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.

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)	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

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.

We will first use the phaCAB biobrick BBa_K934001 to produce P3HB. Our aim is the industrial production of P3HB and this require high yields to be economically viable. The results from the metabolic model suggest that to do this the amount of phaB should be increased. To do this we designed constructs with stronger promoters to upregulate the expression of the operon.

The operon was original under the native promoter from Ralstonia eutropha. We designed two promoters to increase transcription. The first uses the constitutive promoter J23104. The second was designed as a hybrid promoter incorporating BBa_J23104 and the original promoter due to recent results of high expression from a hybrid promoter[1].

We chose the MG1655 E.coli strain as chassis because it constitutively expresses genes that make it resistant to the potentially toxic molecule. The AldA and 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.

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 extracellular. 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 . 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 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 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, 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 E.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 E.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 E.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.

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. We also lyse the cells at the end of reactions to harvest our product P3HB, ensuring that no living bacteria escape the bioreactors.

Introduction

Both efficiencies of Polyurethane (PUR) degradation and ethylene glycol production are important for the performance of MAPLE system. We built a mathematical and deterministic model that is based on MATLAB extension Simbiology for Polyurethane degradation. The model contains the kinetic property of degradation enzymes that is helpful for the design of assays. As we scaled up the initial concentrations of all substrates to meet the conditions for a bio-reactor, the model can provide preliminary simulations and predictions for the MAPLE system.

Design

Objective

Here are some specific objectives for the model to achieve:

1. The model should contain the gene expression model of the degradation enzymes because the enzyme concentration determines the rate of plastic degradation. In our case for PUR degradation, we used pBAD strong promoter K206000 for most enzymes. We built the gene expression model based on inducible pBAD promoter, which gene expression rate can be regulated by inducer concentration.

2. The model should show the efficiency of the enzyme secretion to the culture from the cells. It's also important because the enzyme concentration in the culture depends on it. Here we used pelB secretion tag for most enzymes in order to achieve a high efficiency.

3. The model basically predict how long will take to degrade a known concentration of soluble polyurethane. It is assumed that the enzyme in our assays has the same kinetic properties as the enzyme used in the literature. The model can suggest a suitable concentration of the plastic to use in order to get good results from the assays.

4. It is known that ethylene glycol is toxic to E.coli. However, it has no clear effect on the growth of our MG1655 strain when the concentration of ethylene glycol is below 200mM. Therefore, the model should suggest a safe range of PUR concentration to avoid a high concentration (>200mM) of ethylene glycol produced.

The Model

Polyurethane (PUR) degradation involves 5 different degradation enzymes:

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

However, 4 of the 5 enzymes are not well characterised before, so we could't find enough kinetic data from the literature. The only well-characterised PUR degradation enzyme PudA is used in the model as an illustration of all PUR degradation enzymes. The model will be more complete when the kinetic data of the other enzymes are defined. The finished PUR degradation model is shown as below:

Key

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 PudA

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	[2]	Taking the "switch point" as the activation coefficient
dmRNA	mRNA degradation rate	0.10	1/min	[3]	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	[4]	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 (PudA)	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	-	-
[PudA]	Concentration of PudA	-	mM	-	-

1.Derivation of the maximal expression rate,β

• Average molecular weight (Mw) of a base pair = 660g/mol[5][6]
• Average mass of a base pair = 660g/mol x 1.66x10^-24 = 1.1x10^-21g
• Volume of an E.coli cell = 1µm³[7] = 1x10^-15L
  • ∴Mass concentration =
  • ∴Molar concentration of 1 base pair in the volume of E.coli = = 1.66x10^-6 mM
• BioBrick assembly plasmid pSB1C3 is a high copy number plasmid (100-300 copies per cell)[8]
  • 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[9] = 80 x 1.66x10^-6mM/s = 80 x 1.66x10^-6 x 60mM/min = 7.97x10^-3mM/min
• ∴ Rate of mRNA_BDH2 production under the control of pBAD = 7.97x10^-3 ÷ 0.25 = 0.032/min

2.Protein production rate of BDH2, k₂

• Average molecular weight(Mw) of an amino acid(aa)= 110g/mol[10][11]
• 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
• Translation rate = 20aa/s = (20 x 1.66x10^-5 x 60)mM/min = 0.020mM/min
• BDH2 comprises of 256aa[12]
  • ∴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

PUR degradation module

The reaction equation of the PUR degradation is:

[Polyurethane]+[PudA]= 5 [ethylene glycol] + 5 [polyisocyanate] + [PudA]

Assumptions:

We assumed 1 mole of polyurethane dispersion can produce 5 moles of ethylene glycol.

