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

Are you planning an iGEM project with E.coli that involves protein secretion? You are at the best place for finding all the information you need for making it happen.

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 <a href="www.2009.igem.org/Team:METU-Gene/EGF_Transportation" "target="_blank">EGF hormone in a previous iGEM project</a> or be important in bioremediation such as in <a href="https://2013.igem.org/Team:Dundee/Project/Mop" "target="_blank"> Dundee Team’s toximop system</a> 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 (1). The pathway transports to the periplasm and additional mechanism are usually necessary to further export the protein to the extracellular space (2). 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 (3). 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 (4). 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 (5). 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 (6). 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 (7). 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 <a href="http://parts.igem.org/Protein_domains/Localization"target="_blank"> page</a> in the registry that lists localisation tags] was 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.

Extracellular Secretion in E.coli:

Outer Membrane Anchors and Periplasmic Expression in E.coli:

Our choice is to use the pelB secretion tag as it has been demonstrated to work in many cases (3) with sometimes as high as 90% transport efficiency (8). 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 and see our result in the lab book. (make a nice and good protocol for Infusing secretion signal/fusion protein) We have constructed a biobrick where LARD1 is after a constitutive promoter and RBS in order to facilitate quicker cloning with less assembly steps.

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

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Tolerance to waste

MG1655 has AldA and FucO. We have these genes from xxxteam and future work would be move these into 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