Design
Project 
DESIGN
TARGET GENES:
The goal of Oak Shield is to control the population of the Oak Processionary Caterpillar (OPC) by inhibiting the expression of its vital proteins. Our team started by identifying the genes of these vital proteins, a non-trivial task that brought along several challenges. Among these challenges, it was especially difficult to find the DNA sequences of these genes, as the OPC genome has not been fully sequenced yet. Thus, the team gathered information from several research papers about the OPC in order to find any of the possible target genes. The priority was to identify genes that are highly conserved between the closely related species but vary in more distant species (i.e. barcode genes) to achieve our specificity goal.
SMALL INTERFERING RNA (siRNA):
Once the target genes were identified, the next step involved the search for the specific short 18-20 base pairs sequence on the antisense DNA strand.
All the short sequences had to align with the specific design guidelines (Thermo Fisher Scientific, n.d.) which include:
- 30-50% GC content
- Absence of stretches with 4 or more A’s or T’s in a row
- 3`AA nucleotide forming 5´UU overhang of our shRNA.
These short sequences were selected at different target sites for the same gene to have many potential targets, some of which will be more effective than others".
BASIC LOCAL ALIGNMENT SEARCH TOOL (BLAST):
In order to make a specific insecticide, all the short sequences that were identified were run through the NCBI BLAST algorithm to assess their specificity. Finally, only the sequences that were present exclusively in the OPC genome were left. Our priority was to ensure that these sequences do not occur in eukaryotic, domestic species, or in other species closely related to the OPC.
SHORT HAIRPIN RNA (shRNA):
The goal is to transform bacteria in a way that it would produce siRNA sequences to reduce the OPC population. However, the siRNA sequences are initially transcribed in a form of short hairpin RNA (shRNA) with a double complementary RNA fragment, which is connected on one side to a loop. The other end of these hairpin RNAs usually has a UU overhang. Subsequently, this hairpin RNA is cleaved in a cell to form a single stranded siRNA, which assembles with cytoplasmic protein complexes in order to silence its target mRNA found in the OPC.
The DNA from which the shRNA was transcribed, had to be made from our short DNA target sequences. For this, a loop sequence CAAGA was chosen, as it showed to be the most effective (McIntyre, G. J., 2011)
We used the Thermofisher Design Tool in order to create the DNA sequences for our vector.
COMPUTATIONAL MODEL:
To determine the effect of gene silencing, a metabolic model for the OPC using the IMAT algorithm was created. This model was built with the COBRA toolbox and, since the expression of a gene or protein is directly linked to the flux of the associated reactions, it works with expression data of one cell in an organism. The model used the Gurobi solver to perform flux balance analysis and single gene deletion analysis to identify the effects of gene silencing.
In order to check whether the silencing of the chosen gene has any effect, a fake BIOMASS reaction was added into the model that takes into consideration the materials needed by the cell to survive and duplicate. The model was then set to optimize for the BIOMASS reaction. Once the flux of the reaction is significantly lowered it can be concluded that it is harming the cell. As a framework, the mammalian RECON3D framework was chosen for practical reasons, as there is more data available and no arthropod framework exists. Furthermore, our targets were highly evolutionary conserved (Teo R., Möhrlen F., Plickert G., Müller W., Frank U., 2015; Hisayuki N., Yoshie O., 2015). It is important to highlight that these genes are not "conserved" in the traditional genetic sense but more in the functional pathway sense - meaning the genes are different, but they perform the same function and use similar pathways. Hence, they should remain functionally intact using mammalian analogues of a drosophila melanogaster gene expression data set. The model did not directly output the interaction of the siRNA but the expected effect: a gene knockout.
MATHEMATICAL MODEL:
With the aim to better understand which factors influence the spread of the OPC, we made a mathematical model of the OPC population changes in an area. This model explores how an initially small, yet stable population of OPC in an area can suddenly lead to an outbreak. The model is heavily based on D. Ludwigs famous Qualitative Analysis of Insect Outbreak Systems model, which is better known as the Spruce Budworm Model. The formula consists of a dynamical system model with multiple steady states; the main formula is split into two sections: a growth section consisting of an intrinsic growth rate and carrying capacity, and a second aspect consisting of the predation. When these sections are equal to each other, the growth rate becomes 0 and the population remains stable. However, depending on the environmental conditions, there are between 1-3 steady states, which are states that occur when the population reaches (or decreases to) the stable population level and the ecosystem is in balance.
PARTS
EXPERIMENTS
LAB BOOK
