Part:BBa_K5160121
pGD-35S promoter-SPS-NTPP-thaumatin-EGFP-NOS terminator
Overview
This year, we did something interesting - we created a new type of tomato sugar substitute production system. Using tomato as a chassis, we successfully expressed the target protein Thaumatin in tomato induced by a tomato fruit specific ripening promoter. However, we noticed that cell lysis during tomato ripening would fail the target protein Thaumatin to stabilize. Therefore, we used SPS-NTPP, a vesicle signal peptide from sweet potato spores, to localize Thaumatin into tomato vesicles for storage. We then constructed the pGD-35S promoter-SPS-NTPP-thaumatin-EGFP-NOS terminator vector with the addition of EGFP fluorescent protein. By localizing the fluorescence emitted by the EGFP fluorescent protein, we could know whether Thaumatin was successfully directed to the vesicle for localization, thus verifying whether SPS-NTPP has the function of vesicle localization.
Sequence and Features
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- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 1966
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 911
- 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 911
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 66
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Here is some information related to pGD-35S promoter-SPS-NTPP-thaumatin-EGFP-NOS terminator plasmid.
CaMV 35S promoter
The CaMV 35S promoter refers to the 35S promoter from cauliflower mosaic virus (CaMV). This promoter guides the synthesis of 35S RNA during the infection of plants by CaMV and enables efficient expression in many dicotyledonous plant tissues. As a constitutive promoter, the CaMV 35S promoter can initiate gene expression in all tissues. It is persistent, with relatively constant RNA and protein expression levels, but lacks spatial and temporal specificity. For detailed information on this component, please refer to BBa_K788000.
SPS-NTPP
Sweet potato sporamin and N-terminal pre-peptide (SPS-NTPP) is a vacuolar signal peptide derived from the sweet potato tuber, targeting and storing sporamin protein in the vacuoles of sweet potatoes. It contains a 21-amino acid signal peptide and a 16-amino acid pre-peptide (the 16 pre-peptides will be cleaved after translation). After the protein is processed in the endoplasmic reticulum, it is further processed in the Golgi apparatus through COPⅡ, and then targeted to the vacuoles through the VSD of the SPS-NTPP vacuolar sorting decision cluster, binds to the specific sorting receptor (VSR) on the vacuole, and then transfers the protein to the vacuole for storage. For detailed information on this component, please refer to BBa_K5160009.
Thaumatin
Thaumatin is derived from the aril of the tropical plant Thaumatococcus daniellii (Benth). Thaumatin contains two types: thaumatin I and thaumatin II. Thaumatin II has a higher sweetness, 3000 times that of sucrose. It is composed of 207 amino acids, and the amino acids are folded into eight disulfide bonds. Therefore, its protein structure is stable, with heat and acid stability. For detailed information on this component, please refer to BBa_K5160003.
EGFP
EGFP is a fluorescent protein obtained by genetically engineering the original green fluorescent protein (GFP). EGFP can emit bright green fluorescence under ultraviolet or blue light, with a high fluorescence quantum yield and significantly better fluorescence intensity than the original GFP. It also has strong fluorescence stability and high photobleaching resistance, which allows it to maintain fluorescence intensity during long-term observation. EGFP can be stably expressed in various organisms, including bacteria, yeast, plants, insects, and mammalian cells. It has a minimal impact on cell growth and function and does not interfere with normal biological processes. EGFP has been used in fields such as gene expression research, protein localization, cell differentiation research, and bioimaging technology. For detailed information on this component, please refer to BBa_K4251013.
NOS terminator
The nopaline synthase terminator (NOS terminator) plays an important role in genetic engineering, especially in plant expression vectors. It ensures that the transcription process of the target gene in plants terminates at a specific location, thus avoiding unnecessary gene expression extension and ensuring the accuracy and efficiency of gene expression. Therefore, it is commonly used to terminate the transcription of the target gene in plant vectors, thereby improving expression efficiency. For detailed information on this component, please refer to BBa_P10401.
Source
35S promoter
The CaMV 35S promoter is the 35S promoter derived from cauliflower mosaic virus (CaMV), used to initiate the expression of the target gene.
SPS-NTPP
Sweet potato sporamin and its N-terminal pre-peptide (SPS-NTPP) is a vacuolar targeting signal peptide originating from the sweet potato tuber, which targets and stores sporamin protein in the vacuoles of sweet potatoes.
Thaumatin
Thaumatin is derived from the aril of the tropical plant Thaumatococcus daniellii (Benth). Two types of thaumatin are found in the plant: thaumatin I and thaumatin II.
EGFP
The precursor of EGFP, GFP, was originally discovered in a jellyfish scientifically known as Aequorea victoria. GFP can emit green fluorescence under blue light or ultraviolet light, and EGFP further enhances its fluorescence intensity and stability.
NOS terminator
The nopaline synthase terminator (NOS terminator) is primarily derived from Agrobacterium tumefaciens. In genetic engineering, especially in plant genetic engineering, the NOS terminator is often used as an important component in plant expression vectors to mark the end of the transcription of the target gene
Design consideration
In our design, we added the SPS-NTPP vacuolar signal peptide to the N-terminus of Thaumatin to increase the storage of Thaumatin in tomatoes. To compare with the experimental group and prevent experimental errors caused by accidental factors, we set up a control group and constructed the corresponding plasmids.
Characterization
Pathway Design
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.
Result
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)
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