Part:BBa_K5160120
pCAMBIA1301-E8 promoter-brazzein-HA-NOS terminator
Description
Transgenic technology can introduce exogenous gene sequences into the host genome, and thus cause heritable changes in biological traits. Therefore, it is often used to improve agricultural production efficiency, enhance crop stress resistance, improve agricultural product quality and protect the environment. For a long time, this method is not only a traditional breeding method, but also an important help for the production of bioreactor products. We decided to use transgenic methods to introduce Brazzein gene into tomato callus by utilizing the integration ability of Agrobacterium Ti plasmid to achieve heterologous expression.
When we were looking for a sweet protein, we noticed Brazzein, a sweet protein that is not inferior to Thaumatin. But as it is not certified for safety, it is not currently available on the market. But we have also characterized it in transgenic tomato and proposed a novel production system for Brazzein, creating a richer sweet world and providing inspiration and advice to the interested iGEM team thereafter.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 2187
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 2187
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 2536
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 2187
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 2187
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 2260
Illegal BsaI.rc site found at 2539
Usage and biology
The transgene technology is based on a segment of T-DNA present on Agrobacterium plasmids. Agrobacterium tumeto inserts T-DNA into the genome of the target plant after invading the cell through the wound of the plant.
Agrobacterium Ti plasmid is a double-stranded covalently closed circular DNA molecule. It contains 4 functional regions, namely T-DNA region, Vir region, Con region and Ori region. The T-DNA region is a fragment that can be transferred and integrated into the plant genome. The protein encoded by Vir region is involved in the processing and transfer of T-DNA. Con region was associated with plasmid binding and transfer. The Ori region is the plasmid replication initiation site.
When the plant gets damaged, cells at the wound site secrete large amounts of phenolic compounds. These phenolics can induce the expression of the Vir gene in Agrobacterium sp. Agrobacterium, through chemotactic movement, recognizes and attaches to plant injured sites. The expression product of the Vir gene cuts a single strand of T-DNA from the Ti plasmid to form single-stranded T-DNA. Subsequently, the single-stranded T-DNA is combined with the expression products of the Vir gene and the Con-gene to form a complex. This complex transfers T-DNA to plant cells through contact between agrobacterium and plant cells. Upon entry into plant cells, T-DNA is able to randomly integrate into plant chromosomes. This process can be single-copy or multi-copy, but will usually be preferentially selected near the coding or regulatory regions of genes.
E8 promoter
The E8 promoter has a highly specific expression pattern in tomato fruits. It is mainly activated at specific stages of tomato fruit development, driving the preferential expression of downstream genes in fruit tissues. E8 promoter activity is significantly higher in fruit compared to other tissues such as roots, stems, and leaves. It can be used to achieve the precise regulation of specific genes in fruits. By linking the target gene to the E8 promoter, the target gene can be expressed only in the tomato fruit and unwanted effects in other tissues can be avoided. Please see BBa_K5160002 for more information.
Brazzein
Brazzein is derived from the pulp of Pentadiplandra brazzeana Baillon (P. brazzeana), which grows in the African rainforest. Each fruit has a red shell-like exocarp, beneath which three to five kidney-shaped seeds are surrounded by a thick layer of soft red flesh, which contains Brazzein. Brazzein is the smallest sweet protein with a peptide chain consisting of 54 amino acids and a molecular weight of only 6.5Kda.
Brazzein induced sweet taste perception by binding to T1R2 and T1R3 sweet taste receptors on the human tongue. At the same time, the flavor characteristics of Brazzein are similar to those of sucrose, and the sweetness of brazzein is 500-2000 times that of sucrose. In addition, Brazzein is a protein with excellent thermal stability and acid resistance. It was able to maintain its protein identity during 2 hours incubation at 98°C, 4.5 hours incubation at 80°C, and 4 hours incubation at 80°C in the 2.5-8pH range. See BBa_K5160004 for details.
HA
HA-tag is a protein tag based on Human influenza hemagglutinin antigen. The chemical essence of HA-tag is a short amino acid sequence derived from amino acids 98-106 of human influenza hemagglutinin, namely YPYDVPDYA. The HA tag is widely used as an epitope tag in expression vectors and is beneficial for protein detection, isolation, and purification because it does not interfere with the biological activity or distribution of the protein. By adding the HA tag to the target protein, specific antibodies against the HA tag can be used to bind to the recombinant protein, so that immunohistochemistry (IHC), Western Blotting and other experiments can be performed conveniently. HA antibodies can also be immobilized on agarose beads for protein purification to improve purification efficiency. Many recombinant proteins can express HA tags, which can help to quickly obtain information related to their localization, expression or biological function.See BBa_K5160012 for details.
NOS terminator
A terminator on a plant expression vector that terminates the transcription of a gene.See BBa_P10401 for details.
Structural design
We used tomato fruit ripening specific promoter E8 promoter to express the Brazzein gene. The pCAMBIA1301_Brazzein plasmid (Fig 3.) was constructed using the binary vector pCAMBIA1301 and transformed into Agrobacterium Tumefacium GV3101. To verify the successful transformation, we performed pcr using specific primers and demonstrated the successful entry of our expression vector into Agrobacterium tumefaciens GV3101 by agarose gel electrophoresis.
Characterize
Agarose gel electrophoresis
Colonies were amplified by PCR with specific primers, and the resulting products were subjected to agarose gel electrophoresis. From the results (Fig 4.), it can be seen that the band of Brazzein (176bp) appears with a band near 100bp. This can verify that our transformed agrobacterium has carried the target sequence.
DNA assay
We selected positive bacteria to infect the callus, and when the callus grew to a certain extent, it was inoculated in rooting medium to cultivate. After the plant had grown leaves, the leaves were harvested to test whether the brazzein gene was present in the tomato genome. The DNA of brazzein (176bp) was detected(Fig 5.).
Proteins expression
We collected samples of leaves, flowers, and fruits, extracted proteins for Western Blot (WB) detection, and successfully obtained positive results near the 10 kDa band in the lanes for leaves and fruits (Fig. 6). This indicates that Brazzein (9.75 kDa) in the transgenic micro-TOM was successfully expressed, and this expression is specific to mature fruits. This marks the first milestone in our experimental process, demonstrating that transgenic tomatoes capable of stably inheriting and specifically producing sweet proteins in mature fruits have been successfully cultivated.
A: Results of brazzein protein detection in leaves of transgenic tomatoes with E8 promoter; B: Results of brazzein protein detection in flowers of transgenic tomatoes with E8 promoter; C: Results of brazzein protein detection in fruit of transgenic tomatoes with E8 promoter.
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