Part:BBa_K5160122
35S promoter-thaumatin-EGFP-NOS terminator
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 950
Illegal NotI site found at 987
Illegal NotI site found at 1089 - 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 2557
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 983
Illegal NgoMIV site found at 1149 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 66
Illegal BsaI site found at 2281
Illegal BsaI.rc site found at 2560
Description
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 target Thaumatin into tomato vesicles for storage. By targeting the fluorescence of the EGFP fluorescent protein, we can determine whether Thaumatin has been successfully directed into the vacuoles for localization. To exclude the impact of accidental factors and better judge whether Thaumatin has been successfully targeted to the vacuoles, a blank control group was set up, and the pGD-35S promoter-thaumatin-EGFP-NOS terminator vector was constructed on this basis. Here is some related information.
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.
Thaumatin
Thaumatin is derived from the aril of the tropical plant Thaumatococcus daniellii (Benth). There are two types of thaumatin found in the plant: 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, it has stable protein structure, 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.
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 known as Aequorea victoria. GFP can emit green fluorescence under blue or ultraviolet light, and EGFP has further enhanced 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.
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