The molecular weight of a single polyurethane monomer is 470 g/mol whereas the molecular weight of the polyurethane dispersion is around 2000 g/mol.[13]Therefore, the short-chain polyurethane consists approximately 5 monomers. 5 molecules of ethylene glycol will be produced by degrading one chain of polymer.

We also assumed a simple Michaelis-Menten mechanism for PudA

The parameters for the kinetic equations are:

Parameter	Description	Value	Units	Sources
Km	Michaelis constant	51.5	mM	[14]
Kcat	Turnover number	141.75	1/min	[15]

The efficiency of Secretion is assumed to be 90% secretion over 2 hours.[16] The rate of secretion in the model is therefore:

rate of secretion = 0.9[concentration of PudA]/120 (mM/mins)

Simulations and Results

In terms of ethylene glycol toxicity, if we can't let the concentration of ethylene glycol over than 200mM, the maximum initial concentration of polyurethane dispersion is 40mM because one mole of polyurethane dispersion can produce 5 moles of ethylene glycol. (from the derivation above) If we put 40mM as the initial concentration in the model, the simulation result is:

40mM of polyurethane dispersion can be efficiently degraded in 20 mins. In case if we keep the concentration of the plastic below 40mM. The ethylene glycol won't affect the cell growth in our assays. 40mM is converted to 80g/L which suggests no more than 80 grams of polyurethane to be put in the system in order to avoid toxicity.

As for the implementation of MAPLE system, the polyurethane degradation enzymes in our system are very efficient. Therefore, we need an efficient filter which removing ethylene glycol from the system.

This section is about modelling the plastic-synthesising E.coli developed in our project. The plastic that this module synthesises is the bioplastic poly-3-hydroxybutyrate, also known as P(3HB). The content below shows the process of building, designing and optimisation of the model. Also, the considerations, assumptions and limitations will be discussed. Specifications for the model

• Input = monomer (3HB)
• Output = polymer (P(3HB))

<p align="justify"> The model was built and simulated in Simbiology, a Matlab package designed for modelling biological systems. In our model we have a compartment (labelled "E.coli_1") that represents our engineered cell. The compartment contains the reactions and species that interact with the polymer synthesis pathway.

The pathways on the right half of the cell are genetic expressions of the 3 enzymes involved in the plastic synthesis pathway. The pathways on the left represent the actual P(3HB) production pathway.

Genetic regulations and expressions

BDH2 (3-hydroxybutyrate dehydrogenase)

PhaB (Acetoacetyl-CoA reductase)and PhaC (P(3HB) synthase)

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.
n	Hill coefficient	2.0	dimensionless	[17]	Rounded to 2.0 from 2.26 as Simbiology wouldn't allow a non-integer value for such parameter.
K	Activation coefficient	0.0031	mM	[18]	Taking the "switch point" as the activation coefficient
dmRNA	mRNA degradation rate	0.10	1/min	[19]	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	[20]	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 (BDH2)	4.7	1/min	Please see derivation 2 below.	Please see derivation 2 below.
k3	Protein production rate (PhaCB)	0.58	mM/min	Please see derivation 3 below.	Please see derivation 3 below.
[Arabinose]	Concentration of arabinose	Initial: 0.008	mM	see section 1.7 "Initial concentrations of metabolites"	see section 1.7 "Initial concentrations of metabolites"
[mRNA]	Concentration of mRNA	-	mM	-	-
[BDH2]	Concentration of BDH2	-	mM	-	-
[PhaB]	Concentration of PhaB	-	mM	-	-
[PhaC]	Concentration of PhaC	-	mM	-	-

1.Derivation of the maximal expression rate,β

• Average molecular weight (Mw) of a base pair = 660g/mol[21][22]
• Average mass of a base pair = 660g/mol x 1.66x10^-24 = 1.1x10^-21g
• Volume of an E.coli cell = 1µm³[23] = 1x10^-15L
  • ∴Mass concentration =
  • ∴Molar concentration of 1 base pair in the volume of E.coli = = 1.66x10^-6 mM
• BioBrick assembly plasmid pSB1C3 is a high copy number plasmid (100-300 copies per cell)[24]
  • 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[25] = 80 x 1.66x10^-6mM/s = 80 x 1.66x10^-6 x 60mM/min = 7.97x10^-3mM/min
• ∴ Rate of mRNA_BDH2 production under the control of pBAD = 7.97x10^-3 ÷ 0.25 = 0.032/min

