eyGFP_uv

BBa_K4844000 - eyGFP_UV

Usage: the next-generation Plant report gene: eyGFP_UV

Green fluorescent protein (GFP) has been widely used for monitoring gene expression and protein localization in diverse organisms. However, highly sensitive imaging equipment, like a fluorescence microscope, is usually required for the visualization of GFP, limiting its application to fixed locations in samples. This is due to the wave Excitation length and emission wavelength being too close, therefore an emission filter is needed.

Thus, we are excited to introduce a new plant report gene, called eYGFP-uv (BBa_K4844000), in transient expression we observed bright fluorescence under UV light on tobacco leaves. GFPuv (a GFP variant) was optimized for maximal fluorescence to be observed by naked eyes under UV light instead of using a fluorescence microscope.

Biology: transient expression of eyGFP(UV)

To transiently express our eyGFP, a patented carbon nanodot-based tracked, transformation, translation, and trans regulation (TTTT) technique invented by our team members and Jianhuang's lab at Soochow University was used to deliver our vector into plants. (More about TTTT can be found at https://2023.igem.wiki/sz-shd/plant ; Our vector sequence can be downloaded at the supplementary material page https://2023.igem.wiki/sz-shd/experiments)

Characterization

Once the tobacco plant has been transformed with carbon nanodots for three days, bright fluorescent can be visualized with the naked eye under the light source of a 398nm UV flashlight. We also ran a western blot using the protein extraction of tobacco tissue samples and anti-flag-tag antibodies. A clear band can be observed on the membrane (27.9 kDa). (Protocols can be downloaded at the supplementary material page https://2023.igem.wiki/sz-shd/experiments.)

Further Application

Therefore, the successful design and construction of our report gene are the solidary part of our low phosphate phytosensor. More potential applications of this report gene are waiting for us to explore.

Based on this report gene, a multi-level low noise amplifier gene circuit has been designed and tested by our team.

More info on this part:

https://2023.igem.wiki/sz-shd/engineering#construction

References:

Chin, D.P., Shiratori, I., Shimizu, A. et al. Generation of brilliant green fluorescent petunia plants by using a new and potent fluorescent protein transgene. Sci Rep 8, 16556 (2018). https://doi.org/10.1038/s41598-018-34837-2

Sequence and Features

Assembly Compatibility: