Team:Hong Kong HKUST/Model

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Laccase Secretion Efficiency Model

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 team's [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 mm3 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. Average fold change of enzyme secretion of E. coli using OmpA.

 

Figure 1 shows 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 [3].

Since we want to try to put the OmpA sequence before the laccase sequence, We try to collaborate the data obtained from Figure 1 with the available characterization result of the laccase we are using on the iGEM Registry for BBa_K863006 [4].

 

Figure 2. Comparison of activity expectation of BBa_K863005 with (orange) and without (blue) OmpA with ABTS as the substrate.

 

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.

This figure helps our team to be aware of the overall impact of OmpA to the secretion of the laccase enzyme.

 

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Alkane and MFC

Approach using Flux Balance Analysis

 

REFERENCES:

  1. Sowmya, H., Ramalingappa, B., Nayanashree, G., Thippeswamy, B. and Krishnappa, M. (2014). Polyethylene Degradation by Fungal Consortium. IJER, [online] 9(3), pp.823-830. Available at: https://ijer.ut.ac.ir/article_969.html.
  2. 2012.igem.org. (2012). Team:University College London/Module 3/Modelling. [online] Available at: http://2012.igem.org/Team:University_College_London/Module_3/Modelling [Accessed 5 Oct. 2018].
  3. Pechsrichuang, P., Songsiriritthigul, C., Haltrich, D., Roytrakul, S., Namvijtr, P., Bonaparte, N. and Yamabhai, M. (2016). OmpA signal peptide leads to heterogenous secretion of B. subtilis chitosanase enzyme from E. coli expression system. SpringerPlus, [online] 5(1), p.1200. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4963352/ .
  4. Parts.igem.org. (2012). Part:BBa_K863006. [online] Available at: http://parts.igem.org/Part:BBa_K863006 [Accessed 5 Oct. 2018].