2.Protein production rate of BDH2, k₂

• Average molecular weight(Mw) of an amino acid(aa)= 110g/mol[26][27]
• 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
• Translation rate = 20aa/s = (20 x 1.66x10^-5 x 60)mM/min = 0.020mM/min
• BDH2 comprises of 256aa[28]
  • ∴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

3.Protein production rate for PhaB and PhaC, k₃

• Relative promoter strengths: J23104 = 1.3RPU, J23101 = 1.0RPU.
  • ∴ 104 is 1.3x stronger than 101.
• In absolute units: take GFP synthesis rate (molecules per min per cell) and approximate that as a generic protein synthesis rate for the promoter.
  • GFP synthesis rate of 101 = 2232 molecules per min per cell.
  • ∴ GFP synthesis rate of 104 = 1.3 x 2232 = 2902 molecules per min per cell
• Assume 1 molecule in an E.coli cell gives a concentration of 1nM.
  • ∴ GFP synthesis rate of 104 = 2902 x 1nM = 2.9x10^-6nM/min per cell
• Plasmid copy number assumed as 200 (as in derivation 1)
  • ∴ GFP synthesis rate of 104 in our E.coli = 200 x 2.9x10^-6 = 0.00058nM/min = 0.58mM/min

BDH2 (3-hydroxybutyrate dehydrogenase)

Reaction R1:

Reaction R1r:

Values, sources and assumptions

Parameter	Description	Value	Units	Sources	Assumptions
kcat,BDH2_f	Turnover number of BDH2 in forward reaction	22200	1/min	[29]
kcat,BDH2_r	Turnover number of BDH2 in reverse reaction	7200	1/min	[30]
Ki,NAD+	Inhibition constant of BDH2 with NAD+	2.5	mM	[31]
Ki,NADH	Inhibition constant of BDH2 with NADH	1.1	mM	[32]
Km,AcAc	Michaelis constant for AcAc	initial: 0.37	mM	[33]
Km,NAD+	Michaelis constant for NAD+	0.24	mM	[34]
Km,NADH	Michaelis constant for NADH	0.010	mM	[35]
Km,3HB	Michaelis constant for 3HB	0.80	mM	[36]
[BDH2]	Concentration of BDH2	-	mM
[NAD+]	Concentration of NAD+	initial: 1.6x10-13	mM	see section 1.7 "Initial concentrations of metabolites"	see section 1.7 "Initial concentrations of metabolites"
[3HB]	Concentration of 3HB	initial: 0.0010	mM	see section 1.7 "Initial concentrations of metabolites"	see section 1.7 "Initial concentrations of metabolites"
[AcAc]	Concentration of AcAc	initial: 1.0x10-13	mM	see section 1.7 "Initial concentrations of metabolites"	see section 1.7 "Initial concentrations of metabolites"
[NADH]	Concentration of NADH	initial: 2.5x10-14	mM	see section 1.7 "Initial concentrations of metabolites"	see section 1.7 "Initial concentrations of metabolites"

atoAD (Acetyl-CoA:acetoacetyl-CoA transferase (α and β subunits))

Reaction R2:

Reaction R2r:

Values, sources and assumptions

Parameter	Description	Value	Units	Sources	Assumptions
Vmax,atoAD_f	Maximum rate of atoAD in forward reaction	0.00244	mM/min	[37]
Vmax,atoAD_r	Maximum rate of atoAD in reverse reaction	0.0108	mM/min	[38]
Km,AcAc	Michaelis constant for AcAc	1.86	mM	[39]
Km,Ac-CoA	Michaelis constant for Ac-CoA	0.26	mM	[40]
Km,Acetate	Michaelis constant for Acetate	53.1	mM	[41]
Km,AcAc-CoA	Michaelis constant for AcAc-CoA	0.035	mM	[42]
[AcAc]	Concentration of AcAc	initial: 1.0x10-13	mM	see section 1.7 "Initial concentrations of metabolites"	see section 1.7 "Initial concentrations of metabolites"
[Acetate]	Concentration of Acetate	initial: 1.0x10-13	mM	see section 1.7 "Initial concentrations of metabolites"	see section 1.7 "Initial concentrations of metabolites"
[AcAc-CoA]	Concentration of AcAc-CoA	initial: 1.0x10-13	mM	see section 1.7 "Initial concentrations of metabolites"	see section 1.7 "Initial concentrations of metabolites"

phaB (Acetoacetyl-CoA reductase)

