Part:BBa_K5293015:Experience

Validating the NSs RNA silencing suppressor protein

One of the most important features of the pHREAC vector collection backbone is the NSs RNA silencing suppressor protein sequence. This component enables higher expression levels in engineered plants by defying their immune system and increasing the success rate of the Agrobacterium transformation. In the initial phases of our research, we were taking advantage of this interesting feature of the original pHREAC plasmid, which contained the NSs protein gene, by co-infiltration in our agroinfiltration media containing our localization-targeting plasmid. In an effort to reduce complexity and steps in our protocol, we searched for a system in which a single plasmid contained both the localization tags as well as transgenic silencing inhibition. As no such thing exists or is easily findable, we set out to build our own library of plasmids: our very own pHREAC vector collection - containing all the necessary features for biofarming.

Using a preliminary version of the chloroplast-localized pHREAC vector (BBa_K5293016), we compared the difference in expression of His-mScarlet-TEV-Semaglutide (BBa_K5293005) with the pCLGGx one. As shown in Figure 1 and 2, the band intensities suggest higher levels of expression with the preliminary pHREAC vector containing the NSs protein gene compared to the pCLGGx that does not.

Figure 1. Anti-His Western blot analysis of crude extract lysates from N. benth. leaves engineered through Agrobacterium-mediated transformation to express His-mScarlet-TEV-Semaglutide (BBa_K5293005) in pCLGGx, co-infiltrated with the original pHREAC.

Figure 2. Western blot analysis of crude extract lysates from N. benth. leaves engineered through Agrobacterium-mediated transformation to express His-mScarlet-TEV-Semaglutide (BBa_K5293005) in a similar version of the pHREAC vectors (BBa_K5293016)

Validating the localization tags in pCLGGX with confocal fluorescence microscopy (His-mScarlet-TEV-Semaglutide)

We validated the efficient targeting of the PR1b + CTPP sequences for vacuolar compartmentalization with confocal fluorescence microscopy. Although the proper targeting investigation was performed with the pCLGGx vectors, this result still provides evidence of the proper targeting of PR1b + CTPP sequence.

Figure 1. Confocal microscopy 3D image of Agrobacterium-mediated transformed pavement cells in Nicotiana benthamiana, expressing the fusion protein His-mScarlet-TEV-semaglutide in pCLGGx localized to the vacuole. The construct was co-infiltrated with the pHREAC backbone for its suppressor of silencing feature. Images acquired with a Nikon A1 Plus microscope using a Prior Z Drive, with slides loaded with purified water. The image has a resolution of 1024x1024 pixels and a Z-stack of 48 slices (0.6 um step) at 60 fps. The numerical aperture was 1.1 and the refractive index was set to 1.333. The imaging zoom was 1.381 with a pinhole size of 20.434 um. The modality employed was widefield fluorescence and laser scan confocal.

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