We as the MSP-Maastricht team have identified four main OPC target genes. These include, the gene for the subunit 2 of the allergenic protein Tha p2 that can be found in the urticating setae of the caterpillars (Berardi, Battisti & Negrisolo, 2015); Pro2 photolyase gene that is involved in proline metabolism; the EF-1a gene for elongation factor-1 alpha which takes part in the protein synthesis elongation phase; as well as, Wg gene for wingless protein, that is thought to be involved in numerous processes of the caterpillar's development (Simonato et al., 2013).
siRNA + BLAST
Based on the designing guidelines and BLAST research for the specificity, the following sequences within target genes were identified (Table 1).
Table 1. For Tha p2, 5 DNA sequences were identified as target sites for the siRNA. 2 DNA sequences were identified for Wg and 5 for the Pro2 partial gene. For EF-1a gene 2 DNA sequences were found.
shRNA
Based on the obtained siRNA the following DNA sequences for the shRNA were designed.
Table 2. For Tha p2, 5 DNA sequences were identified as target sites for the siRNA. 2 DNA sequences were identified for Wg and 5 for the Pro2 partial gene. For EF-1a gene 2 DNA sequences were found.
PLASMID DESIGN
T7-polymerase is prone to read-through transcription which is why we decided to include a termination signal in order to increase the yield of effective product RNAs produced by our strain (Sturm, Saskoi, Tibor, Weinhardt, & Vellai, 2018). Furthermore because the shRNA sequences were not possible to produce as dsOligos (because of their secondary structure) we decided to go with a universal insert (inserted with Gibson assembly) to which we can add any shRNA by using restriction enzymes. Finally, this design allows us to add multiple of the expression cassettes (consisting of promotor, shRNA and terminator) together into one construct, so we can later on create an “oakshield” strain expressing all the promising shRNAs of different targets.
MODEL RESULTS
DYNAMICAL SYSTEMS
From the model it is found that when 3 steady states exists, at least one of the steady states is unstable and, when environmental conditions change in just the right way, a stable point can merge with an unstable one and become a single half stable point before disappearing, thus leaving only one steady state. This explains how the population in an area can suddenly explode.
Another interesting observation when not parameterising the model is that due to the small effect of predation (β), (R) will always be relatively large. This implies that the system will be more sensitive to changes in (Q).
One of these factors is (α), in which a reduction of alpha increases (Q). However, since reducing (α) causes an increase in (R), it means that it cancels itself out. The only factor remaining then is (K’), the carrying capacity.
It can be concluded that the biggest factor in determining the population is not predation or death rate, but the maximum carrying capacity of the ecosystem. This demonstrates that the population, if left alone, would keep increasing until the ecosystem collapses. Furthermore, it implies that the best approach to combat the OPC would be to reduce the general carrying capacity of the caterpillar instead of trying to directly combat the caterpillar. For more in-depth explanation of the model visit our contribution page.
METABOLIC MODEL
A model containing 5407 reactions, 3526 metabolites, and 2248 genes was created.
In this model 23 genes had a BIOMASS reduction greater than 10%, which is considered lethal (Brunk et al., 2018).. 2 out of our 4 proposed genes were in that list: Pro2 and Acyl-CoA desaturase were classified as lethal, with a reduction of 16% and 13% in biomass production respectively. The WG gene was not lethal as there seemed to be many backup genes and alternative pathways. The model was not able to test the EF-1a since it was not programmed to take into account transcription.
WET LAB RESULTS
Transfections
The final constructed plasmids with our siRNA inserts were first cloned into DH5α high copy number cells and subsequently into HT115(DE3) cells. Both times pUC19 plasmids were used as positive control and showed colony growth on the plates, indicating a successful transfection process. The final HT115(DE3) cells capable of siRNA expression were plated on to ampicillin and tetracycline containing selection plates. To confirm the success of our transfection and construction method, we decided to first only transfect one siRNA per gene (Pro2_A, Wg_A, EF1a_A, Tha p2_A). Colonies grew and overnight cultures were prepared from single colonies for further analysis (Figure 1-4).
