UNL 2018 Improving Early Detection of The Emerald Ash Borer

Model

Motivation:

The model created for the TPS4-B73 biological part is mainly based on the peak production of 7-epi-sesquithujene over experimental time. This model represents the levels of production of each major substrate along the pathway to 7-epi-sesquithujene. This is done to identify possible bottlenecks in the system and to refine the model to match experimental results. It is important to improve these models and increase their predictive power to identify possible ways of increasing production that are not immediately obvious.

Creation

TPS4-B73:

The major part of what this model is trying to represent it the kcat and km of the Michaelis Menten equation describing the function of TPS4-B73. The model was first created with characteristics found in proteins that are analogous. A gene that is similar to another terpene synthase gene which originate in tomatoes, was found to have a km of 9.7 μM and a turnover rate of 53 s^-1 [4]. These values are understood to be very relative, but are a good starting point for the model. The degradation rate of TPS4-B73 was found to be 22 min in yeast, this number accounts for direct degradation but also dilution through cell growth [2].

DNA:

The DNA of TPS4-B73, labeled as TPS4_DNA is on a medium copy count plasmid, about 15 total plasmids. In experiments the cell’s growth was inhibited by FPP concentration. This is effect is considered to limit the DNA concentration by 36% (extrapolated from [1]) when FPP is at a peak concentration.

mRNA:

The mRNA is modeled by a transcription rate and a half-life degradation rate. With a transcription rate of 42 nt/s and a translation rate of 14 aa/s [5], the characteristics of mRNA can be fully modeled. A half-life of 3-8 min was found for mRNA [3].

FPP:

The production of FPP is estimated as 2.2 * 10 ^(-5) μM/s (extrapolated from [1]).

7-epi-sesquithujene:

The production rate of 7-epi-sesquithujene is considered to be the Michaelis Menten relation with TPS4-B73.

Degradation Rates:

Species	Half-life / Production limit
mRNA	3 min
TPS4-B73	22 min
TPS4_DNA	36% inhibition

Production Rates:

Species	Rate
mRNA	42 nt/sec
TPS4-B73	14 aa/s
FPP	2.2 *10^(-5) μM/s

Figure 1: Initial Model, Showing a Constant Bottleneck

As can be seen in Figure 1 the initial figure is basically a straight line. It was concluded that this was from a possible bottleneck from the FPP production. Therefore, we predicted that FPP production in our experiments would become the bottleneck in 7-epi-sesquithujene production.

When we received back the data from the experiments though, there was not bottleneck from FPP production. Therefore, the model was changed to fit the curve given. This is the side by side comparison of our revised model with the experimental production (Figure 2/3).

Figure 2: Experimental Data

Figure 3: Revised Model, Closer Fit to Experimental Data

The differences in the model characteristics included FPP production increase from 2.2 * 10^(-5) μM/s to 5 *10^(-4) μM/s, and the efficiency of TPS4-B73 was greatly reduced. Adjusted values for TPS4-B73 include an increase in km from 9.7 μM to 1mM, and an increase in kcat from 50 1/s to 250 1/s. These model results, like the experimental results reflect a need to further identify the factors to increase efficiency.

Works Cited

[1] Anthony, J. R.; Anthony, L. C.; Nowroozi, F.; Kwon, G.; Newman, J. D.; Keasling, J. D. Optimization of the mevalonate-based isoprenoid biosynthetic pathway in Escherichia coli for production of the anti-malarial drug precursor amorpha-4,11-diene. https://www.ncbi.nlm.nih.gov/pubmed/18775787 (accessed Oct 17, 2018).
[2] Belle, A.; Tanay, A.; Bitincka, L.; Shamir, R.; O'Shea, E. K. Quantification of protein half-lives in the budding yeast proteome. https://www.ncbi.nlm.nih.gov/pubmed/16916930 (accessed Oct 17, 2018).
[3] Bernstein, J. A.; Lin, A. B. K. P.-H.; Cohen, S. N. Global analysis of mRNA decay and abundance in Escherichia coli at single-gene resolution using two-color fluorescent DNA microarrays. http://www.pnas.org/content/99/15/9697 (accessed Oct 17, 2018).
[4] Falara, V.; Akhtar, T. A.; Nguyen, T. T. H.; Spyropoulou, E. A.; Bleeker, P. M.; Schauvinhold, I.; Matsuba, Y.; Bonini, M. E.; Schilmiller, A. L.; Last, R. L.; Schuurink, R. C.; Pichersky, E. The Tomato Terpene Synthase Gene Family. http://www.plantphysiol.org/content/157/2/770 (accessed Oct 17, 2018).
[5] Proshkin, S.; Rahmouni, A. R.; Mironov, A.; Nudler, E. Cooperation between translating ribosomes and RNA polymerase in transcription elongation. https://www.ncbi.nlm.nih.gov/pubmed/20413502 (accessed Oct 17, 2018).

Team:UNebraska-Lincoln/Model