Reaction R3:

Reaction R3r:

Values, sources and assumptions

Parameter	Description	Value	Units	Sources	Assumptions
kcat,PhaB_f	Turnover number of PhaB in forward reaction	6120	1/min	[43]
kcat,PhaB_r	Turnover number of PhaB in reverse reaction	3600	1/min	[44]
Km,NADPH	Michaelis constant for NADPH	0.15	mM	[45]
Km,AcAc-CoA	Michaelis constant for AcAc-CoA	0.0057	mM	[46]
Km,NADP+	Michaelis constant for NADP+	0.0060	mM	[47]
Km,3HB-CoA	Michaelis constant for 3HB-CoA	0.026	mM	[48]
[PhaB]	Concentration of PhaB	-	mM
[AcAc-CoA]	Concentration of AcAc-CoA	initial: 1.0x10-13	mM	see section 1.7 "Initial concentrations of metabolites"	see section 1.7 "Initial concentrations of metabolites"
[NADPH]	Concentration of NADPH	initial:3.8x10-14	mM	see section 1.7 "Initial concentrations of metabolites"	see section 1.7 "Initial concentrations of metabolites"
[NADP+]	Concentration of NADP+	initial:1.5x10-13	mM	see section 1.7 "Initial concentrations of metabolites"	see section 1.7 "Initial concentrations of metabolites"
[3HB-CoA]	Concentration of 3HB-CoA	-	mM

phaC (P(3HB) synthase)

Reaction R4:

Values, sources and assumptions

Parameter	Description	Value	Units	Sources	Assumptions
kcat,PhaC	Turnover number of PhaC	1680	1/min	[49]
Km,PhaC	Michaelis constant of PhaC with 3HB-CoA	0.14	mM	[50]
[PhaC]	Concentration of PhaC	-	mM
[3HB-CoA]	Concentration of 3HB-CoA	-	mM

atoB (acetyl-CoA acetyltransferase)

Reaction R5:

Reaction R5r:

Values, sources and assumptions

Parameter	Description	Value	Units	Sources	Assumptions
Vmax,atoB_f	Maximum rate of atoB in forward reaction	3.8x10-5	mM/min	[51]
Vmax,atoB_r	Maximum rate of atoB in reverse reaction	8.5x10-4	mM/min	[52]
Km,atoB	Michaelis constant of atoB with Ac-CoA	0.47	mM	[53]
Km,AcAc-CoA	Michaelis constant for AcAc-CoA	0.47	mM	[54]
Km,CoA	Michaelis constant for CoA	0.25	mM	[55]
[Ac-CoA]	Concentration of Ac-CoA	initial: 1.0x10-13	mM	see section 1.7 "Initial concentrations of metabolites"	see section 1.7 "Initial concentrations of metabolites"
[AcAc-CoA]	Concentration of AcAc-CoA	initial: 1.0x10-13	mM	see section 1.7 "Initial concentrations of metabolites"	see section 1.7 "Initial concentrations of metabolites"
[CoA]	Concentration of CoA	initial: 5.5x10-13	mM	see section 1.7 "Initial concentrations of metabolites"	see section 1.7 "Initial concentrations of metabolites"

blablabla

P(3HB) production

Sensitivity analysis: species concentrations

What is sensitivity analysis and why do it?

In order to look at how we could increase the production of P(3HB) we decided to run a sensitivity analysis (also in Simbiology) to identify what species in the model P(3HB) is sensitive to, given the specific conditions with which we set the model. In other words, we calculated the time-dependent sensitivity of P(3HB) with respect to the initial conditions of other species (species formed along the synthesis pathway and enzymes involved).[56]

For a species x, whose concentration depends on time, it can be expressed as x(t). To calculate the sensitivity of x(t) with respect to another species y(t), the sensitivity with normalisation relative to the numerator x(t) can be calculated as:

Therefore, the underlying algorithm for this sensitivity analysis of P(3HB) is:

Scan with different levels of PhaB

The plot below is to show that increasing PhaB concentration within the engineered organism would have a positive effect on the concentration of the P(3HB) synthesised. As so many parameters within the model were taken from different sources, it would be difficult to give a simulation plot that is accurate quantitatively. It was decided to show the single cell simulation for this because the scans were carried out up to the doubling time of 20mins, hence avoided the issue of scaling-up which would have otherwise subjected the simulation to more errors. Therefore, this plot should be interpreted from a qualitative perspective and the trend should be observed.

Difference between promoter expressions after optimisation

In our Module 1: Resourceful Plastic, we uses glucose as the product from degradation to feed in the system where P3HB is produced. The synthetic pathway needs to be reconsidered if we are using glucose as a solo input to the system. Here, 3HB monomer is no longer the source so BDH2 should be removed from the pathway. Acetyl-coA becomes the key resource to produce P3HB. A metabolic model is needed to show the production of Acetyl-coA from glucose. Furthermore, our synthetic pathway involved several key metabolites from the pathway such as NADPH, NADP+, NADH and NAD+. The initial concentration of those metabolites can be determined from the metabolic model by assuming they are in their steady states. More features and results of the metabolic model can be found in our Module 2.

Initial concentrations of metabolites

The metabolic model involves two pathways, Glycolysis pathway and TCA cycle. Where glucose is used as the single source of carbon. The reason is that Glucose is directly taken by Glycolysis pathway and there are some interactions between Acetyl-coA and TCA cycle. The model is based on original metabolic model by Angela Dixon.[Predictive Mathematical Model for Polyhydroxybutyrate Synthesis in Escherichia coli] The metabolic model is built in MATLAB Simbiology extension as well. All reactions and pathways are based on ODEs and kinetic data. The model can dynamically predict the concentration of all metabolites involved by a defined duration. <p>The metabolic model for determining steady state concentrations is shown as below:

The initial concentrations of all metabolites and kinetic data are referenced in the paper [57]

Production of P3HB with glucose as the only source

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

These assays were designed to test whether our chassis, E.coli (MG1655) could grow directly with waste over a long period of time.

Waste media

LB-waste assay(A) Waste media (B) E.coli containing mCherry stress biosensor (BBa_K639003) were grown in mixed waste (A) over 3 days, then streaked in a qualitative assay to check for growth. (C) mCherry stress biosensor (BBa_K639003) transformed E.coli were streaked again after 7 days growth in SRF. Figure made by Imperial College London 2013 iGEM.

PBS-waste assay(A) waste media made up in PBS (phosphate buffered saline). (B) E. coli expressing mCherry stress biosensor (BBa_K639003) grown in waste media (A) over 3 days, then streaked onto an antibiotic containing plate to qualitatively assess whether the E.coli had survived. (C) Streaked again after 6 days growth in SRF. Figure made by Imperial College London 2013 iGEM.

Conclusion: MG1655 E.coli are viable and grow on mixed waste alone. Therefore we have established that our chassis could survive in a mixed waste bio-reactor context, which is validation of our concept to industrially implement our system.

Waste conditioned media

These assays were designed to test whether our chassis, E.coli MG1655 strain could grow with waste conditioned media (WCM) over a period of 24-48 hours. Waste conditioned media is a filter sterilised version of the waste media and was designed for several reasons; Firstly we were unsure whether mixed waste would be toxic to E.coli and hence a less concentrated version may be more suitable and secondly large chunks of waste would prevent accurate OD600 measurements and therefore we decided to filter out the largest chunks.

Photograph of waste conditioned media cultures mCherry stress biosensor (BBa_K639003) transformed MG1655 were grown with LB-WCM at 37ºC, with shaking for 48 hours. Figure made by Imperial College London 2013 iGEM.
Growth assay in waste conditioned media (WCM).E.coli MG1655 strain transformed with mCherry stress biosensor. were grown with LB-based waste conditioned media at 37ºC, with shaking. Error bars represent S.E.M., n=4. Figure made by Imperial College London 2013 iGEM.	Growth assay in waste conditioned media (WCM). E.coli MG1655 strain transformed with either empty vector control (EV) or mCherry stress biosensor (BBa_K639003) were grown in WCM for 5 hours, with shaking at 37ºC. Error bars are SEM, n=4. Figure made by Imperial College London 2013 iGEM.

