Part:BBa_K5160009
SPS-NTPP
Overview
In this year's project, SZU-China did something interesting - it innovatively created a new type of tomato artificial sweetener production space station. By using the E8 promoter, we successfully expressed the sweet protein Thaumatin in tomatoes. However, we noticed that even with the use of fruit maturation-specific promoters, it was difficult to prevent the degradation of the sweet protein Thaumatin in tomato fruits, especially during the period of cell rupture when tomatoes mature. Therefore, on the original basis, we innovatively used the sweet potato sporamin-N-terminal prepeptide (SPS-NTPP) vacuolar targeting signal peptide. SPS-NTPP is a vacuolar targeting signal peptide derived from sweet potato sporamin. By adding SPS-NTPP at the N-terminus of Thaumatin, we successfully transported Thaumatin into the vacuoles for storage without any impact. Moreover, SPS-NTPP is derived from plants and can be digested by the human body into common amino acids for absorption, which is harmless to human and environmental health. It is evident that SPS-NTPP is a promising signal peptide. We have filled in the information about Thaumatin here and hope to provide inspiration and reference for iGEM teams interested in this.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 70
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 70
- 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 70
- 1000COMPATIBLE WITH RFC[1000]
Biological Knowledge
Sweet potato sporamin and N-terminal prepeptide (SPS-NTPP) is a vacuolar targeting signal peptide derived from the tubers of sweet potatoes, which targets sporamin to the vacuoles for storage in sweet potatoes. It consists of a 21-amino acid signal peptide and a 16-amino acid prepeptide (the 16 prepeptides will be cleaved after translation). After the protein is processed in the endoplasmic reticulum, it is further processed in the Golgi apparatus through COPII vesicles, and then targeted to the vacuoles through the SPS-NTPP vacuolar sorting determinant (VSD). It binds to specific sorting receptors (VSR) on the vacuolar membrane to transfer the protein into the vacuoles for storage.
Currently, SPS-NTPP has been successfully demonstrated to target the correctly expressed and active recombinant human α(1)-proteinase inhibitor to vacuolar storage in tomato leaves. However, it did not alter any morphology of the tomatoes, and all transgenic plants were phenotypically normal, healthy, and fertile. This indicates that the vacuolar signal peptide SPS-NTPP we selected can safely achieve the intended goal—targeting the heterologously expressed protein to vacuolar storage in tomatoes.
Design
Pathway Design
Platform Selection
Due to time constraints, we were unable to experimentally verify SPS-NTPP on tomatoes. Therefore, we shifted our focus to a commonly used model organism in plant science—tobacco (Nicotiana benthamiana). As a plant frequently utilized in scientific research, tobacco (Nicotiana benthamiana) has the advantages of rapid growth, clear genetic background, and a variety of genetic editing tools. Moreover, what is more important is that tobacco (Nicotiana benthamiana), as an annual herb of the Solanaceae family, shares a high degree of homology with tomatoes. Additionally, the number, shape, and function of organelles in tomatoes and tobacco (Nicotiana benthamiana) are essentially the same. Thus, choosing tobacco (Nicotiana benthamiana) as the plant for the proof-of-concept of SPS-NTPP is, to a certain extent, credible and reliable.
Design
Therefore, to verify that SPS-NTPP can guide Thaumatin to target vacuoles, we added the SPS-NTPP signal peptide to the N-terminus of Thaumatin and connected an EGFP fluorescent protein behind it, whose fluorescence can indicate the location of the sweet protein Thaumatin. Under a confocal microscope, compared with the control group, we can determine whether SPS-NTPP can target Thaumatin to vacuolar storage.
Construction
Based on the aforementioned concept, we constructed two plasmids, pGD_SPS-NTPP-Thaumatin-EGFP and pGD_Thaumatin-EGFP, and introduced them into Agrobacterium GV3101 (Figure 2). However, since tomatoes are not traditional model organisms, and considering the time cost and our immediate objectives, we chose to 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.
Characterization
The results show that the green fluorescence, which seems to evade the red fluorescence (autofluorescence of chloroplasts), is roughly distributed in the central vacuole of the cell, preliminarily proving that SPS-NTPP has the function of guiding the directional transport of proteins to the vacuole. (Figure 3)
Application Prospects
In summary, the SPS-NTPP vacuolar targeting signal peptide is a useful tool in plant synthetic biology. In our design, SPS-NTPP is used to target Thaumatin to the vacuoles for storage, thereby increasing the content of Thaumatin in tomatoes. We have characterized SPS-NTPP this year as described above, and we hope to provide inspiration and suggestions for future iGEM teams interested in this, creating a more diverse and colorful iGEM community.
Reference
[1] Matsuoka K, Nakamura K. Propeptide of a precursor to a plant vacuolar protein required for vacuolar targeting. Proc Natl Acad Sci U S A. 1991 Feb 1;88(3):834-8. doi: 10.1073/pnas.88.3.834. PMID: 1992474; PMCID: PMC50908.
[2] Differential subcellular targeting of recombinant human a1-proteinase inhibitor influences yield, biological activity and in planta stability of the protein in transgenic tomato plants,Plant Science,Volume 196, November 2012.
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