Difference between revisions of "Team:Newcastle/Model"
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Revision as of 11:54, 19 September 2018
IGEM 2018
Modelling
Rationale and Aim
We have chosen to use Pseudomonas fluorescens (DSM 25356) as a chassis organism due to its rapid and abundant colonisation abilities within the rhizosphere. To ensure P. fluorescens can colonise effectively, the metabolic load and resource drain of our system on the cell must be kept to a minimum in order to conserve natural homeostasis and minimise waste. As our system utilises enzymes found in flavonoid production, our naringenin synthesis pathway will be most taxing on resources used to maintain the natural homeostasis of flavonoid pathways. Some of these resources (ATP, CoA and Malonyl CoA, for example) are included but not restricted to flavonoid production. If we are to program a cell to produce an amount of naringenin per unit time, we need to consider what range of output works best in terms of maintaining the cells homeostasis.
On top of maintaining the cells homeostasis, sufficient naringenin needs to be produced by the pathway to attract the desired nitrogen fixing bacteria. The results of our chemotaxis experiments and previous research have shown higher concentrations of naringenin to inhibit growth of multiple soil microbes (1). It is therefore important that production by our system is stable in variable environmental conditions such that there are no detrimental effects on the rhizosphere community. By creating an enzymatic model of the pathway, we aimed to alter the operon design in order to minimise resource drain and stabilise naringenin production as a means of increasing system optimisation and robustness.
The chemotaxis modelling and wetware element to our project characterise the production rate of naringenin required for chemotaxis to occur. When the production rates of naringenin by our endophytic chassis have been characterised, the pathway model will give us the tools to alter the operon in order to optimise production.
Model Design
Core Enzyme Kinetics
The core of the model uses enzymatic turnover rates (Kcat) to simulate the relative rate (molecules per second) of conversion from substrate to product for each enzymatic reaction in the pathway. Each reaction is split into two parts, formation of the enzyme substrate complex (Substrate binding) and conversion of the complex to the reaction products (Catalysis). The formation of the substrate enzyme complex is reversible whilst the conversion of the complex to the reaction products is not. In the case of 4CL and CHS, for which more than one substrate is required for catalysis of the product, additional steps are coded as separate reversible reactions to emulate these circumstances.
Background
Naringenin, our chemoattract of choice, is synthesised from L-tyrosine via the enzymatic action of tyrosine ammonia lyase (TAL), 4-coumarayl ligase (4CL), chalcone synthase (CHS) and chalcone isomerase (CHI) (Figure1). These four enzymes are contained within the iGEM part BBa_1497017. Within this part all four enzymes are under the control of a single strong T7 promoter (BBa_I712074), with each enzyme having the same strong RBS (BBa_B0034). This system gives equal expression of each of the four enzymes. Previous research has identified that changing relative expression of each enzyme in the pathway improved naringenin production (2). We intended to optimise our system by using a model to explore how we could redesign the operon in order to maximise flux through the pathway whilst minimising resource consumption and waste.