Part:BBa_K4251013
EGFP
EGFP
Contribution by Team 2024 SZU-CHINA
Group: SZU-CHINA iGEM 2024
Summary
EGFP is a fluorescent protein derived through genetic engineering modifications of the original Green Fluorescent Protein (GFP). It emits bright green fluorescence under ultraviolet or blue light, exhibiting a high quantum yield and significantly enhanced fluorescence intensity compared to the original GFP. EGFP also features strong fluorescence stability and high resistance to photobleaching, allowing it to maintain its fluorescence intensity during extended observation. It can be stably expressed in various organisms, including bacteria, yeast, plants, insects, and mammalian cells, with minimal impact on cell growth and function, thereby not interfering with normal biological processes. Currently, EGFP has been applied in areas such as gene expression research, protein localization, cell differentiation studies, and bioimaging technologies.
In our project, we added the SPS-NTPP vacuolar targeting signal peptide to the N-terminal of Thaumatin. To verify whether SPS-NTPP could successfully express in tomatoes and target Thaumatin to the vacuole, we appended the EGFP fluorescent protein sequence to the C-terminal of Thaumatin. The fluorescence emitted by EGFP can indicate the location of Thaumatin. Subsequently, we utilized a confocal microscope to observe the distribution and focusing of fluorescent foci, thereby validating the function of SPS-NTPP.
Source
GFP, the precursor of EGFP, was originally discovered in a jellyfish species known as Aequorea victoria. GFP can emit green fluorescence under blue or ultraviolet light, and EGFP further enhances its fluorescence intensity and stability based on this property.
Design
Based on the aforementioned concept, we constructed plasmids, pGD_SPS-NTPP-Thaumatin-EGFP , and introduced them into Agrobacterium GV3101 (Figure 2). We perform transient infiltration in Nicotiana benthamiana, commonly known as Benthamiana tobacco, and then used confocal microscopy to compare the distribution and aggregation of fluorescence foci.
Fig 1. pGD_SPS-NTPP-Thaumatin-EGFP plasmid map.
Characterization
The results show that EGFP can emit green fluorescence well under blue or ultraviolet irradiation, and its fluorescence intensity and stability are higher than GFP. (Figure 2).
Fig 2. The first and second rows consist of confocal microscopy images of tobacco leaf sections post-infection, and the third and fourth rows consist of confocal microscopy images of tomato leaf sections post-infection.
The first column displays the green fluorescent protein (GFP) fluorescence images, the second column presents the autofluorescence images of the chloroplasts, the third column comprises the bright-field illumination images, and the fourth column illustrates the superimposed images.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |