethylene-glycol

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.

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]

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

A major component of mixed waste is cellulose, in form of textile fibres, paper and wood. This can be potentially broken down to glucose by cellulase enzymes. Therefore we designed an optimal system for the production of PHB from glucose. The modellers figured out and told us that phaB is key in the system and therefore we are changing the native promoter to a strong E.coli one in front of the operon.

Results

Protocols

Safety