Conclusion: MG1655 transformed with either empty vector (EV) control or mCherry stress biosensor (BBa_K639003) vector are viable and can grow in waste conditioned media. Therefore waste conditioned media is an appropriate and novel experimental media with which to characterise biobricks within a mixed waste/landfill context. These data are also characterisation of an existing biobrick (BBa_K639003)

PUR Esterase enzyme activity

Cell lysate assay

Since the PUR Esterases were not secreted, initially the cells were lysed to obtain crude cell extracts in order to test whether the enzymes are active. The Western Blot results showed that the constructs EstC2 BBa_K1149002, PueB BBa_K1149004 and PulA BBa_K1149006 were being expressed. The cultures expressing these three constructs were grown, lysed by sonication and utilised in a colourimetric assay with the substrate analog para-Nitrophenyl butyrate. The data shows that PUR Esterase EstC2 BBa_K1149002 is definitely active.

para-Nitrophenyl butyrate (p-NP) is commonly used to indicate an enzyme’s esterase activity. The enzyme cleaves the ester bond and releases the 4-nitrophenol (4-NP), thus causing a colour change from colourless to yellow and an increased absorbance at wavelength 405 nm.

The assay was run in the Eppendorf BioSpectrometer was used to automatically read the absorbance of the reaction mixture every 30 seconds. The concentration of 4-Nitrophenol produced from the reaction was calculated using the Beer-Lambert Law; the extinction coefficient of 4-NP is 18,000 M-1 cm-1 at 405 nm. [58]

The results below show the concentration of 4-NP produced by the three different PUR esterases and compare them to the Empty Vector and Substrate alone as negative controls.

The above graphs clearly show that PUR Esterase EstC2 is active and cleaving p-NP. The recorded concentrations of 4-NP, in the presence of this PUR Esterase, are much greater than with the Empty Vector or the Substrate alone. Is PUR Esterase EstC2 active? Yes!

The enzymes expressed by both PueB and PulA do not appear to have esterase activity for this substrate. There is a tiny increase in 4-NP concentration for PueB, PulA, Empty Vector and Substrate alone. This probably indicates that the substrate is slowly degrading by itself. Other constituents of the cell lysates are likely to be causing a slight increase in p-NP degradation, as PueB, PulA and Empty Vector show higher concentrations of 4-NP than the Substrate alone.

Conclusion: PUR Esterase EstC2 is active!

Purified enzyme assay

Stay tuned

Ethylene glycol

Reduced growth at 30°C likely due to decreased efficiency of MG1655 ethylene glycol break down enzymes. These enzymes (see UC Davis 2012) are endogenously expressed and detoxify Ethylene Glycol.

E.coliMG1655 containing the PhaCAB construct accumulates the bioplastic P3HB inside its cells. P3HB can be visualised through staining with Nile Red, which fluoresces strongly in a hydrophobic environment, such as when it binds to the P3HB. However staining will be much lower in cells where P3HB is not present. To see whether P3HB was being produced we grew E.coli containing the phaCAB operon under the native promoter from Ralstonia eutropha. This produced the results we hoped for as seen in the image below.

This also provides a preliminary qualitative way in which to investigate the effect of changing the promoter to a stronger version in order to upregulate the amount of P3HB produced. The darker staining of the streaks of the constitutive and hybrid promoter containing constructs suggests the production of higher levels of P3HB in these strains.

E.coli transformed with the phaCAB operon(native, hybrid, constitutive - different promoters expressing the operon) fluoresce when grown on LB-Agar plates with Nile Red stain as the P3HB produced within them is stained. Those without the operon do not(EV- transformed with empty vector). When the promoter is changed from the native form to the our hybrid or the constitutive J23104 then the fluorescence is more intense, suggesting more P3HB is created.

Streaking the bacteria on Nile Red plates cannot be used to accurately decide the difference between P3HB production due to the effect differing thicknesses of cell layers will have on fluorescence intensity. We imaged the cells using the fluorescent microscope to qualitatively understand how much P3HB is produced per cell. As indicated by other authors, we saw that P3HB is found in localised granules within the bacteria. Empty vector containing cells show little fluorescence. In those that do, the staining is qualitatively different than in the phaCAB containing strains. Nile Red is also used as a lipid stain and this is indicated by its staining of the membranes of EV cells. In contrast, in phaCAB containing cells, staining is restricted to points within the cells.

Each fluorescent image was created by exciting the stain at 530nm for 100ms. Despite this standard treatment of samples the intensity of fluorescence produced by the cells containing the hybrid promoter is greater than from the constitutive ones. This could indicate a difference in the strength of the promoters which lead to phaCAB expression and therefore the amount of P3HB produced. The images also suggest that not every single E.coli is stained. This may be due to the conditions in which the cells were grown or may be dependent upon the stage in the lifecycle of the E.coli.

