~Our Experimental Design~

In our quest to provide a solution for V. dahliae and create THAELIA, our primary goal was to assess all the different parts of the design experimentally, separately, in order to demonstrate the overall feasibility of our design. We approached our system step by step to gain insights and improve our approach. While our experiments provided insights into different aspects of our design, more experimental and biological repeats are needed to fully characterize the system and ensure the reliability of our data. A larger number of repeats would reduce variability and provide a clearer picture of how our system behaves under various conditions. However, within the time constraints, we tried to gather as much data as possible. Our work provides a starting point for future studies to expand upon.

Experiment	Short Description
Experiment 1: Characterization of regulatory components of T7 polymerase production system and eTEV production device	We conducted plate reader assay using E. coli BL21 DE3 cells containing the T7 expression system, which share similar traits with P. putida cells (our Project Design chassis). Our aim was: 1) to identify the most suitable promoter for the T7 polymerase production system, 2) to evaluate various RBSs for the P3.1 stationary promoter. We measured sfGFP levels and OD600.
Experiment 2: Further characterization of BG37 promoter and evaluation of T7 polymerase system	We conducted experiments with E. coli DH5a cells, which do not contain the T7 polymerase system. Using these cells, we aimed to better characterize our BG37 promoter by testing different sources of carbon and backbones and measuring the differences observed. We also aimed to determine if our T7 polymerase production device functions as a level 2 construct. We measured sfGFP levels and OD600
Experiment 3: Further characterization of BG37 promoter in our proposed chassis	We conducted experiments with P. putida KT2440, which is our Project Design chassis. Our goal was to further characterize the BG37 promoter by measuring the sfGFP levels and OD600
Experiment 4: Evaluation of gene silencing in Verticillium dahliae	We conducted experiments in order to determine efficiency of our gene silencing system targeting V. dahliae. We produced dsRNA in vitro to treat fungal cultures. We measured OD595 and used RT-qPCR to compare fungal growth and gene expression levels.

~Experiments~

Experiment 1: Characterization of regulatory components of T7 polymerase production system and eTEV production device

Our system is built on two main functions: 1) dsRNA production via T7 polymerase, and 2) OMV generation through TEV protease activity. To ensure optimal performance, our first objective was to test and identify the most suitable promoters for regulating T7 polymerase and TEV production. Given that our design requires distinct regulation across growth phases, we aimed to find the best promoters to drive T7 polymerase expression during the exponential phase and eTEV production in the stationary phase.

Figure 1: The regulation of our system: 1) T7 polymerase is produced during the exponential phase, leading to dsRNA production, 2) eTEV is produced during the stationary phase, leading to OMVs production

Experiment 2: Further characterization of BG37 promoter and evaluation of T7 polymerase system

Our next goal was to further characterize the BG37 promoter to ensure that it is activated specifically during the exponential phase. In our design, the BG37 promoter regulates T7 production, which drives the production of dsRNA. To evaluate its functionality, we first tested the BG37 promoter in a Level omega construct, where it controls T7 production, with an sfGFP reporter gene positioned downstream of the T7 promoter. This step was important, as testing in a Level 2 construct is as close to the proposed design as possible for a first evaluation of the functionality of the promoter.

Figure 2: BG37 promoter is responsible for T7 polymerase production

Additionally, understanding that various growth conditions can significantly influence promoter activity, we focused on testing the BG37 promoter under different carbon sources. The choice of carbon source can affect cellular metabolism, growth rates, and energy availability, all of which may impact the promoter’s functionality [2]. To evaluate this, we used an sfGFP reporter gene positioned downstream of the BG37 promoter to assess how different carbon sources, specifically citrate and glucose, influence its performance [1]. Moreover, since various plasmid backbones have different characteristics (e.g. origin of replication) that are needed to work in different chassis, we compared the pSEVA23g19g1 vector with the Golden Braid pDGB3a1 vector, starting with E. coli DH5α as an intermediate step before transitioning to P. putida. Simultaneously, we validated that its function is consistent across different genetic backbones, which reduces the likelihood of vector-specific behavior or artifacts [2].

Experiment 3: Further characterization of BG37 in our design chassis: P. putida

We chose to further characterize the BG37 promoter in P. putida, our proposed chassis, to verify its autoinducible properties during the exponential phase. By using sfGFP as a reporter gene and comparing BG37 with well-characterized promoters: J23119 (Anderson constitutive promoter) and OsmY (stationary phase-specific promoter) , we aimed to assess its strength and activation across different growth stages in P. putida. This allows us to determine how effectively BG37 performs in our system during key phases of cell growth [3].

Experiment 4: Evaluation of gene silencing in vitro

While designing our project and reading about RNAi our interest was heightened. Could we actually observe the impact of dsRNA on the fungus? Fascinated by this possibility, we designed this experiment with the aim to assess the effect that dsRNA has on the fungus. To be able to do that though we first have to create the DNA template needed for the in vitro transcription for the production of dsRNA and cultivate V. dahliae. To obtain our results we measured OD595 and performed an RT-qPCR reaction. In the Read More button below you can see in more detail the process of this experiment and our results. All protocols and photos are also provided on this page.

The other part of our system that we wanted to test was the effect of dsRNA on V. dahliae. We designed this experiment to investigate how dsRNA influences both fungal growth and expression levels of the target genes. We chose genes that according to literature are necessary for the correct development and virulence of the fungus. To determine whether dsRNA targeting these genes impacts fungal growth, we measured OD595, while gene expression levels were assessed using RT-qPCR.

~Materials~

~Protocols~

General Recipes

General Protocols

Working with E. coli

Working with P. putida

Evaluation of gene silencing

~Lab Book~

Throughout our journey, we made sure to keep a detailed lab book to reference our past experiments and tweak any steps we deemed important. When conducting a protocol, each member of the Experimental Wetlab Team took note of their actions. This facilitated the improvement of our experiments and ensured cohesion in our practices, as each member could reference the notes of another member at any time.

References

[1] Blázquez, B., et all. (2023). Golden Standard: a complete standard, portable, and interoperative MoClo tool for model and non-model proteobacteria. In Nucleic Acids Research (Vol. 51, Issue 19, pp. e98–e98). Oxford University Press (OUP). https://doi.org/10.1093/nar/gkad758

[2] Zobel, S., Benedetti, I., Eisenbach, L., de Lorenzo, V., Wierckx, N., & Blank, L. M. (2015). Tn7-Based Device for Calibrated Heterologous Gene Expression in Pseudomonas putida. In ACS Synthetic Biology (Vol. 4, Issue 12, pp. 1341–1351). American Chemical Society (ACS). https://doi.org/10.1021/acssynbio.5b00058

[3] Esteban Martínez-García, Angel Goñi-Moreno, Bryan Bartley, James McLaughlin, Lucas Sánchez-Sampedro, Héctor Pascual del Pozo, Clara Prieto Hernández, Ada Serena Marletta, Davide De Lucrezia, Guzmán Sánchez-Fernández, Sofía Fraile, Víctor de Lorenzo, SEVA 3.0: an update of the Standard European Vector Architecture for enabling portability of genetic constructs among diverse bacterial hosts, Nucleic Acids Research, Volume 48, Issue D1, 08 January 2020, Pages D1164–D1170, https://doi.org/10.1093/nar/gkz1024

[4] Pédelacq, J.-D., Cabantous, S., Tran, T., Terwilliger, T. C., & Waldo, G. S. (2006). Engineering and characterization of a superfolder green fluorescent protein. Nature Biotechnology, 24(1), 79–88. doi:10.1038/nbt1172