Making Mixed Waste Useable

Overview

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 secrete the extracellular degrading enzymes

Our Bacteria should be able to degrade mixed waste

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

Our Bacteria should be able to synthesise P(3HB) from mixed waste

Modelling

Design

Survival in waste

Story about the Choice of MG1655 as chassis.

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 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 (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 page 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.

	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 (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.

Plastic degradation

enzyme	source organism	biobrick	reference
EstCS2	uncultured unknown bacterium (GU256649.1)	BBa_K1149002	Kang et.al 2011
pueA	Pseudomonas chlororaphis	BBa_K1149003	Stern et al., 2000
pueB	Pseudomonas chlororaphis	BBa_K1149004	Howard et al., 2001
pudA	Comamonas acidovorans	BBa_K1149005	Allen et al. 1999
pulA	Pseudomonas fluorescens	BBa_K1149006	Vega et al., 1999

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

see the appropriate section under PHB for design.

Results

Protocols

Safety