Team:Hong Kong HKUST/Model

iGem HKUST 2018 Hielo by TEMPLATED

Laccase module

Introduction

According to our project modules, we try to categorize our modelling into three parts.

The first one is from the Laccase Module, where we attempt to characterize our laccase construct from previous iGEM data.

The second one is from the Alkane Metabolism Module. We are fitting the kinetic parameters of fumarate addition mechanism to observe the activity of ASS and observe the rate of conversion from alkane to succinate.

While for the MFC module, we focus our modelling to establish data to find the optimum concentration for Shewanella oneidensis MR-1 growth, as well as estimating the voltage and power density that can be produced.

Laccase Module

As there is little documentation about the usage of laccase from E. coli, we rely on the literature reviews [1] of laccase secreted by fungi to correlate with the number of alkane and alkene chains that can be formed. Using simple calculation, it can easily be translated that 2900 alkane chains (30%) and 900 alkene chains (10%) should be formed after polyethylene is treated with laccase for every centimeter squared.

Using UCL iGEM 2012 teams' [2] findings on polyethylene degradation, we assume that the rate of degradation will eventually be linear with 0.9 PE molecule degraded per second, of which 28 molecules of alkane and 9 molecules of alkene will be formed in 100 seconds. Using the weight and the density of polyethylene, there would be 3.86*10^-12 mm^3 or 82,000 polyethylene molecules degraded within one day..

To improve BBa_K863006 obtained from the Bielefeld iGEM 2012, we try to characterize it by adding OmpA and his-tag. However, due to time constraint, our wet lab experiment could not acquire any valuable data and the modelling team will only rely on literature findings.

OmpA itself is a major protein that can be found in the outer membranes of most gram-negative bacteria, including E. coli. It will signal and facilitate E. coli to secrete enzyme more efficiently, in this case, laccase.

Figure 1

From figure 1, it is known that the average secretion efficiency of E. coli using OmpA can be increased by 3% during the first hour of incubation, and the fold changes by 2.11 within the first 20 hours [3]. Here, fold change indicates the relative values of the yields or secretion efficiencies of constructs from OmpA Sp divided by native Sp. We collaborate this data with the available characterization result on iGEM Registry for BBa_K863006 [4].

Figure 2

Figure 2 denotes the estimated activity with ABTS as substrate. Here, the measurement of 308 ng ECOL (BBa_K863005) was done in pH 5 at 25° C (Bielefeld, 2012). The 8mM concentration of ABTS was assigned to be substrate saturated. The bars on the right indicates the assessed amount of oxidized ABTS when ECOL is ligated with OmpA.

CO₂ to Methane

Carbon dioxides can be converted into methane after undergoing reduction process, in which the molecule uses the energy from the sun/catalyst to break up the CO₂ molecule into carbon and oxygen atoms, then combine with hydrogen to form methane and water, as explained on the chemical equation below.

Using irreversible Henri-Michaelis-Menten Kinetics, we try to consolidate an enzyme-catalyzed reaction with a single reaction and reaction rate equation with Vmax of 0.8 ± 0.07 nmol/min and a Km for CO₂ of 23.3 ± 3.7 mM [1].

Figure 3

From figure 3, it can be seen that it takes over 3 hours to fully convert 10 nmol of CO₂ into methane. It appears to verify that common features of homogeneous catalysts for CO₂ reduction to CH₄ are low reaction rates (e.g., turnover frequencies) and a limited number of turnovers (e.g., turnover number) before inactivation of the catalyst [2].