Fluorescent microscope images of E.coli stained with Nile Red. 1 -Empty vector, 2 - constitutive promoter phaCAB, 3 - hybrid promoter phaCAB. Images labelled a are fluorescent images excited at 530nm ovelaid on the bright field images shown in the b set of images. Images by Imperial College iGEM Team

A comparison between the amount of plastic produced by bacteria containing an empty plasmid(EV), native promoter-phaCAB and hybrid promoter-phaCAB after plastic extraction. The EV does not produce P3HB while the hybrid produces much more than the native promoter.

A summary of the improved production of P3HB by our hybrid promoter-phaCAB construct(BBa_K1149051) over the native promoter-phaCAB.

Comparison of P3HB production (left) 1.5ml tube, 90mg produced by native promoter-phaCAB (BBa_K934001), (right) 5ml tube, hybrid promoter-phaCAB produced 2.05g P3HB, (BBa_K1149051).

3HB Assay: Confirming production of 3HB

The chemical analysis of the produced bioplastic. The samples break break down to 3HB monomers after treatment with our PhaZ1 enzyme (BBa_K1149010). We synthesised P(3HB) using our improved Biobrick part (hybrid promoter phaCAB, BBa_K1149051). Our engineered bioplastic producing E.coli synthesised P(3HB) directly from waste. Imperial iGEM data

Please see data here.

Growth assays with different experimental media

In additional to standard LB and minimal media, several novel experimental media were developed in order to characterise Biobricks within a mixed waste/landfill setting. These media were characterised through an examination of pH and through an array of growth assays with the project chassis, E.coli MG1655 strain.

Media characterisation. E.coli strain MG1655 were transformed with a control plasmid and grown in different experimental media over a period of 5 hours. LB media, minimal media (M9M), supplemented minimal media (M9S), as described here or waste conditioned media (WCM), which is made from sterile filtrated mixed waste, see here. OD600 measured, error bars are S.E.M., n=4. Figure made by Imperial College London 2013 iGEM.

pH of experimental media. pH measurements of experimental media were made both before and after several experiments. The time periods refer to the duration MG1655 transformed E.coliwere cultured in the media before a pH measurement was made. Figure made by Imperial College London 2013 iGEM.

Conclusion: E.coli strain are viable and grow in all of our experimental medias. We have established a novel media that is optimised for characterisation of biobricks within a mixed waste/landfill context.

Waste Conditioned Media (WCM)

We added 1g [http://en.wikipedia.org/wiki/Refuse-derived_fuel SRF] (Solid Recovered Fuel) /50 mL LB and then autoclaved the mixture. Once autoclaved, large waste chunks were removed through filter sterilisation (0.2 µm filters) before being used in growth assays as waste conditioned media (WCM). SRF refers to the refuse from recycling facilities that has no value currently and is incinerated to produce power at a cost to the recycling facility, it is composed of 30% plastics while the rest is cellulosic waste

Waste Growth Assay

Overnight (O/N) cultures of MG1655 transformed with (BBa_K639003) were diluted to OD 0.05 in either fresh LB or waste conditioned media and plated into 96 well plates (200 µl/well). OD600 was read at the indicated time points and media only (LB) was taken away as background signal. In addition to this a qualitative assay as performed. 9 mL mCherry bacteria were transferred into 300 mL of autoclaved SRF in LB, which were placed in a shaking incubator at 37°C. After 2 days 200 µL of this solution was plated out on chloramphenicol plates, this was then repeated a week later to gauge whether or not the bacteria were still alive. To show that the MG1655 could grow on waste solely, they were grown in PBS (phosphate buffered saline) and autoclaved waste. In 100 mL PBS + waste, 1 mL MG1655 mCherry E.coli were added. They were then grown in a shaking incubator at 37°C and samples were plated after 2 days and 6 days.

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 rarely survives outside laboratories, although minor symptoms may show if infected. Meanwhile, the parts we designed are specialised for degrading PUR and they should not 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. Tributyrin

2. Poly(diethylene glycol adipate).

3. Nile red.

4. Arabinose.

5. 4-Nitrophenyl butyrate.

6. 4-Nitrophenyl acetate.

7. N-Succinyl-Ala-Ala-Pro-Leu p-nitroanilide.

8. Lipase from porcine pancreas.