Polymerase Chain Reaction
Touchdown PCRs were run on OPC DNA-extract samples resulting in amplicons of our desired target genes. Specific amplicons of the correct lengths were produced (Pro 2=651 bp, Wg= 164 bp, EF1a=541 bp, Tha p2= 269 bp). The bands of the Wg and Tha p2 were shorter than expected and after multiple PCRs and changes of primers, the bands did not become more intense. Therefore, we decided to sequence the two most promising bands and prepared these genes for sequencing, together with the other amplicons.
Sequencing
The PCR amplicons of our target genes were sent to Macrogen for targeted sequencing (Figure 5):
The Pro2 amplicons were almost 100% identical to our reference sequence and the amplicon spanned all 5 siRNA target regions (Pro2_A, B, C, D, E siRNAs) with 100% identity.
The EF1a amplicons were almost 100% identical to our reference sequence and the amplicon spanned both of the 2 siRNA target regions with 100% (EF1a_A) and 86% (EF1a_B) identity.
The Wg amplicon only yielded results from the reverse primers sequencing. However, the short amplicon produced spans includes both siRNA target regions with 100% identity for both of the 2 siRNA target regions (Wg_A) and (Wg_B).
The Tha p2 amplicon was split into two amplicons spanning the whole gene but only one primer produced sequencing results. Thus, only 3 out of 5 potential siRNA target regions were included in the produced amplicon (Tha p2_C, D, E siRNAs) However, all of these 3 siRNA target regions were 100% identical to our reference sequence.
Confirmation of Successful Gibson Assembly of Empty Expression Vector
To confirm that our expression cassette was indeed inserted into the L4440 Plasmid, we performed a restriction digest with the two enzymes PciI and NgoMIV on the plasmid isolated from the bacterial strain used for transfections. Since the Gibson assembly removed the the PciI & NgoMIV restriction sites, the empty siRNA expression vector should not be cut, while the empty L4440 vector would be cut. This would leave us with the predicted bands on the gel. Our gel perfectly matches the predicted gel, confirming that the Gibson assembly of our siRNA expression vector was successful (Figure 6).
Unfortunately, our final HT115(DE3) E. coli strain did not seem to produce shRNAs after IPTG induction. To confirm that our siRNA inserts were assembled into our vector, we performed a restriction digest with EcoRI & BamHI to remove and detect our shRNA inserts. However, the predicted bands were not seen on the gel. This could be possibly due to low shRNA insert concentration and/or low ligation efficiency. However, we will sequence the produced plasmids to confirm the shRNA insert presence in the vector and repeat the insertion step if necessary (Figure 7).
Testing the Lifetime of Our Bacteria on Oak Trees
To determine the efficacy of our bacteria we wanted to test how long our bacterial strain could survive on the oak trees. To do this we collected oak branches and sprayed these with our HT115(DE3) E. coli strain. SRK hygiene monitoring swabs were used to swab our branches every 24 hours and 500 μL of isotonic solution were plated on to tetracycline selection plates. Spraying was performed twice, once with medium containing tetracycline and once with regular LB medium. The bacteria sprayed in LB could not be detected after 48 hours. However, the bacteria with tetracycline medium survived on the branches for 4 days. Of course, the tetracycline would have an effect on the microbiome of oak trees (Figure 8-9).
Figure 1. Selection plates with EF1a_A insert in HT115(DE3)E. coli strain.
Figure 2. Selection plates with Pro2_A insert in HT115(DE3)E. coli strain.
Figure 3. Selection plates with The p2_A insert in HT115(DE3)E. coli strain.
Figure 4. Selection plates with Wg_A insert in HT115(DE3)E. coli strain.
Figure 5. Schematic overview of siRNA target sequences in Wg, Pro2, EF1a and The p2 genes.
Figure 6. Predicted and obtained gels for Gibson assembly confirmation. Well 1: plasmid with expression cassette. Well 2: uncut plasmid without expression cassette.
Figure 7. Gel for shRNA DNA insertion, containing plasmids with EF1a_A, Pro2_A, The p2_A and Wg_A.
Figure 8. Oak branch testing set up for spraying with HT115(DE3) cells.
Figure 9. Spray bottle used in branch testing, filled with HT115(DE3) in LB medium.
